NMDAR antagonists to prevent aging and aging-related conditions and diseases by increasing 20S proteasome activity

Abstract

The use of NMDAR antagonists provides compositions and methods for the treatment of dysfunction of protein homeostasis networks that interfere with the key signaling pathways and are often associated with a variety of human diseases. Importantly, the present invention provides information comprising at least one NMDA receptor blocker for use in the prevention and / or treatment of aging and aging-related conditions and diseases by increasing 20S proteasome activity.

Description

[0001] Instruction Manual Summary

[0002] The use of NMDAR antagonists provides compositions and methods for treating malfunctions of the protein homeostasis network, which interfere with key signaling pathways and are often associated with a variety of human diseases. Importantly, the present invention provides information comprising at least one NMDA receptor blocker for preventing and / or treating aging and aging-related conditions and diseases by increasing 20S proteasome activity. Background Art

[0003] Aging is associated with malfunction of protein homeostasis networks and perturbs key signaling pathways, and is often associated with a variety of human diseases. However, it is difficult to separate the effects of aging itself from those of age-related diseases (Vilchez et al., 2014).

[0004] Over the past few decades, there has been a dramatic increase in a group of medical conditions collectively referred to as “protein misfolding disorders,” which include Alzheimer’s disease (AD) and type 2 diabetes (Dobson, 2017). These protein misfolding disease conditions—virtually unknown a century ago—are now becoming frighteningly common (Chiti and Dobson, 2017). Many of these “modern diseases” can be attributed to the fact that we now live in unprecedented times in human history, and as we age, we are at greater risk for our protective mechanisms to fail (Hipp et al., 2014).

[0005] It is rapidly becoming increasingly apparent that protein misfolding disorders have enormous implications not only for individuals and their relatives, but also for society as a whole.

[0006] Furthermore, the economic burden of these conditions (due to the need for ongoing care of patients, often for years) is reaching levels that even the healthcare system cannot afford to ignore. For example, in the United States, the cost of AD alone was estimated at $200 billion in 2015 and is projected to exceed $1 trillion by 2050; thus, over the next 35 years, the cost of caring for AD patients in the United States alone is projected to reach $20 trillion (Alzheimer's Association - https: / / www.azm.org ).

[0007] More than 30% of newly synthesized proteins are misfolded due to errors in translation or post-translational processes (Chiti and Dobson, 2017). Oxidative stress is another factor that causes protein unfolding. After oxidative damage, proteins unfold and expose hydrophobic regions, which makes these proteins prone to aggregation.

[0008] The role of oxidative stress in age-related organ changes is generally considered to be the increase in reactive oxygen species (ROS) that accompanies aging, leading to functional changes, increased morbidity, and shortened lifespan (Kregel and Ziliang, 2007; Jung and Grune, 2013). Aerobic organisms frequently encounter ROS. Cells continuously generate oxidants and produce antioxidants. Although they try to achieve a healthy balance, organisms encounter many situations in which oxidant levels are no longer synchronized with the cell's detoxification systems, resulting in a potentially lethal condition known as oxidative stress.

[0009] There is a large body of information in the literature on increased amounts of oxidized proteins in skeletal muscle, heart, skin, liver, lymphocytes of various animal species (Jung and Grune, 2013). Oxidative stress has also been shown to play a major role in the initiation and progression of cardiovascular diseases, including atherosclerosis and hypertension, cerebrovascular disease (stroke), type 2 diabetes, cancer, several chronic neurodegenerative diseases (including AD and Parkinson's disease), osteoarthritis, osteoporosis, hearing loss, age-related macular degeneration, etc. Heart failure is the leading cause of hospitalization in people aged 65 years and older, and almost all patients with heart disease or at risk for heart disease have increased levels of oxidative stress (Jung and Grune, 2013; Jung et al., 2014).

[0010] According to generally accepted views, misfolded and aggregated proteins generated by oxidative stress are key pathological findings even in the natural aging process (Reichmann et al., 2018).

[0011] The accumulation of ROS has long been considered a major contributing factor to aging and age-related diseases, and the fact that aged tissues show increased oxidative damage remains indisputable.

[0012] Proteins are one of the major cellular targets of ROS. This is largely due to the large number of oxidation-sensitive amino acid side chains present in proteins. The most commonly observed oxidative modifications involve cysteine ​​and methionine, which react at very high rates with hydroxyl radicals or HOC1. In addition to the mostly reversible thiol and methionine oxidation reactions that are commonly used for redox regulation of protein activity, proteins tend to undergo a number of irreversible side chain modifications, including thiol peroxidation, carbonylation, and dityrosine formation. Newly synthesized polypeptide chains are particularly vulnerable, presumably because side chain modifications directly affect nascent protein folding. However, mature proteins are also sensitive. ROS react with residues during local unfolding events, shifting the equilibrium of proteins toward unfolding and aggregation. Hydroxyl radicals are considered to be among the most reactive ROS and are responsible for most of the oxidative damage experienced by proteins, lipids, and DNA once ROS levels are elevated. Extensively cross-linked and hydrophobically bound protein aggregates are combined with other oxidation products of lipids, carbohydrates, and nucleic acids and tend to accumulate within cells, giving rise to entities such as inclusion bodies, Heinz bodies, ceroid pigments, ‘senile’ pigments, and lipofuscin. Large aggregates, including amyloids, amorphous assemblies, or native-like assemblies, have been implicated in many human diseases and physiological processes, including aging and aging-related diseases (Reichmann et al., 2018).

[0013] A range of proteins involved in these processes (e.g., transthyretin, insulin, (β-2-microglobulin, superoxide dismutase) are natively folded. However, the vast majority of these proteins involved in protein misfolding disorders are particularly those that are relatively small intrinsically disordered proteins (IDPs). Disorder resides primarily in intrinsically disordered regions (IDRs) within otherwise well-structured proteins. Thus, the term IDP includes proteins containing IDRs as well as completely disordered proteins. Intrinsically disordered systems can also arise following proteolysis of otherwise larger proteins such as amyloid-β peptide and amyloidogenic fragments of gelsolin. Due to their lack of a stable 3D structure, IDPs are extremely sensitive to their environment, much more so than ordered proteins. Furthermore, this lack of fixed structure is often associated with extreme binding promiscuity of IDPs (Breydo et al., 2017).

[0014] Illustrative examples of amyloidogenic IDPs important for human diseases are listed in Table 1. The most notable examples of amyloidogenic IDPs include amyloid β (Aβ) peptide, tau (AD), α-synuclein (Parkinson's disease (PD) and other synucleinopathies), TAR DNA binding protein 43 (TDP-43), fusion in sarcoma (FUS) protein (amyotrophic lateral sclerosis (ALS)), and huntingtin (Huntington's disease). Many human functional amyloid proteins (cytoplasmic polyadenylation element binding protein (CPEB), T cell restricted intracellular antigen-1 (TIA-1), premelanosomal protein 17 (Pme117), secreted peptide hormones) and cell cycle related proteins including p53, p27, p21 (cell cycle, apoptosis).

[0015] Twenty percent of cellular proteins are classified as IDPs, and up to 41% of the eukaryotic proteome is predicted to contain IDRs. Proteins share no significant similarity in sequence, structure, or function.

[0016] The different conformational states adopted by proteins involve a series of highly complex equilibria that are thermodynamically and kinetically determined in properly functioning living systems by their intrinsic amino acid sequence and by degradation processes and other complex quality control mechanisms.

[0017] Protein quality control in cells is maintained by a protein homeostasis (proteostasis) network consisting of a system of protein synthesis, protein folding complexes, and protein degradation machinery.

[0018] Proteome imbalance is accompanied by widespread protein aggregation, in which abundant proteins exceeding solubility contribute most to the total load.

[0019] (Walther et al., 2017)

[0020] Therefore, the clearance of misfolded proteins is critical for cell survival because misfolding alters the three-dimensional structure of proteins, thereby impairing their biological activity and increasing their propensity to form toxic aggregates (Hartl, 2017).

[0021] Modulation of protein concentrations by regulating the proteolytic machinery has long been demonstrated to be a promising setting for developing therapeutic strategies against different aging and aging-related human diseases, such as neurodegeneration, cancer and autoimmunity, aging and aging-related diseases.

[0022] Therefore, increasing proteolytic capacity provides an important approach to maintain protein homeostasis (Njomen and Tepe, 2019).

[0023] The main pathway for the proteolytic machinery in cells is the proteasome system. Most enzymatic protein decomposition was originally thought to occur in the relatively harsh acidic environment of lysosomes. However, in the 1970s, Goldberg et al. demonstrated that cells lacking lysosomes were still able to carry out protein turnover through an autonomous ATP-stimulated proteolytic pathway. Both the 20S proteasome and the 26S proteasome were discovered, both named according to their Svedberg sedimentation coefficients (a reflection of their relative molecular mass), and both were thought to represent two independent entities within the cell until it was demonstrated that the 26S proteasome was composed of two 19S regulatory factors bound to the 20S core (Hipp et al., 2014).

[0024] Although there is evidence that chaperone-mediated autophagy is activated during oxidative stress responses, the proteasome represents the major proteolytic machinery for the removal of oxidized and misfolded proteins ( Njomen and Tepe, 2019 , Hipp et al., 2014 , Pohl and Dikic, 2019 ).

[0025] The proteasome is the primary protein degradation machinery in eukaryotes. Through its proteolytic activity, it removes damaged proteins and ensures the delivery of amino acids to support ongoing biosynthesis. The proteasome accounts for a staggering 1% to 2% of the entire proteome in healthy cells.

[0026] Proteasomal degradation is divided into two main categories:

[0027] ubiquitin-proteasome system (UPS) and ubiquitin-independent proteasome system (UIPS) (Njomen and Tepe, 2019).

[0028] At the center of the UPS is the proteasome as described by Yu and Matouschek (Yu and Matouschek, 2017). The human 26S proteasome consists of a barrel-shaped 20S core particle (CP) capped by one or two 19S regulatory particles (RP), also known as PA700. The 20S CP is a threonine protease composed of four stacked rings. The two inner β-rings contain three catalytic subunits (β5, β2, and β1) that exhibit chymotrypsin-like, trypsin-like, and caspase-like activities, respectively. The outer α-rings serve as gating channels that regulate the entry of substrates and the exit of products from the inner catalytic chamber. These outer rings also serve as docking surfaces for the 19S RP. The α-ring and β-ring each consist of heteroheptamer subunits; α1 to α7 and β1 to β7. The 26S proteasome (PA700) is formed when the 28-subunit CP is docked at one or both ends via the ATPase 19S cap. The 19S RP is responsible for the opening of the 20S gate, substrate recognition and binding, unfolding, and directing ubiquitinated substrates into the 20S CP (Yu and Matouschek, 2017; Jung et al., 2014)

[0029] Many other non-ATPase regulatory particles such as 11S complex (PA28) and PA200 also reversibly associate with 20S CP by docking on α-ring. 19S RP is composed of two subcomplexes, that is, the base and peripheral lid that directly interact with 20S. The base is composed of hexameric AAA-ATPase subunits (Rpt1 to Rpt6) and tetrameric non-ATPase subunits (Rpn1, Rpn2, Rpn10 and Rpn13). The ATPase activity in the base is essential for the unfolding of protein substrates, the opening of the gate and the translocation of substrates to the 20S core. The lid is composed of nine non-ATPase subunits (Rpn3, Rpn5 to Rpn9, Rpn11, Rpn12 and Seml). The lid, particularly the Rpn11 subunit, serves as a deubiquitinating enzyme. Rpn13, Rpn10, and other reversible-associated proteins, such as radiosensitive abnormal protein 23 (Rad23) and dual-specificity protein kinase 2, act as ubiquitin receptors that direct polyubiquitinated proteins to the proteasome. Rpn subunits also create docking sites for other proteins, including the proteasome-associated deubiquitinase, ubiquitin-specific peptidase 14 (USP14), and ubiquitin C-terminal hydrolase (UCH37) (Collins and Goldberg, 2017; Yu and Matouschek, 2017).

[0030] The 26S proteasome and ubiquitin-ATP-dependent proteolysis.

[0031] The 26S proteasome primarily targets structured proteins for degradation, although a certain proportion

[0032] Misfolded proteins, IDPs, and proteins containing IDRs are also degraded by 26S. Structured proteins produced due to degradation are tagged with chains of polyubiquitin, which act as degradation determinants for structured protein turnover. Polyubiquitinated proteins are recognized by ubiquitin receptors Rpn10 and Rpn13. The ubiquitin tag is then removed by deubiquitinating enzymes such as Rpn11, USP14, and UCH37. The ATPase activity at the base of 19S then unfolds the protein and directs the protein into the catalytic chamber for degradation (Collins and Goldberg, 2017).

[0033] Ubiquitin is a small protein (about 8 kDa) composed of 76 amino acids, consisting of seven lysine (K) residues at positions 6, 11, 27, 29, 33, 48 and 63, through which it can be attached to protein substrates. A series of well-defined enzymes, ubiquitin ligases (E1, E2 and E3) coordinate the attachment of mono- and polyubiquitin to proteins. Ubiquitin is first activated by the E1 ubiquitin activating enzyme in an ATP-dependent reaction, to which it is attached via a thioester bond. Subsequently, the activated ubiquitin is transferred to the cysteine ​​active site of the E2 ubiquitin-conjugating enzyme. Ubiquitin-protein ligase (E3) together with E2 catalyzes the transfer of ubiquitin to proteins destined for degradation (Collins and Goldberg, 2017; Yu and Matouschek, 2017).

[0034] The 20S proteasome and ubiquitin-independent proteolysis.

[0035] Unlike the 26S proteasome, which mainly degrades polyubiquitinated proteins, the 20S directly degrades misfolded proteins, oxidatively damaged proteins, as well as IDPs and IDR-containing proteins, and does not require the unfolding enzyme activity of the 19S base. In addition, 20S-mediated proteolysis does not require polyubiquitination of its substrates. IDPs and IDR-containing proteins that lack this three-dimensional structure are thought to easily pass through the α-ring gate of the 20S proteasome. Twenty percent of cellular proteins are classified as IDPs, and it is estimated that up to 41% of the eukaryotic proteome contains IDRs, indicating that the substrate library of UIPS may be quite large. These substrates are particularly relevant because they include proteins that accumulate in neurodegenerative diseases, such as amyloid beta, tau, TDP-43 and α-synuclein, prion protein (PrP), and polyglutamine repeats.

[0036] In cells, IDPs generally have a shorter half-life relative to structured proteins. Both UPS and UIPS have been shown to promote rapid proteasomal degradation of IDPs such as p21, p53, and p27 (Chiti and Dobson, 2017).

[0037] Although the degradation of oxidatively damaged proteins can occur through both ubiquitin / ATP-dependent (i.e., 26S-dependent) and ubiquitin / ATP-independent (i.e., 20S-dependent) mechanisms, various studies suggest that the 20S proteasome is more critical for the removal of damaged proteins (Jung and Grune, 2008; Jung et al., 2014).

[0038] It has been shown that the 20S proteasome can degrade oxidized proteins (e.g., histones, hemoglobin, superoxide dismutase) in vitro independently of ubiquitin / ATP. This phenomenon has been attributed to the recognition and interaction of the 20S proteasome with abnormally exposed hydrophobic patches in oxidatively damaged and unfolded proteins, which induce conformational changes in the 20S structure and promote the opening of the channel, followed by protein degradation (Davies, 2001, Ishii et al., 2015)

[0039] Therefore, the 20S proteasome complex is able to directly target oxidatively damaged proteins for cellular detoxification.

[0040] In part, this may be because the 20S proteasome is more resistant to oxidative stress than the 26S proteasome, as the 20S complex remains active even when treated with moderate to high concentrations of H2O2, whereas the 26S proteasome is more fragile, probably because the 19S particles have been observed to dissociate from the 20S core in the presence of H2O2 (Ishii et al., 2005; Davies, 2001). Upon incubation with a metal-catalyzed ROS-generating system (Cu2+ / H2O2), the 26S proteasome was converted into lower molecular weight protein species in a Cu2+ concentration-dependent manner, while the protein bands corresponding to the 20S proteasome were virtually unchanged, suggesting that changes in the 26S proteasome may be due to oxidative modifications of the 19S regulatory subunits (Jung and Grune, 2013; Jung et al., 2014; Jung et al., 2014).

[0041] The 20S proteasome plays a key role in the selective recognition and degradation of oxidized proteins, and is responsible for degrading approximately 90% of all intracellular oxidatively damaged proteins. It is noteworthy that the degradation of the 20S proteasome occurs in an Ub-independent manner. The main recognition motif of the substrate appears to be a hydrophobic patch exposed on the surface of the damaged protein. Studies have shown that under oxidative conditions or in response to mitochondrial dysfunction, cells can reversibly decompose the 26S proteasome to increase the level of free 20S particles (Davies, 2001, Aiken et al., 2011).

[0042] Probably the most important problem that can occur due to reduced proteasome activity is the accumulation of potentially toxic unfolded / misfolded proteins and protein aggregates. Impaired proteasomes will eventually affect cytoplasmic processes and all other important functions of organelles due to the accumulation of unfolded and damaged proteins. In addition, the accumulated waste products will also hinder the recycling of amino acids and Ub required for cell survival (Reichmann et al., 2018).

[0043] The accumulation of unfolded, misfolded, or damaged proteins severely impairs the functions of organelles and cells and has been recognized as a key factor in aging and various diseases.

[0044] Several reports indicate that one of the first detectable consequences of proteasome dysfunction is associated with mitochondria, which develop deleterious changes in their proteome. Mitochondria are the cellular powerhouses that produce energy through oxidative phosphorylation. These mitochondria also directly contact most, if not all, other cytoplasmic organelles (e.g., endoplasmic reticulum (ER), plasma membrane, and peroxisomes) to create specialized networks that control several cellular functions such as Ca2+ homeostasis, lipid synthesis, and apoptosis. Importantly, dysfunctional mitochondria are also a major source of oxidative stress through the aberrant production of ROS, which has profound implications for the pathogenesis of various diseases, especially neurodegenerative disorders and cancer, as well as aging.

[0045] Since inactive proteasomes also impair the ER-associated degradation (ERAD) pathway, severe ER stress is another inevitable consequence of sustained proteasome inhibition (Ding et al., 2007).

[0046] The proteasome pathway maintains mitochondrial plasticity and quality by controlling mitochondrial dynamics at several levels. Depending on the degree of stress, mitochondria trigger an adaptive response (moderate stress) and remove damaged organelles by mitophagy (persistent stress) or, if these measures fail, induce cell death (irreparable damage). These activities and processes are tightly linked. During moderate stress, as a first line of defense against mitochondrial dysfunction, the proteasome pathway is responsible for the outer membrane PQC (protein quality control) and the degradation of damaged proteins in a process known as outer mitochondrial membrane associated degradation (OMMAD). In addition, the proteasome pathway directly controls mitochondrial dynamics by ubiquitinating and degrading proteins involved in mitochondrial fusion and fission processes (Roussel et al., 2013).

[0047] Although the 20S proteasome has largely been considered a dormant protease that is activated only upon binding to a regulatory complex, increasing evidence has clearly demonstrated that the 20S core has a major role in overall protein degradation, independent of ubiquitin and ATP (Raynes et al., 2016). The 20S proteasome is the core unit of all proteasome systems and (in mammals) constitutes approximately 1% of total cellular protein (Raynes et al., 2016). The 20S proteasome is most abundant throughout the cytoplasm and nucleus. In addition, the 20S proteasome has been found to be directly attached to various organelle membranes, particularly the endoplasmic reticulum, where it degrades membrane-bound substrates (Hirsch and Ploegh, 2000).

[0048] Therefore, it is generally agreed that the proteasome is the first defense mechanism of the cell against the toxic stress of accumulated proteins induced by oxidative damage ( Njomen and Tepe, 2019 ; Jones and Tepe, 2019 , Korovila et al., 2017 ).

[0049] The role of the proteasome in aging

[0050] The accumulation of unfolded, misfolded, or damaged proteins severely impairs the functions of organelles and cells and has been recognized as a key factor in aging and various diseases.

[0051] During aging, oxidized proteins appear to aggregate and accumulate to abnormally high levels. Such large aggregates, including amyloids, amorphous assemblies, or native-like assemblies, have been implicated in many human diseases and physiological processes, including aging and aging-related diseases (Chondrogianni et al., 2015).

[0052] It is well known that the overall rate of protein degradation decreases as an organism ages. It is also known that a decrease in proteasome activity has been widely associated with aging and age-related diseases, including neurodegeneration. It is speculated that this decrease leads to a serious imbalance in protein homeostasis and the accumulation of damaged and / or misfolded proteins.

[0053] Species with extended lifespan and long-lived individuals within a given species exhibit higher proteasome activity and are less susceptible to diseases, including neurodegeneration (Chondsigianni et al., 2015).

[0054] Several studies have found that increased proteasome activity is beneficial, thereby reducing the accumulation of pathogenic proteins and delaying aging. For example, delivery of purified 20S to cells by direct injection has been shown to accelerate tau clearance. Inactivation of the deubiquitinating activity of the deubiquitinating enzyme USP14, allosterically activated the proteasomal degradation of poly-Ub-conjugated proteins, and identified small molecules that are consistent with the model accelerated tau turnover in vitro (Lee et al., 2010; Boselli et al., 2017).

[0055] Furthermore, a study using human lung fibroblast WI-38 cells as a model of replicative senescence found that senescent cells had reduced activity, which correlated with an increase in carbonylated proteins. Furthermore, blocking the proteolytic activity of the 20S core in low-passage WI-38 cells induced an aging phenotype. In an in vivo study using rat liver tissue, the 20S proteasome peptidylglutamyl peptide hydrolysis activity (caspase-like) was shown to be reduced by 60% in aged rats compared to young animals. Enhancing proteasome activity reduces the proteotoxic burden experienced by cells during aging. In humans, rodents, and cells, increased proteasome activity delays aging and extends lifespan by reducing proteotoxic lesions. Supporting this notion, cells from human centenarians showed enhanced proteasome activity compared to cells from adults of different ages (Raynes et al., 2016).

[0056] While the activity of the 26S proteasome and ubiquitin-dependent proteolysis does increase during aging in a tissue-specific manner, the capacity to degrade oxidized proteins via the 20S proteasome actually decreases with age, leading to an overall decline in proteostasis.

[0057] Aging cells contain a latent pool of free (e.g., unbound) 20S. The relative levels of 20S and PA-bound 20S change with aging and disease. Using unlabeled protein groups in 9 different human cell lines, the results showed that 21% to 35% of the total proteasome pool was PA700-bound (26S), and less than 10% was bound to UIPS-specific PAs, while the remaining 66% was unbound 20S. This suggests that cells may contain a latent pool of 20S that is not bound to any PA. Because PA increases the rate of proteolysis by up to 20 times, these findings indicate that some cells have a pool of 20S ready to be activated.

[0058] In elderly individuals, who are the main victims of this disease type, the proteasome exists mainly as latent 20S, thus making the 20S a better target for these diseases (Pickering and Davies, 2012).

[0059] Since oxidatively modified proteins are known to be more susceptible to proteolytic degradation by the proteasome than native proteins, it seems obvious that the proteolytic activity of the proteasome is increasingly required with age. In contrast, proteasome activity is known to decrease with age, in particular through the accumulation of oxidized and cross-linked cellular proteins as shown experimentally. Therefore, the age-related accumulation of oxidized proteins could be caused by either increased protein oxidation or decreased degradation of oxidized proteins.

[0060] In any case, attempts to pharmacologically improve proteostasis will likely have to be made at an early stage of the disease, before severe manifestations of cellular dysfunction occur (Hipp et al., 2014). Therefore, timely removal of oxidatively damaged proteins is crucial to maintain normal cellular homeostasis and viability.

[0061] Protein oxidation is a factor in many diseases of aging, including Alzheimer's and Parkinson's diseases, atherosclerosis and arteriosclerosis, stroke, cancer, arthritis, cataracts, macular degeneration, frailty, and many others. Loss of protein homeostasis and resilience to oxidative stress during aging could have staggering implications for the world economy. Therefore, finding a new approach that specifically increases 20S activity would be extremely valuable. L-Glutamate is the major excitatory neurotransmitter in the central nervous system and acts at the NMDA receptor. The ionotropic NMDA receptor, which allows both calcium and sodium ions to pass, is located on the neuronal cell surface and has multiple binding sites (i.e., glycine, polyamines, NMDA) as well as ion channels with several internal binding sites (i.e., Mg++, PCP) (Lucia et al., 2019). Unique properties of this receptor include: voltage dependence, high permeability to Ca++, requirement for co-activation by glycine, and blockade by physiological concentrations of Mg++.

[0062] Recently, increasingly convincing evidence has demonstrated that specific glutamate receptors and glutamate transporters are also expressed in non-neural tissues such as the lung, liver, kidney, stomach, and immune system, indicating that glutamate plays an important role in the regulation of physiological functions in various peripheral organs. Glutamate has also been found to play different roles in non-neural diseases, such as renal injury (including ischemia, toxic injury, and renal cell carcinoma), liver inflammation, myocardial ischemia, various types of lung diseases (including pulmonary edema, pulmonary fibrosis, and lung cancer), gastric ulcers, autoimmunity and other diseases associated with the innate immune system and the adoptive immune system (Du et al., 2016).

[0063] Glutamate receptors are key components of the glutamate pathway, functioning as both signal detectors and transmitters. Glutamate receptors are mainly subdivided into two groups: ionotropic glutamate receptors (iGluRs) that constitute ion channels and metabotropic glutamate receptors (mGluRs) that are members of the G protein-coupled receptor (GPCR) superfamily.

[0064] N-methyl-D-aspartate (NMDA) receptors belong to the iGluR family, which includes kainate receptors, (2-amino-3(3-hydroxy-5-methylisoxazol-4-yl)propanoic acid)AMPA, and (N-methyl-D-aspartate receptor)NMDA receptors (Paoletti et al., 2013, Petit-Pedrol and Groc, 2021, Vieira et al., 2020; Groc et al., 2009)

[0065] Unlike other iGluRs, NMDA receptors are well known for their high Ca2+ ion conductance, voltage-dependent channel blockade, and mandatory binding of two endogenous ligands for receptor activation. Although glutamate synaptic concentrations are tightly regulated, excessive glutamatergic signaling is a key feature of neurodegeneration, leading to a dramatic increase in intracellular Ca2+ ion levels and subsequent neuronal apoptosis. Given the relatively high calcium permeability, NMDA receptors are primarily associated with these deleterious events, collectively referred to as excitotoxicity.

[0066] NMDA receptor is a plastic heterotetrameric structural complex located at synapse or synaptic membrane (such as cell body and dendritic stem). Subunits belong to GluN1 class, GluN2 class or GluN3 class. Specifically, there are eight isoforms of GluN1 (a to h, different splicing variants of a single gene), and four GluN2 (A to D, encoded by four different genes) and two GluN3 (A to B, encoded by two different genes) subunits have been identified. Functional receptors require two GluN1 subunits and GluN2 / GluN3 subunit combinations (heterotrimeric tetrameric assemblies). The activation of NMDA receptors depends on the combination of two agonists, namely glutamate and glycine or D-serine. Typically, NMDA receptors are composed of two glycine-bound GluN1 subunits and two glutamate-bound GluN2 subunits. There are multiple ligand binding sites on the NMDA receptor, including the glutamate binding site, the glycine binding site, the ion channel pore, and the allosteric binding site on the amino-terminal domain (ATD), which regulate receptor activity in a subtype-selective manner. More and more studies have shown that different subtypes of the NMDA receptor produce different functional outputs.

[0067] NMDA receptor antagonists or "N-methyl D-aspartate receptor antagonists" relate to compounds that are able to completely or partially block the action and / or function of NMDA receptors or NMDA receptor complexes in vivo and / or in vitro. Several glutamate receptor antagonists (NMDA receptor antagonists) and their neuroprotective effects have been described.

[0068] There are three classes of NMDAR antagonists: competitive antagonists, uncompetitive channel blockers, and noncompetitive allosteric inhibitors. Although ultimately leading to similar results, each antagonist class has a different mechanism of action. Competitive NMDAR antagonists act by occupying either the glycine binding site or the glutamate binding site located on the GluN1 subunit or the GluN2 subunit, respectively. This class of antagonists prevents endogenous ligand binding, thereby preventing activation of the receptor. Competitive and uncompetitive antagonists tend to have poor selectivity for specific GluN1 / GluN2 subunits, while noncompetitive allosteric inhibitors exhibit much higher selectivity. Uncompetitive channel blockers act by blocking the NMDAR ion channel pore (Das 2020; Omar et al., 2020).

[0069] Uncompetitive NMDAR channel blockers are typically positively charged and require depolarization to remove the Mg2+ blockade and expose the binding site. Once bound, these channel blockers prevent ion influx / out of the channel, resulting in inhibition. Of note, glutamate may still bind to the NMDAR when the channel blocker is in the pore, potentially leading to Ca2+-independent intracellular signaling; although the significance of this phenomenon is not fully understood. Finally, noncompetitive allosteric modulators inhibit NMDAR activity by acting on the N-terminal or “agonist binding” domain of the GluN1 / GluN2 subunit, thereby reducing receptor activity by reducing binding of endogenous ligands.

[0070] This class of uncompetitive ion channel blockers are ketamine, dizocilpine (MK-801), and memantine (Das 2020; Omar et al., 2020).

[0071] A variety of NMDA receptor antagonists have been developed. Some NMDA (receptor / complex) antagonists block ion channels, while others act at the glycine site. Others are selective for the NR2B NMDA receptor subtype. Non-limiting examples of agents that can act as antagonists have been described and patented. NMDA receptor and receptor-specific antagonists, molecular formulas, other activities of NMDA receptor antagonists are described in detail, for example, by Futerman et al., U.S. Patent No. US2017 / 0273917Al. Recently, Ahmed et al. have described patented NMDA receptor antagonists (Ahmed et al., 2020)

[0072] Memantine (1-amino-3,5-dimethyladamantane) is a systemically active uncompetitive NMDA receptor antagonist with moderate affinity, strong voltage dependence and fast blocking / unblocking kinetics for receptors. Memantine has been shown to be useful for alleviating various progressive neurodegenerative disorders such as dementia in patients with moderate to severe Alzheimer's disease, Parkinson's disease and spasticity (see, e.g., U.S. Patent Nos. 5,061,703; 5,614,560 and 6,034,134). Memantine is approved by the FDA and EMA for the treatment of moderate to severe Alzheimer's disease. Increasing evidence shows that in addition to being beneficial for neurological disorders, memantine is also beneficial for cancer, cardiovascular disorders and renal disorders (Shafiei-lrannejad et al., 2021; Cauli et al., 2014).

[0073] Memantine has multiple mechanisms of action that are hypothesized to generate its safety and efficacy profiles. These mechanisms include at least: (1) use-dependent channel blockade or agonist-gated binding and blockade of open channels more rapidly than closed channels; (2) low binding affinity or faster effective blockade rate; (3) fast intrinsic association kinetics; (4) fast dissociation kinetics and voltage dependence, which allow blockade during synaptic depolarization but allow physiological neuronal activity; (5) NMDA subunit selectivity, where memantine preferentially blocks NR2C and NR2D subunits and, to a lesser extent, NR2B subunits; (6) partial capture or mechanisms whereby a portion of the blocker can escape from the closed channel; (7) multiple actions or allosteric noncompetitive actions at the NMDA receptor; and (8) actions at other receptor targets.

[0074] In humans, a 20 mg daily dose of memantine produces serum levels ranging from 0.5 μM to 1.0 μM. However, mice tolerated memantine well at a dose of 30 mg / kg (see, Futerman et al., U.S. Patent No. US2017 / 0273917 Al). Brain microdialysis with in vivo recovery showed that free rat brain concentrations were 20% to 30% lower in plasma, while CSF sampling of human subjects showed concentrations 30% to 40% lower. There were no adverse drug interactions when memantine was combined with AchE (acetylcholinesterase inhibitor). Induction of HSPs (heat shock proteins) or neuronal vacuolation and necrosis has not been observed in animal studies.

[0075] At levels of 1 μM to 10 μM, the mechanism of action of memantine is specific for antagonizing NMDA receptors and does not affect other ligand-gated or voltage-gated channels. Importantly, these concentrations (6 μM to 10 μM) do not impair LTP or alter the function of postsynaptic excitatory currents in hippocampal slices.

[0076] Memantine preferentially blocks the NR2C and NR2D subunits and has moderate potency at NR1A / 2B of the NMDA receptor, but is weakly active at NR2A.

[0077] At the onset of pathological hyperactivity, memantine rapidly enters open channels at NMDA receptors, thereby attenuating the progression of this pathological hyperactivity. The high index of therapeutic efficacy and safety of memantine is due to its ability to block tonic low-level pathological activation of NMDA receptors by agonists and mild membrane depolarization in chronic neurodegenerative diseases, while allowing physiological NMDA activation following synaptic release of glutamate.

[0078] Proteasome activity measurement assay

[0079] Traditionally, increased ubiquitin levels measured in total cell lysates have been used as an indicator of decreased proteasome function. Given that ubiquitination plays a role in cell signaling that is independent of proteasomal degradation, assessing changes in ubiquitin levels can be a deceptive indicator of proteasome activity (Gan et al., 2019).

[0080] The lack of a good assay has hampered the identification of compounds that increase proteasome activity. In order to properly assess the effects of compounds on the UPS, a solid phase method is needed to accurately measure the total amount of 26S proteasomes in cells and their activity. A common method for measuring proteasome activity is to use a fluorescent substrate. In this type of experiment, a small peptide is linked to a 7-amino-4-methylcoumarin (AMC) group. When a protease such as the proteasome cleaves the group, the AMC group becomes fluorescent and the signal can be measured over time. When appropriate controls are included, the rate of conversion can be derived from these data and interpreted as a measure of proteasome activity. The advantage of this method is that each of the catalytic subunits can be measured separately by using a substrate-AMC conjugate that is preferentially cleaved by one of the three catalytic subunits. One disadvantage is that only the activity of a single subunit can be measured, and not the total proteasome activity, because not all subunits contribute equally to the total proteasome activity. In addition, this type of experiment is almost always performed with cell lysates or purified proteasomes. Therefore, data from this type of experiment may not be relevant to more complex environments such as whole cells or in vivo situations. The use of cell-permeable formats of fluorescent substrates can help overcome some of these limitations (Gan et al., 2019).

[0081] Compounds that clear senescent cells

[0082] Cellular Senescence

[0083] Mammalian cells have a unique ability to adapt to perturbations in the extracellular and intracellular environment. Perturbations in the cell microenvironment promote the activation of metabolic changes and molecular changes to ensure cell survival. Despite these adaptive mechanisms, chronic or severe, irreversible damage will terminate damaged cells to protect the life of the organism. Termination of damaged cells refers to the end of the normal physiological state of the cell. This can occur through the inactivation of cell division, a state called senescence.

[0084] Intracellular and extracellular signals that can cause cells to enter the fate of senescent cells mainly include signals related to tissue or cell damage and / or cancer development. These include DNA damage, telomere uncapping or dysfunction, exposure to extracellular DNA, oncogene activation, replicative stress or proliferation inducers (such as growth hormone / IGF-1), protein aggregates, misfolded proteins, reduced proteasome activity and failure to remove proteins, the presence of AGEs due to the reaction of reducing sugars with amino groups in proteins (for example, hemoglobin A1c is an AGE), saturated lipids and other bioactive lipids (bradykinin, certain prostaglandins, etc.), reactive metabolites (for example, ROS, hypoxia or hyperoxia), mechanical stress (for example, bone-to-bone stress in osteoarthritis or shear stress such as shear stress occurring on the venous side of an AV fistula for hemodialysis or around atherosclerotic plaques), inflammatory cytokines (for example, TNFa), damage-associated molecular patterns (DAMPs, for example, released intracellular contents signal neighboring cells to rupture) and pathogen-associated molecular patterns (PAMPs, for example, bacterial endotoxins). These inducers activate one or more pro-senescence transcription factor cascades, in some cases involving p16INK4a-retinoblastoma protein (Rb), and in other cases involving p53 and p21CIP1, both of these pathways, or other pathways.

[0085] Therefore, when the injury is severe enough but not fatal, the cells switch to a permanent non-proliferative state. This state is characterized by an inflammatory phenotype, which is called the senescence-associated secretory phenotype (SASP). The SASP consists of a large number of cytokines, chemokines, growth factors and proteases, which trigger inflammation, wound healing and growth responses in nearby cells. In young healthy tissues, the SASP is usually short-lived and tends to contribute to the maintenance or restoration of tissue homeostasis. However, senescent cells increase with age, and chronic SASP is known or suspected to be a key driver of many pathological hallmarks of aging, including chronic inflammation, tumorigenesis and impaired stem cell renewal.

[0086] Data from several laboratories strongly support the view that senescent cells and the SASP drive multiple age-related phenotypes and pathologies, including atherosclerosis, osteoarthritis, cancer metastasis, cardiac dysfunction, bone marrow skewing, renal dysfunction, and an overall reduction in healthspan (Saez-Atienzar and Masliah, 2020).

[0087] Senescent cells can accumulate at the etiological sites of a variety of diseases throughout the lifespan. For example, in humans, senescent cells accumulate in adipose tissue in diabetes and obesity, in the hippocampus and frontal cortex in Alzheimer's disease, in the substantia nigra in Parkinson's disease, in the bone and bone marrow in age-related osteoporosis, in the lungs in idiopathic pulmonary fibrosis, in the liver in cirrhosis, in the retina in macular degeneration, in the plaques in psoriasis, in the kidneys in diabetic kidney disease, in the endothelium in preeclampsia, in the heart and major arteries in cardiovascular disease, and in many other conditions.

[0088] The SASP is one of the three main characteristics of senescent cells, the other two being cell growth arrest and resistance to apoptosis. SASP factors can include the anti-apoptotic protein Bcl-xL, but growth arrest and SASP production are independently regulated. Although the SASP from senescent cells can kill adjacent normal cells, the resistance of senescent cells to apoptosis protects these cells from the SASP.

[0089] Senescent cells are highly metabolically active and produce large amounts of SASP, which is why senescent cells, which make up only 2 or 3% of tissue cells, may be the main cause of aging-related diseases. SASP factors cause non-senescent cells to become senescent. SASP factors induce insulin resistance. SASP disrupts normal tissue function by producing chronic inflammation, inducing fibrosis, and suppressing stem cells. Chronic inflammation associated with aging has been called inflammaging, although SASP may be only one of the possible causes of this condition. Chronic inflammation caused by SASP can suppress immune system function, which is one reason why older people are more vulnerable to COVID-19.

[0090] The SASP induces an unfolded protein response in cells due to the accumulation of unfolded proteins, which results in proteotoxic impairment of cellular functions.

[0091] Although cellular senescence may have evolved as a defense against cancer early in life, the SASP promotes the development of cancer later in life. Cancer aggressiveness is promoted primarily through the actions of the SASP factors metalloproteinases, chemokines, interleukin 6 (IL-6), and interleukin 8 (IL-8). In fact, the SASP from senescent cells has been implicated in many age-related diseases, including not only cancer but also atherosclerosis and osteoarthritis. For this reason, therapies to eliminate senescent cells have been proposed as a general treatment option for these and many other diseases.

[0092] Clearing established senescent cells, many of which carry oncogenic mutations or cause cancer-promoting inflammation due to their SASP, prevents or slows cancer development. In fact, drugs that clear senescent cells, that is, agents that selectively eliminate senescent cells, slow the development of multiple forms of cancer in old mice as well as mice that are susceptible to cancer (Atienzar and Masliah, 2020).

[0093] Targeting cellular senescence

[0094] Ablation of senescent cells has been postulated as a promising therapeutic approach to target the aging phenotype and thereby prevent, delay, or alleviate aging-related diseases. Compounds that clear senescent cells aim to rejuvenate the organism by selectively killing senescent cells, and the efficacy of these compounds is based on the ability of senescent cells to resist apoptosis. These cells exhibit upregulation of prosurvival pathways to protect themselves from the damaging (and pro-apoptotic) effects of the SASP. To date, six prosurvival pathways have been detected in senescent cells: the BCL-2-BCL-xL pathway, the MDM2-p53-p21Cip 1-serine element pathway, the ephrins-dependent receptor-tyrosine kinase, the PI3K-AKT-ceramide metabolism pathway, the hypoxia-inducible factor 1a (HIF-1a) pathway, and the SP-90-dependent pathway. Compounds that clear senescent cells interfere with these prosurvival pathways to allow senescent cells to die by apoptosis (Atienzar and Masliah, 2020).

[0095] Therefore, p53, p21, BCL-2, AKT, SP-90, pRb, and other proteins involved in these pathways are targets for compounds that eliminate senescent cells.

[0096] Geriatric hypothesis

[0097] The geroscience hypothesis posits that fundamental aging mechanisms are the “root cause” contributors to the increasing burden of diseases and illnesses that occur with aging and that are responsible for much of the morbidity, mortality, and health costs in both developed and developing countries. These fundamental aging processes include: (1) chronic low-grade “sterile” (free of bacteria, fungi, etc.) inflammation often accompanied by fibrosis; 2) macromolecular dysfunction (e.g., DNA damage, telomere uncapping, protein misfolding and aggregation, decreased proteasome activity, increased advanced oxidation-glycation end products, lipotoxicity, and accumulation of bioactive lipids) and organelle dysfunction (nuclear membrane alterations associated with lamin B deficiency, mitochondrial dysfunction resulting in decreased fatty acid metabolism, increased glucose utilization, depletion of NAD+, and increased ROS production, etc.); 3) stem cell, progenitor, and immune cell dysfunction (including altered proliferation capacity and impaired differentiation, failure to develop into functional mature cells, decreased “anti-aging” factors [e.g., a-Klotho], leading to stem cell and progenitor cell dysfunction); and 4) cellular senescence. The disease monism of fundamental aging processes assumes that these fundamental aging processes may be interconnected. Therefore, targeting any one fundamental aging process genetically or pharmacologically (e.g., reduced proteasome activity, protein misfolding, cellular senescence) should affect many or perhaps all of the remaining processes. Indeed, consistent with the disease monism theory, senescent cells contribute to inflammation, fibrosis, DNA damage, development of protein aggregates, autophagy failure, lipotoxicity, mitochondrial dysfunction, depletion of NAD+, ROS generation, and stem, progenitor, and immune cell dysfunction (Austad, 2016). Summary of the invention

[0098] The accumulation of unfolded, misfolded, or damaged proteins severely impairs the functions of organelles and cells and has been recognized as a key factor in aging and various diseases.

[0099] During the aging process, oxidized proteins appear to aggregate and accumulate to abnormally high levels. Such large aggregates, including amyloids, amorphous assemblies, or native-like assemblies, have been linked to many human diseases and physiological processes, including aging and aging-related diseases.

[0100] It is well known that the overall rate of protein degradation decreases as an organism ages. It is also known that a decrease in proteasome activity has been extensively associated with aging and age-related diseases, including neurodegeneration.

[0101] Since oxidatively modified proteins are known to be more susceptible to proteolytic degradation by the proteasome than native proteins, it seems obvious that the proteolytic activity of the proteasome is increasingly required with age. In contrast, proteasome activity is known to decrease with age, in particular through the accumulation of oxidized and cross-linked cellular proteins.

[0102] Therefore, the age-related accumulation of oxidized proteins could result from either increased protein oxidation or decreased degradation of oxidized proteins.

[0103] Therefore, timely removal of oxidatively damaged proteins is crucial for maintaining normal cellular homeostasis and viability. In any case, attempts to pharmacologically improve proteostasis will probably have to be made at an early stage of the disease, before the manifestation of severe cellular dysfunction (Hipp et al., 2014).

[0104] The proteasome is the first defense mechanism of the cell against toxic stress induced by accumulated proteins due to oxidative damage.

[0105] The present invention is based on the discovery that NMDA (N-methyl-D-aspartate) receptor complex antagonists increase proteasome activity ( FIG. 3A to FIG. 3C ; FIG. 6A to FIG. 6C ). The present invention also provides the effects of NMDA receptor antagonists on activating proteasome 20S subunits, including chymotrypsin-like activity, trypsin-like activity and caspase-like activity (Figures 6, 7 and 8). According to some embodiments of the present invention, the NMDA receptor antagonists activate proteasome 20S subunits mainly independently of UPS (ubiquitin-ATP-dependent proteolysis). Results from a series of experiments support this conclusion (Figures 12 to 22).

[0106] Thus, without wishing to be bound by any theory or mechanism of action, the present invention claims that NMDA receptor antagonists increase proteasome 20S activity, including chymotrypsin-like activity, trypsin-like activity, and caspase-like activity.

[0107] The decrease in the overall rate of protein degradation leads to a serious imbalance in protein homeostasis and the accumulation of oxidatively damaged proteins and / or misfolded proteins. Therefore, the present invention provides compositions and methods for treating diseases caused by oxidative damage and / or protein misfolding disorders by increasing proteasome activity.

[0108] The present invention encompasses the discovery that NMDAR blockers may provide effective treatment, or even prevention, of certain proteinopathies by activating the proteasome through increased levels and / or increased activity.

[0109] The present invention specifically encompasses the insight that, in some cases, increased chymotrypsin-like activity, trypsin-like activity and caspase-like activity of the proteasome may provide effective treatment (and / or prevention) of certain proteinopathies.

[0110] Therefore, in one aspect, the present invention provides a pharmaceutical composition for treating oxidative damage and / or protein misfolding diseases, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof. Conditions associated with amyloid-promoting intrinsic disordered proteins may include, but are not limited to, amyloid beta peptide, tau (Alzheimer's disease), alpha-synuclein (Parkinson's disease and other synucleinopathies), TARDNA binding protein 43, fusion to sarcoma (FUS) protein (amyotrophic lateral sclerosis (ALS)), huntingtin (Huntington's disease), many human functional amyloid proteins (cytoplasmic polyadenylation element binding protein (CPEB), T cell restricted intracellular antigen-1 (TIA-1), premelanosomal protein 17 (Pme117), secreted peptide hormones) and cell cycle related proteins, including p53, p27, p21 (cell cycle, apoptosis) ( Figure 1A ) and others ( Figure 4A and Figure 4B , FIG. 29A to FIG. 29C and Fig.30 ).

[0111] In some embodiments, the present invention provides novel therapeutic approaches for targeting proteotoxic disorders by activating the proteasome via NMDAR blockers. In some embodiments, the present invention provides novel therapeutic approaches associated with the activation of the proteasome 20S as a target.

[0112] Thus, the present invention provides activation of the 20S proteasome, which is present primarily in a latent form in elderly individuals, who are the primary victims of proteotoxic disorders and diseases. Thus, the present invention targets the 20S proteasome, which is one of the primary targets for preventing aging, which is associated with proteotoxic disorders that are commonly associated with a variety of human diseases. Furthermore, the present invention provides for targeting the 20S proteasome to effectively treat and prevent aging. In part, this may be because the 20S proteasome is more resistant to oxidative stress than the 26S proteasome, as the 20S complex remains active even when treated with moderate to high concentrations of H2O2, whereas the 26S proteasome is more fragile ( Fig.23A and Fig. 23B ).

[0113] According to an aspect of some embodiments of the present invention, there is provided an NMDA receptor antagonist for use in treating aging and / or aging-related conditions or diseases associated with decreased proteasome trypsin-like activity, chymotrypsin-like activity and caspase-like activity.

[0114] Proteins are one of the main targets of oxygen free radicals and other reactive species. During the aging process, there is a continuous accumulation of oxidized proteins.

[0115] Oxidative modification of proteins by oxygen free radicals and other reactive species such as hydroxynonenal occurs in physiological and pathological processes. As a result of the modification, carbonyl groups are introduced into protein side chains through a site-specific mechanism (Jung et al., 2014). The present invention also provides compositions and methods for preventing, reducing or even treating oxidatively damaged proteins in tissues by showing a reduction in protein carbonylation ( Fig.28 ).

[0116] Thus, the compositions and methods provided by the present invention not only treat diseases, but more importantly prevent the causes of diseases associated with malfunction of the protein homeostasis network, including the accumulation of oxidatively damaged proteins and / or misfolded proteins.

[0117] In another embodiment, the present invention demonstrates that both NMDA receptor inhibitors ketamine and memantine prevent cellular senescence, as determined by experiments using β-galactosidase staining and cell proliferation assays of primary fibroblasts ( Fig.24A , Fig. 24B ).

[0118] The present invention also demonstrates that memantine extends the lifespan of C. elegans worms. The present invention demonstrates that at day 21, the number of surviving worms in the group receiving memantine was 4 times higher than in the untreated group ( Fig.25A The present invention further demonstrates that the motor activity of worms is significantly increased in the presence of memantine ( Fig.25C ).

[0119] NAD+ / NADH and NADP+ / NADPH are involved in various biological processes in mammalian cells. Although NAD+ and NADP+ are synthesized in sufficient amounts under normal conditions, supply shortages due to overconsumption and reduced synthesis have been observed with age and in certain disease conditions. Some studies have demonstrated that the levels of NAD+ and NADP+ decrease with age in a wide range of tissues (such as liver, skin, muscle, pancreas, and fat). Therefore, the amounts of NADP+ and NADPH+ and their ratios were studied in C. elegans treated with memantine. It was found that on day 19, the NADP+ detected in C. elegans treated with memantine was up to 2 times higher (40%) than that in C. elegans not treated with memantine. Fig.25F and Figure 25G ).

[0120] Thus, the present invention provides an agent that prolongs both healthspan and lifespan.

[0121] Many biomarkers associated with aging have been identified in humans. These biomarkers have also been identified in mice. Mice are used as models for human aging and many movement disorders, including diseases originating from the body and central nervous system. For all of these models, a comprehensive assessment of their motor deficits must include specific strength tests.

[0122] In some embodiments, the present invention demonstrates that the clinical weakness tests including the Kondziela inverted grid test (latency to fall, movement time, four-finger grasp), weight test (forelimb grasp hold time), and open field maze (OFM) test (total distance walked, total number of upright positions test, number of supported upright positions, number of unsupported upright positions) performed in the group of 2-year-old mice receiving memantine were statistically significantly increased compared to the group of mice not receiving memantine ( FIG. 27A to FIG. 27K ).

[0123] The simplest and most easily identified finding of clinical signs of deterioration in aging mice is the evaluation of changes in the mouse skin. Indeed, the color change and condition of the hair of 22-month-old mice treated with and without memantine were very different, with the color change and condition of the hair being better in the mice receiving memantine ( Fig.26 ).

[0124] The present invention encompasses the discovery that NMDAR blockade, through activation of the proteasome by increasing levels and / or increasing activity, may provide effective treatment, or even prevention, of age-related mood anxiety in addition to providing improvements in motor activity.

[0125] In the above studies, we have shown that memantine extends the lifespan of C. elegans and increases their muscle strength. In mouse studies, we have shown that memantine phenotypically increases muscle strength and reduces amyloid formation ( Fig.29A ), reduced melanin and lipofuscin deposition ( Fig.30 A and Fig.30 B) Fat deposition ( Fig.31A 31D ), and reduced protein oxidation in tissues, hallmarks of aging. Strikingly, we did not investigate the direct effects of memantine on the lifespan of mice.

[0126] The most important study supporting our invention was published and patented by Anthony FUTERMAN, Andres David KLEIN, which showed that memantine extended the lifespan of 15 mice of 4 different strains with GD symptoms.

[0127] Futerman et al. showed that by using NMDAR blockers, including memantine, in mice with all symptoms of Gaucher disease (GD), their lifespan was extended and their motor coordination was significantly improved ( FIG. 32A to FIG. 32D ).

[0128] Multiple proteinopathies are known to be the pathophysiological hallmarks of GD.

[0129] Thus, in fact, Futerman et al. disrupted the proteostasis network by inhibiting protein degradation capacity through inhibition of GC enzymes, which actually led to premature aging associated with interference with key signaling pathways, including the autophagy-lysosomal pathway and the proteasome pathway.

[0130] Further, the present invention provides compositions and methods that are effective in ameliorating the onset and / or progression of aging. In particular, the compositions of the present invention reduce or prevent symptoms of aging by increasing proteasome activity.

[0131] Thus, the present invention provides compositions and methods for treating aging associated with malfunction of the proteostasis network, which perturbs key signaling pathways and is often associated with a variety of human diseases.

[0132] According to one aspect of some embodiments of the present invention, there is provided an NMDA receptor antagonist for detoxifying cells having oxidatively damaged proteins by inducing the activity of a 20S proteasome complex, wherein the 20S proteasome complex is capable of directly targeting oxidatively damaged proteins to detoxify cells ( Fig.23A and Fig. 23B ).

[0133] Thus, the compositions and methods provided by the present invention not only treat diseases, but more importantly treat the causes of diseases associated with malfunction of the protein homeostasis network, including the accumulation of oxidatively damaged proteins and / or misfolded proteins.

[0134] According to an aspect of some embodiments of the present invention, there are provided compositions and methods effective for ameliorating the onset and / or progression of aging associated with malfunction of the proteostasis network, including accumulation of oxidatively damaged proteins and / or misfolded proteins.

[0135] Thus, the present invention provides compositions and methods that prevent malfunction of the proteostasis network, including the accumulation of oxidatively damaged and / or misfolded proteins, which have been shown to be the most important cause of aging, and extend lifespan.

[0136] Many documents show that the amount of oxidized proteins in skeletal muscle, heart, skin, liver, lymphocytes and other tissues of various animal species increases during aging. Therefore, the present invention provides compositions and methods for maintaining and increasing muscle mass, muscle strength and muscle function. In addition, the irreversible loss of myocardial cells due to oxidative stress is the main cause of cardiac dysfunction after ischemia / reperfusion injury and aging-induced cardiomyopathy. Therefore, the present invention provides compositions and methods that are effective in preventing heart disease and improving the onset and / or progression of cardiac dysfunction after ischemia / reperfusion injury and aging-induced cardiomyopathy by maintaining and / or improving the function of the myocardium.

[0137] In another embodiment, the present invention provides a pharmaceutical composition for preventing metabolic syndrome by regulating protein homeostasis and protein quality control, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0138] The present invention encompasses the discovery that NMDAR inhibitors, by modulating the activation of the proteasome via dysfunction of the protein homeostasis network, including the accumulation of oxidatively damaged and / or misfolded proteins, may provide effective treatment, and even prevention, for obesity and obesity-related disorders, including cardiovascular disease, insulin resistance, diabetes, dyslipidemia.

[0139] Therefore, age-related reductions in fat oxidation promote the accumulation of systemic and central body fat. Adipogenesis and lipid accumulation during aging have a great impact on the pathogenesis of the aging process and chronic age-related diseases. Aging is associated with malfunction of the lipid-protein homeostasis network, which interferes with key signaling pathways and is often associated with a variety of human diseases, including PD.

[0140] Thus, the present invention provides compositions and methods for preventing adipogenesis and lipid accumulation during aging by maintaining and / or improving the function of the lipid-protein homeostasis network.

[0141] In addition, the present invention provides compositions and methods for improving the onset and / or progression of adipogenesis and lipid accumulation by maintaining and increasing muscle mass, muscle strength and muscle function, since the size and / or reduction in oxidative capacity of metabolically active muscle mass is an important determinant of reduced fat oxidation. The reduction in muscle mass and function is often closely associated with aging and metabolic syndrome, including (i) mitochondrial dysfunction, which causes various skeletal muscle (SkM) lesions such as sarcopenia and muscular dystrophy; (ii): inflammatory myopathies caused by inflammation and oxidative stress, such as dermatomyositis, polymyositis, necrotizing autoimmune myositis and sporadic inclusion body myositis, and (iii) chronic diseases that cause SkM damage, such as type 2 diabetes, obesity, chronic kidney disease and chronic obstructive pulmonary disease. These syndromes often lead to a decline in the patient's quality of life over time and increase the patient's mortality rate.

[0142] Thus, the present invention also provides compositions and methods for preventing aging and metabolic syndrome by increasing muscle mass and activity.

[0143] Therefore, in another embodiment, the present invention provides a pharmaceutical composition for preventing metabolic syndrome by regulating protein homeostasis and protein quality control, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0144] As described above, the present invention demonstrates that the motor activity of C. elegans and mice treated with NMDAR blockers is significantly increased compared to a control group not treated with an NMDAR inhibitor. In addition, the present invention demonstrates that the amount of lipid droplets in the muscle tissue of C. elegans and mice treated with an NMDAR inhibitor is significantly lower than a control group not treated with an NMDAR inhibitor.

[0145] Therefore, in one aspect, the present invention provides a pharmaceutical composition for preventing metabolic syndrome by increasing muscle mass and activity, thereby regulating lipid metabolism, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0146] In addition, in one aspect, the present invention provides a pharmaceutical composition for preventing obesity and related diseases, which comprises at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof, and these related diseases occur simultaneously with metabolic syndrome caused by changes in lipid metabolism with age.

[0147] Thus, the present invention encompasses the discovery that NMDAR blockers can be used to prevent, ameliorate and / or treat metabolic disorders affecting body weight, particularly in the treatment of obese subjects, especially in the treatment of human subjects, by increasing levels and / or increasing activity of proteasome activation.

[0148] According to the present invention, it is also contemplated that NMDA receptor antagonists, in particular memantine, a pharmaceutically acceptable salt or prodrug of an NMDA receptor antagonist, are used for medical intervention of secondary conditions associated with (pathological) weight gain. These "secondary conditions" may include, but are not limited to, type 2 diabetes, hypertension (high blood pressure), cardiovascular disease, cancer, sexual function problems, and disorders of the muscular or skeletal system.

[0149] The present invention provides compositions and methods for maintaining and / or reducing skin and hair aging by reducing the accumulation of oxidized proteins and lipids and inflammation. Therefore, according to one aspect of some embodiments of the present invention, the present invention provides an NMDA receptor antagonist for detoxifying cells with oxidatively damaged proteins and lipids by inducing the activity of the 20S proteasome complex, which can be used as an anti-aging agent in cosmetics.

[0150] The present invention provides compositions and methods for maintaining and increasing stemness characteristics, which are the potential for stem cell self-renewal, multi-lineage differentiation and lineage reprogramming. In addition, the present invention demonstrates that the use of NMDAR blockers extends the lifespan of human mesenchymal stem cells ( FIG. 36A to FIG. 36D ).

[0151] With the increase of age, the physiological activity of muscle stem cells decreases, the regeneration capacity of SkM decreases, and the efficiency of muscle repair decreases, and this is accompanied by a decrease in muscle mass and muscle strength, as well as irreversible muscle loss, which seriously affects the quality of life of the elderly. Therefore, the present invention provides the following composition and method, which maintains and increases stemness characteristics and prevents the decrease in muscle mass and muscle strength, irreversible muscle loss, and muscle diseases that seriously affect the quality of life of the elderly by increasing proteasome activity.

[0152] The effects of the present invention are also not limited to muscle tissue, but the present invention also provides compositions and methods for maintaining and increasing stemness characteristics and preventing aging and related diseases by ensuring the continuation of the regenerative properties of all tissues, including neurons.

[0153] The present invention provides compositions and methods for preventing and / or treating high-incidence eye diseases such as age-related macular degeneration, cataracts, glaucoma and diabetic retinopathy, which are multifactorial in origin and are related to genetics, environmental factors, age and oxidative stress, etc.; the latter factor is one of the factors that have the greatest impact on eye diseases and directly affects the process of autophagic activity. Depending on the specific circumstances, autophagy and proteasomes share common substrates and regulatory factors. The two systems intersect and communicate at multiple points to coordinate and balance their roles in protein homeostasis and organelle homeostasis.

[0154] Probably the most important problem that occurs due to reduced proteasome activity is the accumulation of potentially toxic unfolded / misfolded proteins and protein aggregates. Impairment of the proteasome will ultimately affect cytoplasmic processes including autophagy and the vital functions of all other organelles due to the accumulation of unfolded and damaged proteins.

[0155] According to an aspect of some embodiments of the present invention, there is provided an NMDA receptor antagonist for use in treating or preventing ocular pathologies associated with impaired protein organelle turnover by enhancing proteasome activity and thereby acting by regulating the autophagy-lysosomal pathway.

[0156] According to one aspect of some embodiments of the present invention, there is provided an NMDA receptor antagonist for treating viral infection by degrading viral proteins such as HepC core protein. Another subject of the present invention is HIV, as proteasome inhibitors are known to activate latent HIV (Cary and Peterlin, 2020; Timilsina et al., 2019).

[0157] As described above, the present invention demonstrates that NMDAR inhibitors reduce carbonylated and carbamylated proteins responsible for structural changes that lead to exposure of abnormally denatured regions on the protein surface, thereby generating new antigens that lead to autoimmune responses.

[0158] Thus, in some embodiments, the present invention provides compositions and methods for preventing autoimmunity against new antigenic structures that arise due to changes in protein structure caused by malfunction of the protein homeostasis network, including the accumulation of oxidatively damaged proteins, carbonylated proteins, carbamylated proteins, and / or misfolded proteins.

[0159] In another embodiment, the present invention provides chemical structures and / or methods that will maintain and / or increase proteolysis, thereby improving the onset and / or progression of or even treating autoimmune responses to protein aggregation caused by oxidatively damaged proteins and / or misfolded proteins.

[0160] The present invention enhances proteolytic capacity by increasing proteasome activity using at least one NMDA receptor antagonist, thereby providing chemical structures and / or methods that will maintain and / or increase proteolysis, thereby improving the onset and / or progression of infectious diseases and autoimmune diseases and even treating them.

[0161] The present invention provides methods and compositions related to various neurodegenerative proteinopathies associated with intracellular and / or extracellular accumulation of specific protein aggregates such as Tau ( Figure 4A In many cases, protein aggregates are thought to be toxic to the brain and contribute to disease pathology.

[0162] On the other hand, the present invention demonstrates that the increase in proteasome activity in the presence of NMDAR blockers reduces serine racemase, thereby reducing the conversion of L-serine to -D-serine ( Figure 4B ), which will effectively prevent or treat cerebral ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, hyperfunctioning brain syndrome, retinal ganglion cell loss, long-term sequelae of concussion, epileptic seizures, retinal ganglion cell loss in diabetic patients, chronic social frustration stress and other

[0163] Neurodegenerative diseases.

[0164] In addition, the present invention provides compositions and methods for reducing the toxic effects of astrocytes by reducing serine racemase. Thus, the present invention further reduces microvascular damage in diabetic retinopathy, synaptic damage after traumatic brain injury, neuronal overactivation, choroidal neovascularization, pathological symptoms of Alzheimer's disease, and neurodegeneration.

[0165] In another aspect, the invention provides compositions and methods for neuroprotection in neurological diseases.

[0166] The present invention provides compositions and methods for preventing and / or treating neurodegeneration caused by pathological insults including anti-cancer therapy, cerebral ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, retinal ganglion cell loss, long-term sequelae of concussion, reducing the neurotoxic effects of astrocytes in Alzheimer's disease, and epileptic seizures (Saez-Atienzar and Masliah, 2020; Glass et al., 2010; Pender et al., 2020)

[0167] On the other hand, the present invention demonstrates that the increase in proteasome activity in the presence of NMDAR blockers reduces serine racemase, thereby reducing the conversion of L-serine to -D-serine, which will effectively prevent or treat schizophrenia and other mental illnesses. Tauopathy is a neurodegenerative disorder characterized by the presence of filamentous deposits composed of hyperphosphorylated tau protein in neurons and glia. Abnormal tau phosphorylation and deposition in neurons and glial cells is one of the main characteristics of tauopathy (Weng and He, 2021).

[0168] In addition, it has been reported that abnormal hyperphosphorylation of tau protein

[0169] Tauopathies play a central role in neurodegeneration caused by traumatic brain injury (TBI) and traumatic spinal cord injury. Such exemplary tauopathies include amyotrophic lateral sclerosis (ALS), Parkinson's syndrome, argyrophilic grain dementia, diffuse neurofibrillary tangles with calcifications, frontotemporal dementia associated with chromosome 17, corticobasal degeneration, Pick's disease, progressive supranuclear palsy, progressive subcortical gliosis, and tangled dementia.

[0170] Accumulating evidence suggests that defects in the ubiquitin proteasome system (UPS) lead to p-tau accumulation and that targeting the UPS alleviates tauopathy.

[0171] The present invention provides methods related to tauopathy. In some embodiments, the present invention provides a method for demonstrating that increased proteasome activity in the presence of an NMDAR blocker reduces the accumulation of pTau protein in a cell ( Figure 4B ), the method comprising administering to the cell a therapeutically effective amount of such provided NMDAR blocker. In some embodiments, the cell is a neuronal cell. In some embodiments, the cell is a non-neuronal cell. In some embodiments, the cell expresses tau protein. In certain embodiments, the tau disease is Alzheimer's disease.

[0172] Therefore, in one aspect, the present invention provides compositions and methods for preventing and / or treating the above-mentioned diseases caused by tauopathy.

[0173] The present invention provides methods for preventing, reducing or even treating cognitive impairment and dementia, which may be caused by any etiology.

[0174] Tedeschi and Dupraz (Tedesci and Dupraz, 2019) previously described that actin turnover increases axon regeneration, thereby preventing and / or treating neurodegeneration caused by pathological damage. In another aspect, the present invention provides the discovery that NMDAR blockers cause actin turnover ( Fig.35 ), and thus can increase axonal regeneration. Therefore, the present invention provides compositions and methods that prevent and / or treat neurodegeneration caused by pathological insults including anticancer treatments, cerebral ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, retinal ganglion cell loss, long-term sequelae of concussion, and reduce the neurotoxic effects of astrocytes in Alzheimer's disease. Therefore, the neurodegenerative disease is selected from the group consisting of: neurodegeneration caused by pathological insults including anticancer treatments, brain

[0175] Ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, retinal ganglion cell loss, long-term sequelae of concussion, neurotoxic effects of astrocytes in Alzheimer's disease, epileptic seizures.

[0176] In one aspect, the present invention provides compositions and methods for preventing and / or treating renal diseases caused by accumulation of oxidatively damaged proteins and / or misfolded proteins (Cauli et al., 2014).

[0177] On the other hand, the present invention provides a composition and method for preventing and / or treating renal insufficiency caused by aging, hyperlipidemia, hypertension, smoking, diabetes, obesity and other factors that lead to protein oxidative damage. Therefore, the present invention improves and / or treats acute kidney disease and chronic kidney disease.

[0178] In another aspect, the present invention provides a method for treating colorectal cancer by reducing serine racemase, which enhances the growth of colorectal cancer by generating pyruvate from serine (Ohshima et al., 2020). The present invention provides a method for preventing, reducing or even treating cell proliferative diseases by reducing or preventing the accumulation of unfolded proteins.

[0179] Therefore, on the other hand, the present invention also provides a method for treating different forms of cancer, in which the protein homeostasis network is disturbed, including increased protein synthesis, misfolded protein production, misfolded protein aggregates, and accumulation of apoptotic signaling proteins in cancer cells. In addition, the present invention also provides a method for increasing the sensitivity of cancer cells to anti-tumor drugs (Madden et al., 2019; Hsu et al., 2019).

[0180] Shortening of telomere length is one of the metabolic disorders that impair normal aging processes, such as loss of muscle mass and muscle strength. Previous studies have shown that inhibiting proteasome activity inhibits telomerase activity (Shalem-Cohavi et al., 2019).

[0181] Therefore, in another aspect, the present invention provides NMDA receptor antagonists that increase telomerase activity by increasing proteasome activity. Therefore, in another aspect, the present invention provides such a method that NMDA receptor antagonists delay cell senescence by increasing telomerase activity, and thus delay the aging process, thereby extending lifespan.

[0182] The present invention encompasses the discovery that NMDA inhibitors can provide effective treatment or even prevention of endoplasmic reticulum (ER)-related diseases by increasing levels and / or increasing activity of proteasomes by reducing endoplasmic reticulum (ER) stress. Metabolic diseases, including insulin resistance, atherosclerosis, diabetes, obesity, alcoholic and non-alcoholic fatty liver disease, hyperlipidemia; cancers, including leukemia, multiple myeloma, breast cancer, prostate tumors; immune system-related diseases, including viral infections, bacterial infections, vitiligo, rheumatoid arthritis, type 1 diabetes and many neurological diseases are known to be associated with ER stress (Ding et al., 2007; Roussel et al., 2013).

[0183] The present invention encompasses the discovery that NMDA inhibitors can provide effective treatment, or even prevention, for lysosomal storage diseases by increasing levels and / or increasing activity of the proteasome. The present invention encompasses the discovery that NMDA inhibitors can prevent malfunction of the protein homeostasis network (including accumulation of oxidatively damaged proteins and / or misfolded proteins associated with lysosomal dysfunction) by activation of the proteasome. Lysosomal storage diseases include Gaucher disease, Fabry disease, Niemann-Pick disease, Hunter syndrome, glycogen storage disease II (Pompe disease), Tay-Sachs disease.

[0184] The present invention may provide effective treatment, symptom reduction, or even prevention of certain mitochondrial diseases, including mitochondrial myopathy, diabetes, Leber hereditary optic neuropathy, Leigh's syndrome, subacute sclerosing encephalopathy, neuropathy, ataxia, deafness, retinitis pigmentosa and ptosis, progressive symptoms of dementia, myoneurogenic gastrointestinal encephalopathy, MERRF syndrome, MELAS syndrome, Huntington's disease, cancer, Alzheimer's disease, Parkinson's disease, bipolar disorder, schizophrenia, aging and senility, and anxiety disorders.

[0185] The autophagy-lysosomal pathway is another degradation pathway in which cytoplasmic components, such as protein aggregates and damaged organelles, are degraded and recycled to maintain normal cellular homeostasis. Depending on the specific situation, autophagy and proteasome share common substrates as well as regulatory factors. The two systems intersect and communicate at multiple points to coordinate and balance their roles in protein homeostasis and organelle homeostasis (Kocaturk and Gozuacik, 2018).

[0186] The present invention can provide effective treatment, symptom reduction, and even prevention for certain autophagy-related human diseases, such as adult neurodegenerative disorders, including Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, neuronal ceroid lipofuscinosis, fulminant neurodegeneration, Lewy body dementia; pediatric neurodevelopmental disorders, including spinocerebellar ataxia, cortical atrophy and epilepsy, childhood-onset neurodegeneration, BPAN, spastic quadriplegia and brain abnormalities, primary microcephaly, hereditary spastic paraplegia, ataxia with spasticity, Rett syndrome, Joubert syndrome, leukoencephalopathy, juvenile-onset dystonia, CEDNIK syndrome, Pelizaeus-Merzbacher-like hereditary neuropathies, including sensory and autonomic neuropathy type II, Charcot-Marie-Tooth disease, sensory and autonomic neuropathy type IF, and distal hereditary motor neuropathy; ophthalmologic disorders, including primary open-angle glaucoma and cataracts; cardiac and skeletal myopathy, including Danon cardiomyopathy, distal myopathy with rimmed vacuoles, dilated cardiomyopathy, sporadic inclusion body myositis, and X-linked myopathy with excessive autophagy; inflammatory disorders, including Crohn's disease, ulcerative colitis, and childhood asthma; autoimmune diseases, including systemic lupus erythematosus, diabetes, and other autoimmune diseases; infectious diseases, including Mycobacterium tuberculosis and Mycobacterium leprae; skeletal disorders, including osteopetrosis, Paget's disease of bone, and Kashin-Beck disease; congenital multisystem disorders, including global dysplasia, Vici syndrome, Zellweger syndrome, and glycosylation disorders

[0187] Autophagy deficiency, Zimmerman-Laband syndrome, Hermansky-Pudlak syndrome, multisystem proteinopathy.

[0188] Targeting cellular senescence

[0189] Ablation of senescent cells has been postulated as a way to target the aging phenotype and thereby prevent, delay, or mitigate

[0190] Promising treatments for aging-related diseases. The purpose of compounds that eliminate senescent cells is to rejuvenate an organism by selectively killing senescent cells, and the efficacy of these compounds is based on the ability of senescent cells to resist apoptosis. On the other hand, the present invention demonstrates that NMDA receptor antagonists increase proteasome activity and cause a decrease in p21, p53, pAktl and ppRb, which are the main target molecules of drugs that eliminate senescent cells (Figure 1, Figure 2A and Fig.34 )

[0191] Therefore, in one aspect, the present invention provides a pharmaceutical composition for the ablation of senescent cells, which has been postulated to be a promising treatment method for targeting the aging phenotype, thereby preventing, delaying or alleviating aging-related diseases, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof. Therefore, according to one aspect of some embodiments of the present invention, there is provided an NMDA receptor antagonist for the treatment of aging, which is associated with an increased risk of developing a variety of chronic diseases, geriatric syndromes, impaired physical resilience and mortality (Saez-Atienzar and Masliah, 2020).

[0192] Diseases for which there is emerging evidence of a causal contribution of cellular senescence or a benefit of senescent cell clearance drugs include, but are not limited to, diabetes / obesity, metabolic diseases, cardiac dysfunction, congestive heart failure, myocardial infarction, vascular hyporesponsiveness / calcification, AV fistulas, frailty, age-related muscle loss (sarcopenia), arthritis, osteoporosis, falls, chemotherapy complications, radiation complications, cancer, bone marrow transplant complications, organ transplant complications, myeloma / MGUS (monoclonal gammopathy of undetermined significance), age-related cognitive impairment, Other dementias, Alzheimer's disease, Parkinson's disease, ALS, Ataxia, Obesity related neuropsychiatric dysfunction, Renal dysfunction, Urinary incontinence, Osteoporosis, Osteoarthritis, Age related disc disease, Idiopathic pulmonary fibrosis, Hyperoxic lung injury, COPD, Tobacco, Hepatic steatosis, Cirrhosis, Primary biliary cirrhosis, Progeria, Preeclampsia, Macular degeneration, Glaucoma, Cataracts, Blindness, Prostatic hypertrophy, Incontinence, Psoriasis, Healthy life span, Life span and many other diseases.

[0193] Importantly, in any case, attempts to pharmacologically improve proteostasis must be made at an early stage of the disease, before severe manifestations of cellular dysfunction occur. Indeed, the present invention demonstrates that early initiation of treatment is more effective in extending life expectancy than late initiation of treatment ( Fig.25B ).

[0194] NMDA receptor blockers

[0195] In certain embodiments, the NMDA receptor antagonist is selected from the following group consisting of: NMDA receptor antagonists, which are compounds that antagonize or inhibit the action of NMDA receptors. NMDA receptor antagonists can be competitive antagonists, uncompetitive antagonists, noncompetitive antagonists and / or glycine antagonists (Liu et al., 2020; Vieira et al., 2020).

[0196] According to some embodiments of the invention, the NMDA receptor is an extrasynaptic NMDA receptor.

[0197] Uncompetitive ion channel blockers of this class are ketamine, dizocilpine (MK-801), and memantine (Siala et al., 2020; Das 2020).

[0198] According to some embodiments of the invention, the NMDA receptor comprises a NR2B subunit.

[0199] In certain embodiments, the NMDA receptor antagonist preferentially binds to an extrasynaptic NMDA receptor.In certain embodiments, the extrasynaptic NMDA receptor comprises a NR2B subunit.

[0200] Non-limiting examples of NMDA receptor antagonists are selected from the group consisting of memantine, nitromemantine, neramexane, ketamine, amantadine, dextromethorphan, L-687,384, amitriptyline, 1-benzyl-6-methoxy-6,7'-dihydrospiro[piperidin-4,4'-thieno[3.2-c]pyran], ifenprodil, orphenadrine, kynurenic acid, felbamate, D(-)-AP-5, (+)-CPP, EAA-090, TCN-201, AP-5, AZD6765, SDZ 220-581, (+)-norketamine, eliprodil, dextrorphan, 5,7-dichlorokynurenic acid monohydrate, [G1u3,4,7,10,14]-cono sleep peptide G, D-AP-7, MD-Ada, AP-7, Ro 8-4304, spermine, Ro25-6981, DCOX, traxoprodil, MDL105,519, fanapanel, metaphit, Ro25-6981, NAAG, 5-fluoroindole-2-carboxylic acid, (S)-(-)-4-oxo-2-azetidinecarboxylic acid, (S) (-)-4-oxo-2-azetidinecarboxylic acid benzyl ester and (+)-α-amino-3-methoxycarbonyl-5-methylisoxazole-4-propionic acid and their pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers (Ahmed et al., 2020). Each possibility represents a separate embodiment of the present invention.

[0201] In certain embodiments, the NMDA receptor antagonist is selected from the group consisting of memantine, nitromemantine, neramexane, ketamine, amantadine, dextromethorphan, L-687,384, amitriptyline, 1-benzyl-6-methoxy-6',7'-dihydrospiro[piperidin-4,4-thieno[3.2-c]pyran), eliprodil, ifenprodil, orphenadrine, kynurenic acid, felbamate and their pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers. Each possibility represents a separate embodiment of the present invention.

[0202] In certain embodiments, the NMDA receptor antagonist is selected from the group consisting of memantine, nitromemantine, neramexane, ketamine, and pharmaceutically acceptable salts, hydrates, and pharmaceutically active

[0203] Enantiomers. Each possibility represents a separate embodiment of the present invention.

[0204] According to some embodiments of the invention, the NMDA receptor antagonist is selected from the group consisting of memantine, elirodine and ifenprodil or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0205] In certain embodiments, the NMDA receptor antagonist is memantine (3,5-dimethyl-1-adamantanamine hydrochloride) or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0206] According to some embodiments of the invention, the NMDA receptor antagonist is formulated for oral delivery.

[0207] In certain embodiments, compositions for oral delivery are formulated for sustained release.

[0208] In another embodiment of the present invention, since the effect of NMDAR blockers (memantine and ketamine) on proteasome activity lasts for 4 to 8 hours, it should be clinically evaluated whether the NMDAR blockers are administered every 6 to 8 hours or in a sustained release form.

[0209] According to some embodiments, the NMDA antagonist is a specific inhibitor of glutamatergic neurotransmission. According to some embodiments, the

[0210] NMDA antagonists can be antagonists with lower specificity that act on other neurotransmission pathways. According to some embodiments, the NMDA antagonist can have additional activity, such as serotonergic, cholinergic or dopaminergic antagonist or agonist activity (Paoletti et al., 2013; Petit-Pedrol and Groc, 2021).

[0211] In certain embodiments, the NMDA receptor antagonist having nACHR antagonist activity is selected from the group consisting of amantadine, dextromethorphan and pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers thereof. Each possibility represents a separate embodiment of the present invention.

[0212] In certain embodiments, the NMDA receptor antagonist also has dopaminergic activity. In certain embodiments, the NMDA receptor antagonist is also a dopamine D2 receptor agonist. In certain embodiments, the NMDA receptor antagonist is also a sigma-1 receptor agonist. In certain embodiments, the NMDA receptor antagonist with sigma-1 receptor agonist activity is selected from the group consisting of: L-687,384, amitriptyline and 1-benzyl-6-methoxy-6',7'-dihydrospiro [piperidine-4,4'-thieno [32-c] pyran) and their pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers. Each possibility represents a separate embodiment of the present invention.

[0213] A subject in need of such treatment or prevention is administered a therapeutically effective amount of the NMDA receptor antagonist memantine, less than 10 mg / kg.

[0214] In certain embodiments, an effective amount of an agent of the invention is a dosage of about 0.01 mg to about 10 mg per kilogram of body weight (mg / kg) of the subject, ie, about 0.01 mg / kg body weight to about 10 mg / kg body weight.

[0215] In certain embodiments, the NMDA receptor antagonist is applied to the subject before the stressor. The accumulation of unfolded proteins / misfolded proteins and protein aggregates that are potentially toxic is referred to as a stressor. In addition, proteasome damage is also referred to as a stressor, which depends on or is not dependent on the accumulation of unfolded proteins / misfolded proteins and protein aggregates that are potentially toxic. Therefore, in certain embodiments, the medicament / composition of the present invention is applied to a subject who is 25 years old or older.

[0216] In certain embodiments, agents / compositions of the invention are administered to a subject for life.

[0217] In certain embodiments, the agents / compositions of the present invention are administered to a subject who is a human less than 25 years old.

[0218] Generally, NMDAR blockers can be or include compounds of any chemical class (e.g., small molecules, metals, nucleic acids, polypeptides, lipids and / or carbohydrates). In some embodiments, the NMDAR blocker is or includes an antibody or antibody mimetic. In some embodiments, the NMDAR blocker is or includes a nucleic acid agent (e.g., antisense oligonucleotides, siRNA, shRNA, etc.) or its mimetic. In some embodiments, the NMDAR blocker is or includes a small molecule. In some embodiments, an activator is or includes a naturally occurring compound (e.g., small molecule). In some embodiments, the NMDAR blocker has a chemical structure that is artificially produced and / or modified. Generally, NMDAR blockers increase the level or activity of one or more entity targets present in a cell or an organism and / or produced by the cell or the organism, and in this article, these targets are trypsin-like activity, chymotrypsin-like activity and caspase-like activity of a proteasome. In some embodiments, the target entity is or includes a polypeptide. In some embodiments, the target entity is or comprises a nucleic acid (e.g., a nucleic acid that encodes or modulates [e.g., by altering the expression and / or activity of a polypeptide]. In some embodiments, the target entity is or comprises a carbohydrate. In some embodiments, the target entity is or comprises a lipid. In some embodiments, the target entity is or comprises an enzyme. In some embodiments, the target entity is or comprises a polypeptide involved in cellular trafficking.

[0219] Prevention and / or inhibition of the biological function of the NMDA receptor can be achieved at the protein level, but can also be achieved at the genome and / or transcript level using various molecules that interfere with the transcription and / or translation of the receptor.

[0220] Thus, non-limiting examples of antagonists that may be used according to some embodiments of the present invention include small molecules, antibodies, inhibitory peptides, enzymes that cleave polypeptides, aptamers, homologous recombination agents, site-specific endonucleases, and RNA silencing agents.

[0221] The NMDA antagonists and / or agents of some embodiments of the present invention may be administered to an organism as such or in the form of a pharmaceutical composition mixed with a suitable carrier or excipient.

[0222] Suitable routes of administration may include, for example, oral delivery, rectal delivery, transmucosal delivery, especially transnasal, enteral delivery or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections, as well as intrathecal injections, direct intraventricular injections, intracardiac injections, such as into the right or left ventricle, into the coronary artery, intravenous injections, intraperitoneal injections, intranasal injections or intraocular injections.

[0223] In certain embodiments, the NMDA receptor antagonist and / or the medicament described above is formulated for injection. In certain embodiments, the NMDA receptor antagonist and / or the medicament is formulated for sustained release.

[0224] The scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples are given by way of illustration only when indicating preferred embodiments of the present invention, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on the detailed description. DETAILED DESCRIPTION

[0225] Protein synthesis and turnover must be carefully balanced, and terminal misfolded proteins are efficiently removed by proteolysis to ensure protein homeostasis. The clearance of oxidatively damaged and misfolded proteins is primarily carried out by the ubiquitin-proteasome system (UPS), and increasing evidence has clearly demonstrated that the 20S core plays a major role in overall protein degradation, independent of ubiquitin and ATP (Raynes et al., 2016; Hipp et al., 2014; Jung et al., 2914).

[0226] The present invention is based on the discovery that N-methyl-D-aspartate receptor (NMDAR) complex antagonists increase proteasome activity (Figures 6, 7 and 8). The present invention further demonstrates that NMDAR antagonists activate proteasome 20S subunits primarily independently of UPS (ubiquitin-ATP-dependent proteolysis) (Figures 12 to 22).

[0227] The present invention also provides the effects of NMDAR antagonists on activating the proteasome 20S subunit, including chymotrypsin-like activity, trypsin-like activity and caspase-like activity (Figure 6, Figure 7). Impairment of protein homeostasis is now considered to be a fundamental mechanism by which chronic protein misfolding and toxic aggregation cause cellular dysfunction, thereby promoting the manifestation and progression of many neurodegenerative diseases, other aggregate deposition diseases, and aging and related diseases (Dobson et al., 2015; Chiti and Dobson, 2017).

[0228] Protein aggregates impair the activity of the cellular proteolytic system (proteasome), leading to further accumulation of oxidized proteins. In addition, the accumulation of highly cross-linked protein aggregates leads to further oxidant formation, damage to macromolecules, and ultimately to apoptotic cell death.

[0229] Protein aggregates are observed in a variety of different types of disorders, diseases and / or conditions, including cognitive disorders, proliferative diseases, inflammatory diseases, cardiovascular diseases, immune diseases, eye diseases, mitochondrial diseases, neurodegenerative diseases, lysosomal storage diseases, and aging and related diseases.

[0230] As used herein, the term "protein aggregate" refers to any association of two or more protein molecules in a non-native conformation. Aggregates cover a range of structures, from amorphous assemblies to highly ordered fibrils (amyloid) with cross-b structures. The tendency of specific protein aggregation is mainly determined by the chemical properties of its amino acid sequence, the conformational stability of its folded state, and its cellular concentration. Compared with dilute solutions, extremely high total protein concentrations in cells lead to excluded volume effects (macromolecular crowding) and significantly increase the tendency of non-native protein molecules to aggregate.

[0231] A range of proteins involved in these processes (e.g., transthyretin, insulin, beta-2-microglobulin, superoxide dismutase) are naturally folded. However, the vast majority of these proteins involved in protein-aggregate disorders are particularly those that are relatively small intrinsically disordered proteins (IDPs). Disorder is primarily found in intrinsically disordered regions (IDRs) within otherwise well-structured proteins. Thus, the term IDP includes proteins containing IDRs as well as completely disordered proteins. Intrinsically disordered systems can also be generated following proteolysis of otherwise larger proteins such as amyloid-β peptides and amyloidogenic fragments of gelsolin. Because IDPs lack a stable 3D structure, they are extremely sensitive to their environment, much more so than ordered proteins. Furthermore, this lack of a fixed structure is often associated with extreme binding promiscuity of IDPs. The best-known examples of amyloidogenic IDPs include amyloid β (Aβ) peptide, tau (AD), α-synuclein (Parkinson's disease (PD) and other synucleinopathies), TAR DNA-binding protein 43 (TDP-43), fusion in sarcoma (FUS) protein (amyotrophic lateral sclerosis (ALS)), and huntingtin (Huntington's disease). Many human functional amyloid proteins (cytoplasmic polyadenylation element binding protein (CPEB), T cell-restricted intracellular antigen-1 (TIA-1), premelanosomal protein 17 (Pme117), secreted peptide hormones) and cell cycle-related proteins, including p53, p27, p21 (cell cycle, apoptosis). In addition, twenty to thirty percent of cellular proteins are classified as IDPs, and up to 41% of the eukaryotic proteome is expected to contain IDRs. Table 1 provides a list of several intrinsically disordered or unknown structured peptides or proteins that form intracellular or extracellular amyloid, non-amyloid, and amyloid-like fibril deposits in human diseases.

[0232] As used herein, the term "amyloidosis" or "amyloidogenic" refers to a disease, disorder and / or condition associated with or characterized by the pathological accumulation of any disease-associated protein that exhibits an amyloid conformation (i.e., a 3-fold), including but not limited to Alzheimer's disease, vascular dementia, and cognitive impairment.

[0233] The term "proteostasis" or "protein homeostasis" refers to the concentration, conformation, binding interactions, e.g., quaternary structure, and location of the proteins that make up the proteome. Proteostasis is influenced by the chemistry of protein folding / misfolding as well as numerous regulated networks of interacting and competing biological pathways that affect protein synthesis, folding, conformation, binding interactions, trafficking, dissociation, and degradation.

[0234] Increasing proteolytic capacity offers an alternative approach to maintaining protein homeostasis.

[0235] Thus, the present invention encompasses the discovery that NMDAR blockers, through activation of the proteasome by increasing levels and / or increasing activity, may provide effective treatment, or even prevention, of certain proteinopathies.

[0236] For example, the present invention provides new insights into proteasome activity and its effect on protein degradation, and proves that the NMDAR pathway related to proteasome function regulates the level of protein amount in various cases, specifically including various cell cultures, Caenorhabditis elegans, mammalian organisms (e.g., mice) and mouse brain. The present invention specifically encompasses that the trypsin-like activity, chymotrypsin-like activity and caspase-like activity of proteasomes improved by NMDAR blockers can provide effective treatment (and / or prevention) of certain protein diseases causing cell dysfunction, thereby promoting the performance and progress of many neurodegenerative diseases, other aggregate deposition diseases, even aging and related diseases. Some embodiments of the present invention are applicable to all protein diseases.

[0237] The present invention improves proteasome activity by using at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof, thereby enhancing protein hydrolysis capacity. Therefore, the present invention provides a drug for treating diseases (Table 1) caused by chronic protein misfolding and toxic aggregation.

[0238] In addition to the natural formation of misfolded proteins, oxidative damage is known to be one of the most important factors of protein misfolding (Dobson et al., 2015; Chiti and Dobson, 2017).

[0239] Oxidative stress can lead to nonspecific post-translational modifications of proteins and cause protein aggregation.

[0240] Proteins are one of the main targets of oxygen free radicals and other reactive species. During the aging process, there is a continuous accumulation of oxidized proteins.

[0241] After oxidative damage, proteins unfold and expose hydrophobic regions, which makes these proteins prone to aggregation (Reichmann et al., 20181).

[0242] Oxidative modification of proteins by oxygen free radicals and other reactive species such as hydroxynonenal occurs in physiological and pathological processes. As a result of the modification, carbonyl groups are introduced into protein side chains through site-specific mechanisms and cause protein diseases (Jung et al., 2014). Therefore, protein oxidation plays a major role in the development of various age-related diseases.

[0243] The present invention also provides compositions and methods for preventing, reducing or even treating oxidatively damaged proteins in tissues by showing a reduction in protein carbonylation ( Fig.28 ).

[0244] Thus, the compositions and methods provided by the present invention not only treat diseases, but more importantly prevent the causes of diseases associated with malfunction of the protein homeostasis network, including the accumulation of oxidatively damaged proteins and / or misfolded proteins.

[0245] The inability of cells to maintain proteostasis leads to the toxic effects of protein aggregates in many diseases and is also a major driver of the aging process. In addition to counteracting the toxic effects of aggregated disease proteins, enhancing proteostasis capacity also extends lifespan. Conversely, the long-term presence of aggregates inhibits the ability of cells to respond adequately to stress, supporting the view that protein aggregation is a major driver of the aging process. The decline in proteasome activity during aging would explain why aging is a key risk factor for protein aggregation. Instead, there is evidence that aggregation is not a consequence of a malfunctioning UPS but is actually its cause. This view is supported by the finding that the expression of structurally unrelated aggregation-prone proteins prevents other proteins from being degraded by the proteasome (Chiti and Dobson, 2017).

[0246] Modulation of protein concentrations by regulating the proteolytic machinery has long been shown to be a promising setting for developing therapeutic strategies against different aging and aging-related human diseases, such as neurodegeneration, cancer, autoimmunity and others.

[0247] Therefore, the clearance of misfolded proteins is critical for cell survival because misfolding alters the three-dimensional structure of proteins, thereby impairing their biological activity and increasing their propensity to form toxic aggregates.

[0248] Therefore, it becomes important to investigate chemical structures and / or methods that will maintain and / or increase proteolysis, thereby ameliorating the onset and / or progression of aging and aging-related diseases, or even treating aging and aging-related diseases.

[0249] The method of the present invention comprises administering to a subject a therapeutically effective amount of an NMDA receptor antagonist that is effective in increasing the activity of the proteasome. The present invention relates to means and methods for effectively preventing the onset and / or progression of aging and even treating aging.

[0250] As used herein, the terms "treating" and "treatment" refer to eliminating, substantially inhibiting, slowing or reversing the progression of a condition, substantially delaying the appearance of clinical symptoms of a condition, substantially ameliorating clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms. For example, with respect to aging, the term "treating" may mean reducing or alleviating: susceptibility to infection, risk of heat stroke or hypothermia; thinning of the spine, fractures, joint changes (ranging from mild stiffness to severe arthritis); loss of muscle mass and muscle strength (sarcopenia), stooped posture, slowed and limited movement; decreased strength and dexterity of the hands, including "hand and finger strength and the ability to control submaximal pinching force and maintain a stable, precise pinching posture, manual speed, hand sensation"; frailty, a syndrome of decreased strength, physical activity, physical performance, and energy; maximum oxygen uptake and maximum heart rate slowed thinking, memory and reflection, decreased reflexes and coordination, and difficulty with balance; decreased visual acuity, weakened peripheral vision, and hearing loss; wrinkling and sagging of the skin, whitening or graying of hair, and weight loss, partly due to loss of muscle tissue; loss of strength in the eye's ciliary muscles, leading to difficulty focusing on near objects, or presbyopia; menopause, which usually occurs between the ages of 44 and 58; and loss of elasticity in the arteries, and the resulting hardening of the blood vessels, atherosclerosis, which leads to cardiovascular disease (e.g., stroke and heart attack), the most common cause of death worldwide.

[0251] With respect to aging, the term "protection" as used herein refers to preventing, delaying or treating conditions associated with aging of a human system, or, where appropriate, preventing the development or continuation or worsening of all such conditions. Here are examples of how aging affects some of our major body systems: Cells, Organs and Tissues: Decreased ability of cells to divide, shortened telomeres, accumulation of oxidative products, hardening of intercellular connective tissue, reduced maximal functional capacity of many organs.

[0252] Heart and Blood Vessels: Increased Thickness: The walls of the heart become thicker. The heart muscle becomes less efficient (working harder to pump the same amount of blood). The aorta (the body's main artery) becomes thicker, stiffer, and less flexible. Many of the body's arteries, including those that supply blood to the heart and brain, slowly develop atherosclerosis, although in some people the condition never becomes severe.

[0253] Vital signs: The body has a harder time controlling its temperature. Heart rate takes longer to return to normal after exercise.

[0254] Bones, muscles, joints: Bones become thinner and less strong. Joints become stiffer and less flexible. The cartilage and bone in the joints begin to weaken.

[0255] Muscle tissue becomes less large and less strong

[0256] Digestive system: The movement of food through the digestive system becomes slower. The stomach, liver, pancreas, and small intestine produce smaller amounts of digestive juices.

[0257] Brain and nervous system: Decreased number of nerve cells in the brain and spinal cord. Decreased number of connections between nerve cells. Abnormal structures, called amyloid, plaques and tangles, may form in the brain. Impairment of cognition (such as memory and / or orientation) or global functioning (overall functioning, including activities of daily living) and / or slowing or reversing progressive worsening of global or cognitive impairment.

[0258] Eyes and ears: The retina becomes thinner, the iris becomes harder, and the lens becomes less clear. The walls of the ear canal become thinner. The eardrum becomes thicker.

[0259] Skin, nails, and hair: The skin becomes thinner and less elastic. The sweat glands produce less sweat. Nails grow more slowly. Hair turns gray and some hair stops growing.

[0260] According to the present invention, unless otherwise stated, all molecular and phenotypic changes that occur after using NMDAR blockers, as indicated in the following research results, are the result of NMDAR blockers increasing proteasome activity. Proteasome activation essentially includes activation of the 20S proteasome and its functions, including trypsin-like activity, chymotrypsin-like activity and caspase-like activity.

[0261] The present invention provides compositions and methods effective for ameliorating the onset and / or progression of aging by preventing malfunction of the proteostasis network, including the accumulation of oxidatively damaged proteins and / or misfolded proteins.

[0262] The role of oxidative stress in age-related organ changes is generally considered to be that an increase in ROS accompanies aging, leading to functional alterations, increased morbidity, and shortened lifespan ( Hipp et al., 2014 ).

[0263] The proteasome is the first defense mechanism of cells against the toxic stress of accumulated proteins induced by oxidative damage ( Korovila et al., 2017 ; Shang et al., 1997 ; Grune, 2000 ; McNaought et al., 2001 , Njoman and Tepe, 2019 ).

[0264] Therefore, the present invention provides compositions and methods that effectively improve the onset, progression, and even prevention of aging. In particular, the compositions of the present invention reduce or prevent the symptoms of aging by increasing proteasome activity to maintain proteolysis. In addition, the present invention demonstrates that the use of NMDA receptor complex antagonists not only reduces aging and aging-related diseases, but also prolongs lifespan. In other words, the present invention provides an extension of lifespan and healthspan.

[0265] Simply put, lifespan is the number of years of life, while healthspan is the quality of life. As indicated in the paragraph above, healthspan encompasses the changes in body systems that occur with aging and the improvement, prevention, and / or correction of clinical manifestations associated with those changes in body systems.

[0266] The present invention shows that both NMDA receptor inhibitors ketamine and memantine prevent cellular senescence, as shown by experiments using β-galactosidase staining of primary fibroblasts ( Fig.24A In addition, the present invention also shows that NMDA receptor inhibitors improve the proliferation ability and fat metabolism of primary fibroblasts ( Fig.24C and Fig.24D ).

[0267] The present invention also demonstrates that memantine extends the lifespan of C. elegans worms. The present invention demonstrates that at day 21, the number of surviving worms in the group receiving memantine was 4 times higher than in the untreated group ( Fig.25A and Fig.25B The present invention further demonstrates that the motor activity of worms is significantly increased in the presence of memantine ( Fig.25C ).

[0268] Thus, the present invention provides an agent that prolongs both healthspan and lifespan.

[0269] The present invention further demonstrates that NMDAR blockers increase the amount of NADP+ and increase the ratio of NADP+ / NADPH.

[0270] Coenzyme I (nicotinamide adenine dinucleotide, NAD+ / NADH) and coenzyme II (nicotinamide adenine dinucleotide phosphate, NADP+ / NADPH) are involved in various biological processes in mammalian cells. Although NAD+ and NADP+ are synthesized in sufficient amounts under normal conditions, supply shortages due to overconsumption and reduced synthesis have been observed with age and in certain disease conditions. Some studies have demonstrated that levels of NAD+ and NADP+ decrease with age in a wide range of tissues (such as liver, skin, muscle, pancreas, and fat). The ratios of NAD+ / NADH and NADP+ / NADPH indicate the redox state of the cell. A reduction in this ratio affects cellular anaerobic glycolysis and oxidative phosphorylation functions, which reduces the ability of cells to produce ATP (She et al., 2021).

[0271] This molecule is present in the cell in a reduced (NADPH) form and an oxidized (NADP+) form, thereby reflecting the redox state of the cell. The important cellular role for NADPH is to provide reducing power for the reductive biosynthesis of macromolecules including fatty acids and complex lipids, proteins and nucleotides. NADPH also plays an important role in the control of the redox state of the cell. In the latter function, NADPH provides reducing equivalents for the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), a reaction catalyzed by glutathione reductase. GSH is an important intracellular reductant and plays an important role in cellular antioxidant defense.

[0272] Therefore, the amount of NADP+, NADPH+ and their ratio were studied in C. elegans treated with memantine. It was found that on day 19, NADP+ detected in C. elegans treated with memantine was 2 times higher than that in C. elegans not treated with memantine ( Fig.25F and Figure 25G ).

[0273] Therefore, the age-related reduction in fat oxidation can promote the accumulation of systemic fat and centrosomal fat. Adipogenesis and lipid accumulation during the aging process have a great impact on the pathogenesis of the aging process and chronic age-related diseases. Therefore, on the 20th day, the amount of fat in the organism was studied by Nile red staining in the presence of the memantine group and the control C. elegans group. The results of the present invention show that on the 20th day, the lipid accumulation in the control C. elegans group was more than 2 times higher than that in the C. elegans grown in the presence of memantine ( Fig.25D and Fig.25E ).

[0274] The simplest and most easily determined finding of clinical signs of deterioration in aging mice is the evaluation of changes in the mouse skin. In fact, the present invention further demonstrates that the color change and condition of the hair of 22-month-old mice treated with and without memantine are very different, with the color change and condition of the hair of mice receiving memantine being better ( Fig.26 ).

[0275] Many biomarkers associated with aging have been identified in humans. These biomarkers have also been identified in mice. Mice are used as models for human aging and many movement disorders, including diseases originating from the body and central nervous system. For all of these models, a comprehensive assessment of their motor deficits must include specific strength tests.

[0276] The present invention demonstrates that the clinical weakness tests including the Kondziela inverted grid test (latency to fall, movement time, four-finger grasp), weight test (forelimb grasp holding time) and open field maze (OFM) test (total distance walked, total number of uprights test, number of supported uprights, number of unsupported uprights) in the group of 2-year-old mice receiving memantine were statistically significantly increased compared to the group of mice not receiving memantine ( FIG. 27A to FIG. 27K ).

[0277] As mentioned above, many biomarkers associated with aging have been identified in humans. Importantly, the grip test performed on humans has been defined as a highly predictive indicator of disability, disease, and death that may occur due to aging (Iconaru et al., 2018; Sousa-Santos and Amaral, 2017).

[0278] Open Field Maze (OFM). The OFM is one of the most widely used platforms that allows researchers to measure behaviors ranging from overall locomotor activity to anxiety-related emotional behaviors.

[0279] Besides locomotor activity, OFM remains one of the most widely used techniques in rodent behavioral studies.

[0280] As mentioned above, the total distance traveled test shows the anxiety state of mice in addition to motor activity. Another test that shows anxiety in mice and is included in the OFM experiment is haptotropism. This test includes different measurements such as central area time, peripheral area time, central escape latency, central latency time, and central area entry times in OFM.

[0281] In the next experiment, when the mice's movements in the OFM inner zone were observed for 5 min, it was determined that the mice that received memantine spent 4 times longer in the central zone compared to mice that did not receive memantine (P = 0.01) ( Figure 27G ).

[0282] In addition, it was determined that mice that did not receive memantine remained in the peripheral area more than mice that received memantine (p=0.01) ( Fig.27H ). Central escape latency, central latency time, and number of central zone entries were also studied, which are other studies of OFM. Although the means of all three studies differed from each other by at least two-fold, the P values ​​were determined to be greater than 0.05 in all three studies (P values ​​were 0.4, 0.07, and 0.08, respectively).

[0283] Many biomarkers associated with aging have been identified in humans. However, perhaps more importantly, motor activity is intricately intertwined with complex human activities such as learning and motivation. In fact, the present findings also show that mice receiving memantine have less anxiety as determined by OFM experiments than mice that do not receive memantine.

[0284] Thus, the present invention encompasses the discovery that NMDAR blockade, through activation of the proteasome by increasing levels and / or increasing activity, may provide effective treatment, or even prevention, of age-related mood anxiety in addition to providing improvements in motor activity.

[0285] In the above studies, we have shown that memantine extends the lifespan of C. elegans and increases their muscle strength. In mouse studies, we have shown that memantine phenotypically increases muscle strength and reduces amyloid formation ( FIG. 29A to FIG. 29C ), reduced melanin and lipofuscin deposition ( Fig.30 A and Fig.30 B) Fat deposition ( Fig.31A to Figure 31D), and reduced protein oxidation in tissues ( Fig.28 ), which are hallmarks of aging, it is striking that we did not investigate the direct effects of memantine on lifespan in mice.

[0286] The most important research supporting our invention is the research published and patented by Anthony FUTERMAN, Andres David KLEIN, which showed that memantine extended the lifespan of 4 different strains of mice. The study showed that the lifespan was extended between 2-fold and 5-fold, depending on the strain of mouse ( FIG. 32A to FIG. 32C). In their study, they produced Gaucher disease (GD) in 15 inbred mouse strains from different phylogenetic origins by inhibiting acid β-glucosidase (GC enzyme) using cyclohexene tetraol B-epoxide (CBE). They also demonstrated a correlation between the amount of CBE injected into the mice and the accumulation levels of GC enzyme substrates, glucosylceramide (GlcCer) and glycosphingolipids (GSLs), and showed that the disease pathology represented by the altered levels of pathological markers depended on the levels of accumulated lipids and the time when they started to accumulate.

[0287] They also showed that motor activity was significantly improved in mice that received memantine ( Fig.32D ), which is similar to the findings of the present invention.

[0288] GD is one of the most common lysosomal storage diseases (LSDs) and is caused by mutations in the gene GBA1, which encodes a lysosomal hydrolase (GCase). The resulting GCase deficiency leads to the accumulation of GSLs, GlcCer, and their deacylated forms in the lysosomes of cells.

[0289] There is increasing evidence that GlcCer and Glc-Sph accumulation is directly associated with aging. Haploinsufficiency of the GBA gene, resulting in reduced GC enzyme activity, has recently been found to be one of the most common genetic risk factors for PD. In addition, GC enzyme activity is also reduced in brain regions of sporadic PD patients, with corresponding accumulation of their glycosphingolipid (GSL) substrates.

[0290] We demonstrate that aging of the mouse brain results in a lipid profile similar to that observed in the aging human brain and PD, with elevated levels of GlcCer, Glc-Sph, and lactosylceramide (LacCer). Parallel biochemical analyses reveal that changes in lipid metabolism in the aging brain may precede or be part of abnormal protein processing and may accelerate PD pathophysiological processes in susceptible neurons in PD and other age-related neurodegenerative disorders.

[0291] Furthermore, the fact that ceramide causes an increase in ROS and oxidative stress in many mammalian cells and animal models, which is directly related to aging, is other important evidence showing the relationship between elevated GC enzyme substrates and aging.

[0292] In this regard, findings in mice with GCase inhibition, such as accumulation of insoluble α-synuclein aggregates in the substantia nigra; altered levels of proteins involved in the autophagy / lysosomal system, widespread neuroinflammation, upregulation of complement Clq, abnormalities in synaptic, axonal transport, and cytoskeletal proteins, and neurodegeneration, can be shown as more direct evidence for the relationship between elevated GCase substrates and aging.

[0293] In addition, a significant reduction (28% to 40%) in mitochondrial ATP production and oxygen consumption was detected in neuropathic Gaucher disease (nGD) brain and CBE-treated neurons. These studies suggest that GC enzyme function defects and GlcCer / Glc-Sph accumulation are risk factors for mitochondrial dysfunction and polyproteinopathy (α-synuclein, APP-aggregates, and Ab-aggregates) in nGD.

[0294] nGD has histopathological features that include a proinflammatory response in the CNS and are associated with astrogliosis, microgliosis, and neuronal degeneration in affected humans and mice. Neurodegenerative lesions are often complex, with progressive accumulation of protein aggregates (proteinopathy), for example, α-synuclein, ubiquitin, amyloid precursor protein (APP), β-amyloid (Ab), tau, and other proteins in Parkinson's disease, Alzheimer's disease, and Huntington's disease. In addition to Gaucher disease, other LSDs, such as Tay-Sachs disease and Sandhoff disease and Niemann-Pick disease type C, also show some accumulation of these proteins. Recently, clinical and genetic studies have shown that α-synuclein and ubiquitin protein aggregates in the brains of Gaucher disease patients and mouse models are commonly present in many non-LSD neurodegenerative diseases. Therefore, depletion of GCase polypeptides impairs protein degradation capacity and increases α-synuclein levels in neurons.

[0295] Taken together, these studies suggest that multiple proteinopathies are the pathophysiological hallmarks of nGD.

[0296] These data are important for the prevailing view that proteinopathies are the sole triggers of age-related diseases, including neuronal death in PD, as alterations in GSL homeostasis may occur simultaneously with, or even precede, changes in protein aggregation.

[0297] In support of this, it was shown that C2 and C6 ceramides promote senescence as revealed by increasing the amounts of p53 and p21, important members of the MDM2-p53-p21Cipl pro-survival pathway in mammalian cells.

[0298] Depending on the context, the autophagy-lysosomal pathway and the proteasome share common substrates as well as regulators. The two systems intersect and communicate at multiple points to coordinate and balance their roles in protein homeostasis and organelle homeostasis.

[0299] Probably the most important issue that occurs due to reduced proteasome activity is the accumulation of potentially toxic unfolded / misfolded proteins as well as protein aggregates. Impaired proteasomes will eventually affect cytoplasmic processes including autophagy and all other important functions of organelles due to the accumulation of unfolded and damaged proteins. Futerman et al. showed that by using memantine in mice with all GD symptoms, their lifespan was extended and their motor coordination was significantly improved. On the other hand, they showed that memantine had no effect on the correction of GC enzyme inhibition, which leads to the appearance of GD symptoms.

[0300] Thus, in fact, Futerman et al. disrupted the proteostasis network by inhibiting protein degradation capacity through inhibition of GC enzymes, which actually led to premature aging associated with interference with key signaling pathways, including the autophagy-lysosomal pathway and the proteasome pathway.

[0301] The disease-monism theory of fundamental aging processes posits that these fundamental aging processes include: (1) chronic low-grade “sterile” (free of bacteria, fungi, etc.) inflammation, often with fibrosis; 2) macromolecular dysfunction (e.g., protein misfolding and aggregation, reduced proteasome activity, DNA damage, telomere uncapping, increased advanced glycation end products [AGEs], lipotoxicity, and accumulation of bioactive lipids) and organelle dysfunction (nuclear membrane alterations associated with lamin B deficiency, mitochondrial dysfunction resulting in reduced fatty acid metabolism, increased glucose utilization, depletion of NAD+, and increased ROS production, etc.); 3) stem, progenitor, and immune cell dysfunction (including altered proliferation capacity and impaired differentiation, failure to develop into functional mature cells, decreased “anti-aging” factors [e.g., α-Klotho] that lead to stem and progenitor cell dysfunction), and 4) cellular senescence, and that these fundamental aging processes are likely to be interconnected. Presumably, targeting any one of the fundamental aging processes genetically or with drugs (as described above) should affect many or perhaps all of the remaining processes (Austad, 2016).

[0302] Thus, as described herein, the protein homeostasis provided by memantine through activation of the proteasome plays a major role in preventing many or possibly all of the basic aging processes. Thus, premature aging and even premature death in mice caused by inhibition of the GC enzyme by Futerman et al. in clinical presentations was prevented (see U.S. Patent, Futerman et al., Application Publication No.: US2017 / 0273917 A1, Publication Date: September 28, 2017, and Klein et al., 2016)

[0303] Similar to the results of Futerman et al., the present invention demonstrated that the clinical frailty tests (including falling time, moving time, four-finger grasp, forelimb grasp, total walking distance, and total upright times tests) performed in the group of 2-year-old mice receiving memantine were statistically significantly increased compared to the group of mice not receiving memantine.

[0304] The continuous turnover of intracellular proteins is essential for maintaining cell homeostasis and for regulating a variety of cell functions. All intracellular proteins undergo continuous synthesis and degradation. Among other functions, this constant protein turnover helps to minimize the time that a specific protein is exposed to a dangerous cell environment, and therefore minimizes the possibility of being damaged or changed. At first glance, before the cell components lose functionality, this constant renewal of cell components seems to be a huge waste of cell resources. However, considering the harmful consequences of the accumulation of components in damaged cells on cell function and survival, this renewal is very reasonable. In addition, protein degradation rather than simple destruction is actually a recycling process, because the constituent amino acids of the degraded protein are reused for the synthesis of new proteins. The rates of synthesis and degradation of different proteins in cells are different, and can change in response to different stimuli or under different conditions. This balance between protein synthesis and degradation also allows cells to quickly change the protein level in cells to adapt to changes in the extracellular environment. Under conditions that cause extensive cell damage, appropriate protein degradation is also essential for cell survival. In fact, the activation of the intracellular proteolytic system often occurs as part of the cell's response to stress. In this role as a "quality control" system, this proteolytic system is assisted by the 20S proteasome, which ultimately determines the fate of oxidatively damaged / unfolded proteins.

[0305] Maintenance of protein homeostasis has been shown to be a defense mechanism against toxic stress of accumulated proteins induced by oxidative damage, thereby preventing aging and aging-related diseases and extending lifespan.

[0306] The ubiquitin-proteasome system is currently considered to be the major intracellular proteolytic system, and malfunction of this system can lead to different aspects of the aging phenotype and the pathogenesis of several age-related diseases.

[0307] This age-related decrease in proteolytic activity has been observed in almost all organisms analyzed, and specific defects in different proteolytic systems have been reported with age. Considering the myriad intracellular functions in which protein degradation is involved, it is not surprising that the consequences of age-related alterations in proteolytic systems are extensive and lead to a wide variety of pathologies.

[0308] There is much literature showing that the amount of oxidized proteins is increased in skeletal muscle, heart, skin, liver, lymphocytes, and other tissues of various animal species.

[0309] Cardiovascular diseases, disorders and / or conditions are the leading cause of death worldwide. Typically when cardiovascular heart problems are detected, the disease is often already quite advanced, has developed over decades, and is often too advanced to successfully prevent significant permanent disability.

[0310] Generally, cardiovascular disease can be the disease related to heart and / or blood vessel, artery and occasionally related to vein.In some embodiments, the disease is vascular disease.Exemplary specific protein disease cardiovascular disease, disease and / or the patient's condition can include myocardial ischemia, myocardial infarction, coronary heart disease, acute coronary symptoms, unstable angina or stable angina, stroke, ischemic stroke, angiogenesis, cardiac hypertrophy, congestive heart failure, restenosis, arteriosclerosis, atherosclerosis, vasculitis, polyarteritis nodosa, myocarditis, hypertension, inflammation or autoimmune disease related atherosclerosis or restenosis.In addition, the irreversible loss of myocardial cells caused by oxidative stress is the main cause of cardiac dysfunction after ischemia / reperfusion injury and aging-induced cardiomyopathy (Jian et al., 2005; Giordano, 2005; Eltzschig and Eckle, 2011).

[0311] Therefore, the present invention provides compositions and methods for preventing the above-mentioned heart diseases by maintaining and / or improving the function of the myocardium, and these compositions and methods are effective in improving the onset and / or progression of cardiac dysfunction after ischemia / reperfusion injury and aging-induced cardiomyopathy.

[0312] Aging is the main determinant of the high prevalence of metabolic syndrome. With the increasing number of elderly people with metabolic syndrome, a major threat is the prevalence of cardiovascular morbidity and mortality associated with aging, such as atherosclerotic vascular dysfunction. A large amount of clinical evidence shows that the mortality rate of myocardial infarction or stroke is much higher in patients with premature aging.

[0313] Although many theories about the relationship between premature aging and metabolic syndrome have been elucidated, it is currently recognized that protein homeostasis and protein quality control are impaired. Accumulated protein toxic stress induced by oxidative damage and reduced associated proteasome activity are the main causes of protein homeostasis and protein quality control. Therefore, in another embodiment, the present invention provides a pharmaceutical composition for preventing metabolic syndrome by regulating protein homeostasis and protein quality control, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0314] With aging, changes in lipid metabolism occur, leading to increased plasma free fatty acid concentrations or increased non-oxidative processing, which may contribute to various metabolic disorders, including obesity, accumulation of total body fat and central body fat, insulin resistance, inflammation, and loss of muscle mass and muscle strength. Overall, with aging, changes in lipid metabolism occur, leading to increased plasma free fatty acid concentrations or increased non-oxidative processing, which contribute to an increased risk of developing diabetes and cardiovascular disease. A reduction in the size and / or oxidative capacity of metabolically active muscle mass may be a more likely determinant of reduced fat oxidation. Therefore, maintaining muscle mass and activity during aging may prevent metabolic syndrome by preventing changes in lipid metabolism.

[0315] In addition, muscle mass is the processing center of whole-body glucose metabolism, participating in 80% of the body's glucose uptake and treatment. Imbalances in muscle mass metabolism strongly affect glucose homeostasis and insulin sensitivity throughout the body, which can lead to SkM inflammation, induce oxidative stress muscle mass, and impair muscle mass health. In addition to participating in insulin-related glucose metabolism, muscle mass is also involved in multiple metabolic pathways in the body, including lipid metabolism.

[0316] As described above, the present invention demonstrates that the motor activity of both C. elegans and mice treated with NMDAR inhibitors was significantly increased compared to the control group ( Fig.25C , FIG. 27A to FIG. 27K ). In addition, the present invention demonstrates that the amount of lipid droplets in the muscle tissue of Caenorhabditis elegans and mice treated with NMDAR inhibitors is significantly lower than that of the control group not treated with NMDAR inhibitors ( Fig.25D , Fig.25E ; FIG. 32A to FIG. 32E ).

[0317] Therefore, in one aspect, the present invention provides a pharmaceutical composition for preventing metabolic syndrome by increasing muscle mass and activity, thereby regulating lipid metabolism, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof. In addition, in one aspect, the present invention provides a pharmaceutical composition for preventing obesity and related diseases, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof, which are accompanied by the occurrence of metabolic syndrome caused by changes in lipid metabolism with age.

[0318] Thus, the present invention encompasses the discovery that NMDAR blockers can be used to prevent, ameliorate and / or treat metabolic disorders affecting body weight, particularly in the treatment of obese subjects, especially in the treatment of human subjects, by increasing levels and / or increasing activity of proteasome activation.

[0319] According to the present invention, it is also contemplated that NMDA receptor antagonists, in particular memantine, a pharmaceutically acceptable salt or prodrug of an NMDA receptor antagonist, are used for medical intervention of secondary conditions associated with (pathological) weight gain. These "secondary conditions" may include, but are not limited to, type 2 diabetes, hypertension (high blood pressure), cardiovascular disease, cancer, sexual function problems, and disorders of the muscular or skeletal system.

[0320] As we age, muscle mass decreases at a rate of approximately 1% to 2% per year, which greatly increases the risk of muscle diseases, including (i) mitochondrial dysfunction, which causes various muscle pathologies such as sarcopenia and muscular dystrophy; (ii): inflammatory myopathies caused by inflammation and oxidative stress, such as dermatomyositis, polymyositis, necrotizing autoimmune myositis, and sporadic inclusion body myositis, and (iii) chronic diseases that cause SkM damage, such as type 2 diabetes, obesity, chronic kidney disease, and chronic obstructive pulmonary disease. These syndromes often lead to a decline in the patient's quality of life over time and increase the patient's mortality rate.

[0321] Therefore, the present invention provides a method for preventing and / or treating the above-mentioned muscle diseases.

[0322] In another aspect, the present invention provides compositions and methods for maintaining and / or reducing skin and hair aging by reducing the accumulation of oxidized proteins, lipids and inflammation. Therefore, according to one aspect of some embodiments of the present invention, the present invention provides an NMDA receptor antagonist that detoxifies cells with oxidatively damaged proteins and lipids by inducing the activity of a 20S proteasome complex, which can be used in cosmetics, including cosmetics for graying hair, skin wrinkles and age spots on the skin.

[0323] In addition, the present invention provides compositions and methods for maintaining and increasing stemness characteristics. The term "stemness" is a property of cells, i.e., a property of having certain genes, the expression of which enables the potential for self-renewal and multi-lineage differentiation. Recognized stemness is "the highest degree of plasticity of cells within the cell type pool present in an organism."

[0324] Kapetanou et al. showed that proteasome activation enhanced the stemness and lifespan of human mesenchymal stem cells (Kapetanou et al., 2017).

[0325] One indicator of stem cell differentiation is to measure the amount of stemness markers (including CD44, CD73 and CD90) in late cell passages (Ali et al., 2015; Gonzalez-Garza et al., 2018, Yoo et al., 2020). Therefore, in the present invention, these markers were studied in memantine-treated stem cells and untreated stem cells. According to the control cells (not passaged), the amount of CD44, CD73 and CD90 increased on day 14, and on day 14, the increase in markers in memantine-treated cells was statistically significantly lower than that in cells not treated with memantine ( FIG. 36A to FIG. 36C ). Another indicator of stem cell differentiation is to measure the amount of lipid droplets in the cells. Fourteen days of cell passage increased the lipid in the cells. It was observed that memantine treatment had suppressed the amount of lipid droplets ( Fig.36D ). In addition, the present invention prolongs the life span of human mesenchymal stem cells. Fig.36E MTT assay of day 21 cells is shown (P<0.034).

[0326] Thus, in another embodiment, the present invention provides compositions and methods for maintaining and increasing stemness characteristics, which are the potential for stem cell self-renewal, multi-lineage differentiation, and lineage reprogramming.

[0327] With aging, the physiological activity of muscle stem cells decreases, the regenerative capacity of SkM decreases, and the efficiency of muscle repair decreases, and this is accompanied by a decrease in muscle mass and muscle strength, as well as irreversible muscle loss, which seriously affects the quality of life of the elderly.

[0328] Therefore, the present invention provides compositions and methods for maintaining and increasing stemness characteristics and preventing the decrease of muscle mass and muscle strength, irreversible muscle loss, and muscle diseases that seriously affect the quality of life of the elderly by enhancing proteasome activity.

[0329] The effects of the present invention are also not limited to muscle tissue, but the present invention also provides compositions and methods for maintaining and increasing stemness characteristics and preventing aging and related diseases by ensuring the continuation of the regenerative properties of all tissues, including neurons.

[0330] High incidence of ocular diseases such as age-related macular degeneration, cataracts, glaucoma and diabetic retinopathy are multifactorial in origin and are related to genetics, environmental factors, age and oxidative stress. Oxidative stress is one of the factors that have the greatest impact on ocular diseases. The most important problem that may occur due to reduced proteasome activity is the accumulation of potentially toxic unfolded / misfolded proteins and protein aggregates. Impairment of the proteasome will ultimately affect cytoplasmic processes including autophagy and all other important functions of organelles due to the accumulation of unfolded and damaged proteins. Alterations in the normal function of the autophagic process can interrupt organelle turnover, leading to the accumulation of cell debris and causing physiological dysfunction of the eye.

[0331] High incidence of eye diseases such as age-related macular degeneration (AMD) is characterized by the accumulation of extracellular deposits, i.e. drusen, and progressive degeneration of photoreceptors and adjacent tissues. The protein composition of drusen includes apolipoproteins and oxidized proteins. Chronic inflammation, lipid deposition, oxidative stress and extracellular matrix maintenance are closely related to the pathogenesis of AMD.

[0332] According to one aspect of some embodiments of the present invention, there is provided an NMDA receptor antagonist for treating or preventing ocular pathologies associated with impaired protein organelle turnover by increasing proteasome activity and thereby acting by regulating the autophagy-lysosomal pathway (Fleckenstein et al., 2021; Fernandez-Albarral et al., 2021; Blasiak et al., 2021).

[0333] According to one aspect of some embodiments of the present invention, there is provided an NMDA receptor antagonist for treating viral infection by degrading viral proteins such as HepC core protein. Another subject of the present invention is HIV, as proteasome inhibitors are known to activate latent HIV (Cary and Peterlin, 2020; Timilsina et al., 2019).

[0334] As described above, the present invention also demonstrates that NMDAR inhibitors reduce carbonylated and carbamylated proteins responsible for structural changes that lead to exposure of abnormally denatured regions on the protein surface, thereby generating new antigens that lead to autoimmune responses ( Fig.28 )(Jaisson et al., 2018; Shi et al., 2011).

[0335] There is increasing evidence that proteasome dysfunction is associated with (auto)inflammation and autoimmunity (Brehm and Kriger, 2015). Autoimmune responses to protein aggregation caused by oxidatively damaged and / or misfolded proteins are often accompanied by altered cytokine patterns, such as in diabetes or Sjogren syndrome.

[0336] Importantly, one of the key issues that arise in proteasome activity defects is increased functional susceptibility to infection (especially viral infection), delayed recovery, and autoimmunity following infection. For example, LMP7 (PSMB8) knockout mice are physiologically indistinguishable from wild-type mice, but the former are more susceptible to some infections and have prolonged or inadequate pathogen clearance. In particular, an example of the CNS is that by studying autoimmune encephalomyelitis (EAE) in a mouse model, the immunoproteasome (i.e., a specific isoform of the 20S proteasome deficiency) was found to result in a significant increase in clinical scores. In a mouse model for coxsackievirus B3-induced myocarditis, immunoproteasome (IP) deficiency was also associated with severe myocardial damage with large inflammatory lesions and severe myocardial tissue damage. This protective effect has also been shown by studies that IP-deficient mice suffer from severe impairment of stress response, survival and / or clearance after infection or inflammation, or develop severe LPS-induced hepatitis. Furthermore, IP dysfunction is associated with (auto)inflammation and autoimmunity, often accompanied by altered cytokine patterns, such as in diabetes or Sjögren's syndrome (Brehm and Kruger, 2015).

[0337] Several previously described autoinflammatory syndromes, such as Nakajo-Nishimura syndrome (NNS), joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy (JMP) syndrome, Japanese autoinflammatory syndrome with lipodystrophy (JASL), and chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), are now classified into a series of diseases called proteasome-associated autoinflammatory syndromes (PRAAS). Multiple inflammatory manifestations develop including nonspecific lymphadenopathy, hepatosplenomegaly, autoimmune hemolytic anemia, hypertriglyceridemia, and lipodystrophy.

[0338] The most common known cause of the syndrome is mutations in proteasome subunits, including PSMB8 (encoding β1i), PSMA3 (encoding a7), PSMB4 (encoding β7), PSMB9 (encoding β1i), which are genes encoding proteasomes that in turn break down other proteins. The reported mutations have different effects on the proteasome through regulation of gene expression, subunit folding and maturation, assembly of the core complex, and / or structural alterations in the proteolytic pocket, but all result in reduced proteasome activity, which is insufficient to cope with the higher load of damaged proteins and leads to non-degradation of proteins and accumulation of oxidized proteins in cellular tissues, such as crystalline inclusions within mitochondria and in the cytoplasm and myeloid bodies, suggesting that the accumulation of damaged / aggregated proteins ultimately leads to apoptosis, especially in muscle and adipocytes. These findings strongly suggest that interference with overall proteasome activity and capacity to respond to environmental stress is critical (Brehm and Kruger, 2015).

[0339] In certain embodiments, proteinopathic inflammatory diseases, disorders and / or conditions may include one or more of the following diseases: inflammatory pelvic disease, urethritis, sunburn, sinusitis, pneumonia, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis, dermatitis, gingivitis, appendicitis, pancreatitis, cholecystitis, irritable bowel syndrome, ulcerative colitis, glomerulonephritis, dermatomyositis, scleroderma, vasculitis; allergic disorders, including asthma such as bronchial asthma, allergies, Asthma caused by pathogens, such as endogenous asthma, intrinsic asthma, exogenous asthma and dust asthma, especially chronic or refractory asthma (e.g., late stage asthma with airway hyperresponsiveness) and bronchitis, chronic obstructive pulmonary disease, multiple sclerosis, rheumatoid arthritis; gastrointestinal disorders, including but not limited to celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, pancreatitis, Crohn's disease, ulcerative colitis, food-related allergies with effects away from the intestine, such as migraine, rhinitis and eczema. Conditions characterized by inflammation of the nasal mucosa, including acute rhinitis, allergic rhinitis, atrophic rhinitis and chronic rhinitis, including caseous rhinitis, hypertrophic rhinitis, purulent rhinitis, sicca rhinitis and rhinitis medicamentosa; membranous rhinitis, including croupous rhinitis, fibrinous rhinitis and pseudomembranous and adenotic rhinitis; seasonal rhinitis, including neurogenic rhinitis (hay fever) and vasomotor rhinitis, sarcoidosis, farmer's lung and related diseases, fibrotic lung and idiopathic interstitial pneumonia, acute pancreatitis, chronic pancreatitis and adult respiratory distress syndrome and / or acute inflammatory reactions (such as acute respiratory distress syndrome and ischemia / reperfusion injury).

[0340] Other examples of immune-mediated responses and diseases include rejection following transplantation of synthetic or organic transplant materials, cells, organs or tissues to replace all or part of the function of tissues such as the heart, kidney, liver, bone marrow, skin, cornea, blood vessels, lungs, pancreas, intestine, limbs, muscle, neural tissue, duodenum, small intestine, pancreatic islet cells, including xenografts, etc.; treatment of graft-versus-host disease, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, thyroiditis, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetic uveitis, juvenile-onset or recent-onset diabetes, glaucoma, etc. Uveitis, Graves' disease, psoriasis, atopic dermatitis, Crohn's disease, ulcerative colitis, vasculitis, autoantibody-mediated diseases, aplastic anemia, Evans syndrome, autoimmune hemolytic anemia, etc.; and further treatment of infectious diseases that cause abnormal immune responses and / or activation, such as traumatic or pathogen-induced immune disorders, including, for example, those caused by hepatitis B infection and hepatitis C infection, HIV, Staphylococcus aureus infection, viral encephalitis, sepsis, parasitic diseases (e.g., leprosy) in which damage is induced by inflammatory responses. Other immune-mediated responses and diseases involve graft-versus-host disease (especially graft-versus-host disease of allogeneic cells), rheumatoid arthritis, systemic lupus erythematosus, psoriasis, atopic dermatitis, Crohn's disease, ulcerative colitis, and / or multiple sclerosis.

[0341] Examples also include diseases caused or worsened by the host's own immune response. For example, autoimmune diseases and immune responses cause diseases that usually cause organ transplant rejection, these autoimmune diseases such as multiple sclerosis, lupus erythematosus, psoriasis, pulmonary fibrosis and rheumatoid arthritis, these immune responses cause diseases such as atherosclerosis, inflammatory diseases, osteomyelitis, ulcerative colitis, Crohn's disease and graft-versus-host disease (GVHD) that morbidity. Additional exemplary inflammatory disease states include fibromyalgia, osteoarthritis, sarcoidosis, systemic sclerosis, Sjogren's syndrome, skin inflammation (e.g., psoriasis), glomerulonephritis, proliferative retinopathy, restenosis and chronic inflammation.

[0342] The present invention increases proteasome activity by using at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof, thereby enhancing protein hydrolysis capacity. The at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof improves the onset and / or progression of, or even treats, autoimmune responses generated by protein aggregation caused by oxidatively damaged proteins and / or misfolded proteins.

[0343] The present invention provides methods and compositions directed to a variety of neurodegenerative proteinopathies associated with increased D-serine.

[0344] D-serine (DS), an endogenous D-amino acid, is produced by serine racemase (SR) in glial cells and neurons. SR converts free L-serine to DS. Free DS acts as a co-agonist of NMDA receptors in the brain, retina, keratinocytes, and in chondrocytes. Incorporation of D-serine significantly alters the functional properties of many proteins that are harmful to humans. Racemization of serine residues is known to occur in β-amyloid senile plaques of Alzheimer's disease (AD), human crystalline protein α-crystallin, human myelin basic protein, and mitochondria.

[0345] Protein, osteoarthritic joint and meniscus cartilage, funnel-web spider toxins, ovalbumin, and skin. In addition to serine isomerization, SR also undergoes α-elimination and β-elimination reactions with L-serine and D-serine to produce pyruvate and ammonia (Rani et al., 2020).

[0346] One of the main characteristics of the NMDA receptor is that its activation requires the simultaneous binding of its agonist glutamate and its co-agonist glycine. DS binds to the NMDA receptor at the glycine site with three times the affinity of glycine, and DS is 10 times more potent at activating the NMDA receptor than glycine. Overactivation of the NMDA receptor occurs when there is an excess of DS.

[0347] Overactivity of NMDA receptors has been associated with many neurological disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, epilepsy, neuropathic pain, and cerebral ischemia.

[0348] Therefore, inhibition of the SR is another way to modulate NMDA receptor transmission and is the only way to prevent serine isomerization in proteins in certain pathological conditions, including AD. SR inhibition has been shown to be neuroprotective in many cases.

[0349] Thus, in another aspect, the present invention demonstrates that increased proteasome activity in the presence of NMDAR blockers reduces serine racemase ( Figure 4B ), thereby reducing the conversion of L-serine to -D-serine, which will effectively prevent or treat cerebral ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, hyperactivity syndrome, long-term sequelae of concussion, epileptic seizures, retinal ganglion cell loss in diabetic patients, chronic social frustration stress and other neurodegenerative diseases.

[0350] And importantly, in another aspect, the present invention provides compositions and methods for neuroprotection in neurological diseases.

[0351] As used herein, the term "neuroprotective activity" refers to the effect of reducing or ameliorating nerve damage, and protecting or restoring neuronal cells that have suffered nerve damage. As used herein, the term "neural damage" refers to any damage to neuronal cells or tissues caused by various causes such as metabolic causes, toxic causes, neurotoxic causes, and chemical causes. It also refers to aging, which is the process of accumulation of all these causes (Darric et al., 2019).

[0352] Recent evidence suggests that astrocyte d-serine promotes neuronal degeneration in Alzheimer's disease. The present invention provides compositions and methods for reducing the toxic effects of astrocytes by reducing serine racemase. Thus, the present invention further reduces microvascular damage in diabetic retinopathy, synaptic damage after traumatic brain injury, neuronal overactivation, choroidal neovascularization, pathological symptoms of Alzheimer's disease, and neurodegeneration (Balu et al., 2019).

[0353] On the other hand, the present invention demonstrates that the increase in proteasome activity in the presence of NMDAR blockers reduces serine racemase, thereby reducing the conversion of L-serine to -D-serine, which will be effective in preventing or treating schizophrenia and other mental illnesses (Kishi and Iwata, 2013).

[0354] According to an aspect of some embodiments of the present invention, it is demonstrated that NMDA receptor antagonists reduce phosphorylated tau (pTau) ( Figure 4A ). Tau is one of the main components of the microtubule (MTN) network in neurons, and its abnormal phosphorylation and aggregation are closely related to the impairment of axonal transport (Weng and He, 2021).

[0355] MTN is crucial for neurons because neuronal signaling and protein turnover between the cell body and axon are highly dependent on axonal transport along MTN. The function of MTN can be regulated by tau protein. By binding to microtubules, tau regulates the stability and dynamics of microtubules. Abnormal phosphorylation of tau interrupts its binding to microtubules and leads to the destruction of MTN. In this sense, the accumulation of p-tau, a prominent feature of AD, may hinder the normal function of MTN and lead to a series of catastrophic cascades (Weng and He, 2021).

[0356] Tauopathies are neurodegenerative disorders characterized by the presence of filamentous deposits composed of hyperphosphorylated tau protein in neurons and glia. Abnormal tau phosphorylation and deposition in neurons and glial cells is one of the main features of tauopathies.

[0357] The term is now used to identify a class of diseases with extensive tau pathology, in which tau accumulation seems to be directly related to pathogenesis.Main neurodegenerative tau pathology includes sporadic diseases and hereditary diseases characterized by filamentous tau deposition in the brain and spinal cord.In most tauopathies, glial and neuronal tau inclusions are unique or main CNS lesions.This type of exemplary tauopathy includes amyotrophic lateral sclerosis (ALS), Parkinson's syndrome, argyrophilic grain dementia, diffuse neurofibrillary tangles with calcification, frontotemporal dementia related to chromosome 17, corticobasal degeneration, Pick's disease, progressive supranuclear palsy, progressive subcortical gliosis and tangled dementia.

[0358] Additionally, tauopathy characterizes a large class of diseases, disorders, and conditions in which significant Tau protein fibrils and aggregates are found. Such exemplary diseases, disorders, and conditions include sporadic and / or familial Alzheimer's disease, amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-FTDP), argyrophilic grain dementia, dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, and schizophrenia. disease), inclusion body myositis, Creutzfeldt-Jakob encephalopathy (CJD), multiple system atrophy, Niemann-Pick disease (NPC), Pick's disease, prion protein cerebral amyloid angiopathy, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-predominant Alzheimer's disease, corticobasal degeneration (CBD), myotonic dystrophy, non-guanamian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle dementia.

[0359] When tauopathies are found in conjunction with other abnormal protein foci, neurodegenerative diseases can be considered secondary tauopathies. Examples include AD and certain diseases in which prion proteins, Bri, or alpha-synuclein aggregate. Although tau may not be the initial pathological factor, tau aggregates lead to the final degeneration.

[0360] Alzheimer's disease (AD) is a neurodegenerative disease that affects many people worldwide. It is the most common cause of dementia (Alzheimer's Association, 2020), and the number of its victims continues to grow as the population ages. It is now the sixth leading cause of death in the United States (Alzheimer's Association, 2020). Although the pathogenesis and treatment of AD are being extensively studied, great progress has not been made in this field. Intracellular aggregates composed mainly of phosphorylated tau (p-tau) are one of the hallmarks of AD (Alzheimer's Association, 2020; Weng and He, 2021). Pathological proteinopathies are also considered to be part of the protein lesions detected in neurodegeneration with brain iron accumulation type I, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, multiple system atrophy, and familial AD.

[0361] The proteasome system maintains intracellular protein homeostasis by degrading abnormal or excess proteins. There is increasing evidence that proteasome activity defects lead to p-tau accumulation. Moreover, increasing proteasome activity can alleviate tau pathology (Boselli, 2017). In fact, the present invention proves that the increase of proteasome activity in the presence of NMDAR blockers can reduce the amount of pTau.

[0362] Therefore, on the one hand, the present invention provides compositions and methods for preventing and / or treating the above-mentioned diseases caused by tau protein disease. pTau transgenic mice show interrupted mitochondrial dynamics and impaired mitophagy. These studies reveal that the aggregation of APP and its cleavage products (AD and pTau) leads to autophagy and mitophagy defects (Bussian et al., 2018). Therefore, as demonstrated in the present invention, in the presence of NMDAR blockers, the establishment of protein homeostasis should be ensured before the occurrence of lesions such as autophagy and mitophagy defects caused by the appearance of pTau and other cleavage products.

[0363] Cognitive impairment and dementia are generally defined as a progressive decline in cognitive function due to physical injury or disease beyond what is expected from normal aging. Dementia is described as a loss of intellectual function, involving problems with memory, reasoning, attention, language, and problem solving. The higher-level functions are usually affected first. Dementia interferes with a person's ability to function normally in daily life.

[0364] Cognitive impairment or dementia may be caused by any etiology. Specific diseases associated with cognitive impairment or dementia include, but are not limited to, neurodegenerative diseases, neurological diseases, psychiatric disorders, genetic diseases, infectious diseases, metabolic diseases, cardiovascular diseases, vascular diseases, aging, trauma, malnutrition, childhood diseases, chemotherapy, autoimmune diseases, eye diseases, and inflammatory diseases.

[0365] The present invention provides methods for preventing, reducing or even treating cognitive impairment and dementia in normal aging as well as cognitive impairment and dementia caused by any etiology.

[0366] In another aspect, the present invention also provides the discovery that NMDAR blockers induce actin turnover by increasing proteasome activity. Fig.35 ). It has been previously described that actin turnover promotes axon regeneration in the adult CNS (Tedeschi and Dupraz, 2019). In another aspect, the present invention can increase axon regeneration. Thus, the present invention provides compositions and methods that prevent and / or treat neurodegeneration caused by pathological insults including anticancer therapy, cerebral ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, retinal ganglion cell loss, long-term sequelae of concussion, and reduce the neurotoxic effects of astrocytes in Alzheimer's disease.

[0367] In one aspect, the present invention provides compositions and methods for preventing and / or treating renal diseases caused by accumulation of oxidatively damaged proteins and / or misfolded proteins.

[0368] On the other hand, the present invention provides compositions and methods for preventing and / or treating renal insufficiency caused by aging, hyperlipidemia, hypertension, smoking, diabetes, obesity and other factors that lead to protein oxidative damage (Cauli et al., 2014). Therefore, the present invention improves and / or treats acute kidney disease and chronic kidney disease.

[0369] The unfolded protein response (UPR) is an adaptive cellular program used by eukaryotic cells to cope with protein misfolding stress (Madden et al., 2019; Hsu et al., 2019). The UPR is a pro-survival mechanism that is triggered by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), a condition known as ER stress. Over the past decade, evidence has emerged to support a role for the UPR in the establishment and progression of several cancers. In addition, several publications have linked mechanisms of cancer chemotherapy resistance to the activation of the UPR (Madden et al., 2019; Hsu et al., 2019).

[0370] The present invention provides methods for preventing, reducing or even treating cell proliferative diseases by reducing or preventing the accumulation of unfolded proteins and the subsequent UPR.

[0371] Typically, cell proliferative disorders, diseases and / or conditions encompass various conditions characterized by aberrant cell growth, preferably abnormally increased cell proliferation. For example, proteinopathy cell proliferative diseases, disorders and / or conditions include but are not limited to cancer, immune-mediated responses and diseases (e.g., transplant rejection, graft-versus-host disease, immune responses to gene therapy, autoimmune diseases, pathogen-induced immune disorders, etc.), some circulatory system diseases and some neurodegenerative diseases.

[0372] According to the present invention, the term "cell proliferative disorder" also refers to a cancer disease. The term "cancer disease" or "cancer" (medical term: malignant tumor) refers to a class of diseases in which a group of cells exhibit uncontrolled growth (division beyond normal limits), invasion (invasion and destruction of adjacent tissues) and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant characteristics of cancer distinguish them from benign tumors, which are self-limited and do not invade or metastasize. Most cancers form tumors, i.e., swellings or lesions formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some cancers, such as leukemia, do not form tumors. Examples of cancer include, but are not limited to, carcinoma, leukemia, and lymphoma, such as cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, lymphoma associated with human T-cell virus (HTLV), such as adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma, acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, mesothelioma, common solid tumors in adults, such as head and neck cancer ( For example, oral cancer, laryngeal cancer and esophageal cancer), genitourinary cancer (for example, prostate cancer, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, testicular cancer, rectal cancer and colon cancer), lung cancer, breast cancer, liver cancer, colon cancer, small intestine cancer, pancreatic cancer, melanoma and other skin cancers, gastric cancer, tumors of the central nervous system (CNS), neuroectodermal cancer, spinal tumors, gliomas, meningiomas, pituitary adenomas, hepatocarcinoma and thyroid cancer and / or solid tumors of childhood, such as brain tumors, neuroblastomas, retinoblastomas, Wilms' tumors, bone tumors and soft tissue sarcomas. The term "cancer" according to the present invention also includes cancer metastasis.

[0373] In another aspect, the present invention provides a method for preventing chemoresistance mechanisms caused by the UPR.

[0374] On the other hand, the present invention also provides a method for treating different forms of cancer in which the protein homeostasis network is disturbed, including increased protein synthesis, misfolded protein production, misfolded protein aggregates, and accumulation of apoptosis signaling proteins in cancer cells. This can alter the sensitivity of cancer cells to anti-tumor drugs (Madden et al., 2019; Hsu et al., 2019).

[0375] Serine racemase (SR) converts free L-serine into D-serine (DS) in cells. DS acts as a co-agonist of NMDAR. Overactivation of NMDA receptors leads to many neurological disorders such as stroke, amyotrophic lateral sclerosis and AD. NMDAR inhibitors (ketamine) degrade serine racemase (SR) by activating the proteasome ( Figure 4A , Figure 4B ).

[0376] Therefore, in another aspect, the present invention provides a method for treating colorectal cancer by reducing serine racemase, which promotes the growth of colorectal cancer by producing pyruvate from serine (Ohshima et al., 2020).

[0377] Previous studies have shown that inhibition of proteasome activity inhibits telomerase activity (Shalem-Cohavi et al., 2019). Shortening of telomere length is one of the metabolic disorders that impairs the normal aging process, such as loss of muscle mass and muscle strength.

[0378] Therefore, on the other hand, the present invention provides NMDA receptor antagonists to increase telomerase activity by increasing the activity of the proteasome. Therefore, the present invention provides a method for improving / correcting metabolic disorders, and the method impairs the normal aging process. The present invention also provides NMDA receptor antagonists to delay cell aging by increasing telomerase activity, which prolongs life (Shalem-Cohavi et al., 2019).

[0379] NMDR blockers as drugs to eliminate senescent cells

[0380] Mammalian cells have a special ability to adapt to extracellular and intracellular environmental disturbances. Disturbance in the cell microenvironment promotes the activation of metabolic changes and molecular changes to ensure cell survival. Despite these adaptive mechanisms, chronic or severe, irreversible damage will terminate damaged cells to protect the life of the organism. The termination of damaged cells refers to the end of the normal physiological state of the cell. This can occur by cell division inactivation (a state known as aging) or by eliminating damaged cells (a process known as regulated cell death). Even if the mechanism is not fully understood, the cell cycle regulator p53 plays a key role in controlling the activation of pro-apoptotic regulatory factors in response to severe damage or regulating p21Cipl transcription in response to milder but still harmful damage to induce aging. Therefore, when the damage is severe enough but not fatal, the cell is converted into a permanent non-proliferative state. The intracellular and extracellular signals that can cause cells to enter the fate of senescent cells mainly include signals related to tissue or cell damage and / or cancer development. These include protein aggregates, misfolded proteins, reduced proteasome activity and failure to remove proteins, the presence of AGEs (e.g., hemoglobin A1c is an AGE) due to the reaction of reducing sugars with amino groups in proteins, saturated lipids and other bioactive lipids (bradykinin, certain prostaglandins, etc.), reactive metabolites (e.g., ROS, hypoxia or hyperoxia), DNA damage, telomere uncapping or dysfunction, exposure to extracellular DNA, oncogene activation, replicative stress or proliferation inducers (such as growth hormone / IGF-1), inflammatory cytokines (e.g., TNFa). Therefore, cellular senescence is a complex stress response that causes essentially irreversible arrest of cell proliferation and the development of a multicomponent senescence-associated secretory phenotype (SASP).

[0381] The SASP consists of a large array of cytokines, chemokines, growth factors, and proteases that trigger inflammatory, wound healing, and growth responses in nearby cells. In young healthy tissues, the SASP is typically transient and tends to contribute to the maintenance or restoration of tissue homeostasis. However, senescent cells increase with age, and the chronic SASP is known or suspected to be a key driver of many pathological hallmarks of aging, including chronic inflammation, tumorigenesis, and impaired stem cell renewal (Saez-Atienzar and Masliah, 2020). These inducers activate one or more pro-senescence transcription factor cascades, in some cases involving p16INK4a-retinoblastoma protein (Rb), and in other cases involving p53 and p21CIP1, two of these pathways, or other pathways.

[0382] Data from several laboratories strongly support the view that senescent cells and the SASP drive multiple age-related phenotypes and pathologies, including atherosclerosis, osteoarthritis, cancer metastasis, cardiac dysfunction, bone marrow skeletal abnormalities, renal dysfunction, and an overall reduction in healthspan.

[0383] The SASP is one of the three main characteristics of senescent cells, the other two being cell growth arrest and resistance to apoptosis. Senescent cells are highly metabolically active and produce large amounts of the SASP, which is why senescent cells, which consist of only 2% or 3% of tissue cells, may be the main cause of aging-related diseases. SASP factors cause non-senescent cells to become senescent. SASP factors induce insulin resistance. The SASP disrupts normal tissue function by producing chronic inflammation, inducing fibrosis, and inhibiting stem cells. Chronic inflammation associated with aging has been referred to as inflammatory aging, although the SASP may be only one of the possible causes of this condition. Chronic inflammation caused by the SASP can suppress immune system function, which is one reason why the elderly are more susceptible to COVID-19. Due to the accumulation of unfolded proteins, the SASP induces the UPR in cells, resulting in proteotoxic impairment of cellular function (Saez-Atienzar and Masliah, 2020).

[0384] Ablation of senescent cells (therapy to eliminate senescent cells) has been postulated as a promising therapeutic approach to target the aging phenotype and thereby prevent, delay or alleviate aging-related diseases and many other diseases. Compounds that eliminate senescent cells aim to rejuvenate the organism by selectively killing senescent cells, and the efficacy of these compounds is based on the ability of senescent cells to resist apoptosis. These cells exhibit upregulation of prosurvival pathways to protect themselves from the damaging (and pro-apoptotic) effects of the SASP. To date, six prosurvival pathways have been detected in senescent cells: the BCL-2-BCL-xL pathway, the MDM2-p53-p21Cipl-serine element pathway, the ephrins-dependent receptor-tyrosine kinase, the PI3K—AKT-ceramide metabolism pathway, the hypoxia-inducible factor 1a (HIF-1a) pathway, and the SP-90-dependent pathway. Compounds that eliminate senescent cells interfere with these prosurvival pathways to allow senescent cells to die by apoptosis (Saez-Atienzar and Masliah, 2020).

[0385] Clearing established senescent cells, many of which carry oncogenic mutations or cause cancer-promoting inflammation due to their SASP, prevents or slows cancer development. Indeed, drugs that clear senescent cells, that is, agents that selectively eliminate senescent cells, slow the development of multiple forms of cancer in old mice as well as in cancer-prone Erccl- / D mice with DNA repair defects.

[0386] Furthermore, although the ability of cells to become senescent is a cancer defense mechanism, established, long-lived senescent cells can accelerate cancer growth due to suppression of immune responses, growth factors in the SASP, production of proteases that interfere with tumor encapsulation and thus enhance cancer spread, and promotion of cell-mesenchymal transition. Thus, targeting the transcription factor cascade that allows cells to become senescent can exacerbate cancer development, while clearing established senescent cells with senescent cell-clearing drugs can prevent cancer development, growth, and spread (Saez-Atienzar and Masliah, 2020; Thoppil and Riabool, 2020).

[0387] Therefore, p53, p21, pRb, BCL-2, AKT, SP-90, and other proteins involved in these pathways are targets for compounds that clear senescent cells (Saez-Atienzar and Masliah, 2020; Thoppil and Riabowol, 2020).

[0388] The present invention demonstrates that NMDA receptor antagonists increase proteasome activity and cause a decrease in p21, p53, pAkt1, and ppRb, which are the main target molecules for drugs that eliminate senescent cells (Figure 1, Figure 2A and Fig.34 ).

[0389] According to some embodiments, the present invention provides a pharmaceutical composition for ablation of senescent cells, which ablation of senescent cells has been postulated to be a promising treatment method for targeting the aging phenotype, thereby preventing, delaying or alleviating aging-related diseases, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0390] Thus, according to an aspect of some embodiments of the present invention, there is provided an NMDA receptor antagonist for use in the treatment of aging, which is caused by cellular senescence and is associated with an increased risk of developing various chronic diseases, geriatric syndromes, impaired physical resilience and mortality.

[0391] Diseases for which there is emerging evidence of a causal contribution of cellular senescence or a benefit of senescent cell-clearing drugs include, but are not limited to, diabetes / obesity, metabolic diseases, cardiac dysfunction, congestive heart failure, myocardial infarction, vascular hyporesponsiveness / calcification, AV fistulas, frailty, age-related muscle loss (sarcopenia), arthritis, osteoporosis, falls, chemotherapy complications, radiation complications, cancer, bone marrow transplant complications, organ transplant complications, myeloma / MGUS (monoclonal gammopathy of undetermined significance), age-related cognitive impairment, other dementias, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia, obesity-related

[0392] Neuropsychiatric disorders, renal dysfunction, urinary incontinence, osteoporosis, osteoarthritis, age-related disc disease, idiopathic pulmonary fibrosis, hyperoxic lung injury, chronic obstructive pulmonary disease, tobacco, hepatic steatosis, cirrhosis, primary biliary cirrhosis, progeria, preeclampsia, macular degeneration, glaucoma, cataracts, blindness, prostatic hypertrophy, incontinence, psoriasis, health span, life span and many other diseases. (Kirkland and Tchkonia, 2020)

[0393] The 20S proteasome plays a key role in the selective recognition and degradation of oxidized proteins, and is responsible for degrading approximately 90% of all intracellular oxidatively damaged proteins. It is noteworthy that the degradation of the 20S proteasome occurs in an Ub-independent manner. Studies have shown that under oxidative conditions or in response to mitochondrial dysfunction, cells can reversibly decompose the 26S proteasome to increase the level of free 20S particles (Ishiie et al., 2005).

[0394] The accumulation of unfolded, misfolded, or damaged proteins severely impairs the function of the organelle. Several reports have shown that one of the first detectable consequences of proteasome dysfunction is associated with mitochondria, which develop deleterious alterations in their proteome. Mitochondria are cellular powerhouses that produce energy through oxidative phosphorylation. These mitochondria also directly contact most, if not all, other cytosolic organelles (e.g., ER, plasma membrane, and peroxisomes) to generate specialized networks that control several cellular functions such as Ca2+ homeostasis, lipid synthesis, and apoptosis. Importantly, dysfunctional mitochondria are also a major source of oxidative stress through the aberrant production of ROS, which has profound implications for the pathogenesis of various diseases, especially neurodegenerative disorders and cancer, as well as aging (Pan et al., 2021).

[0395] Mitochondria are involved in a range of other processes, such as signal transduction, cell differentiation, cell death, and control of the cell cycle and cell growth. Given the central role of mitochondria in cellular metabolism, mitochondrial damage and subsequent dysfunction are important factors in a variety of human diseases and may play a role in the aging process.

[0396] The proteasome pathway maintains mitochondrial plasticity and quality by controlling mitochondrial dynamics at several levels. These activities and processes are tightly linked. During moderate stress, the UPS is responsible for

[0397] The outer membrane PQC and the degradation of damaged proteins, a process known as outer mitochondrial membrane associated degradation (OMMAD). In addition, the UPS directly controls mitochondrial dynamics by degrading proteins involved in mitochondrial fusion and fission processes. Although the 20S proteasome has largely been considered a dormant protease that is only activated when it binds to regulatory complexes, increasing evidence has clearly demonstrated that the 20S core has a major role in global protein degradation, independent of ubiquitin and ATP (Raynes et al., 2016).

[0398] Therefore, the present invention can directly or indirectly provide effective treatment, symptom relief, or even prevention for certain mitochondrial diseases, including mitochondrial myopathy, diabetes and deafness, Leber hereditary optic neuropathy, Leigh syndrome, subacute sclerosing encephalopathy, neuropathy, ataxia, retinitis pigmentosa, ptosis, progressive symptoms of dementia, myoneurogenic gastrointestinal encephalopathy, MERRF syndrome (myoclonic epilepsy with ragged red fibers), MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes), mitochondrial DNA depletion syndrome, Huntington's disease, cancer, Alzheimer's disease, Parkinson's disease, bipolar disorder, schizophrenia, aging and senescence, anxiety disorders, Alper syndrome, Lowe syndrome, Luft syndrome, Menke curly hair syndrome, Zellweger syndrome, mitochondrial myopathy and proximal limb chondrodysplasia punctata.

[0399] Therefore, in another aspect, the present invention also provides a method for correcting and / or treating the above-mentioned impaired autophagy and impaired mitochondrial autophagy and related diseases.

[0400] The endoplasmic reticulum is an organelle that performs multiple functions such as lipid synthesis and protein synthesis, protein folding, and calcium homeostasis. In this organelle, the increase and accumulation of unfolded or misfolded proteins can disrupt the function of the organelle and cause endoplasmic reticulum stress. The complex signal transduction pathways triggered by ER stress constitute the UPR. Endoplasmic reticulum stress is seen in neurodegenerative diseases, metabolic diseases, atherosclerosis, diabetes, and obesity. Endoplasmic reticulum stress is also associated with cancer (Ding et al., 2007; Roussel, 2013).

[0401] In one aspect, the present invention provides a pharmaceutical composition for treating endoplasmic reticulum (ER) related diseases directly or indirectly by reducing ER stress, the pharmaceutical composition comprising at least one NMDA receptor antagonist or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof. Metabolic diseases include insulin resistance, atherosclerosis, diabetes, obesity, alcoholic and non-alcoholic fatty liver disease, hyperlipidemia; cancers include leukemia, multiple myeloma, breast cancer, prostate tumors; immune system related diseases include viral infection, bacterial infection, vitiligo, rheumatoid arthritis and type 1 diabetes. Many neurological diseases are known to be associated with ER stress, including cerebral ischemia, sleep apnea, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, prion disease, Parkinson's disease and Huntington's disease and neuroserine proteinase inhibitor inclusion body familial encephalopathy.

[0402] The autophagy-lysosomal pathway is another degradation pathway in which cytoplasmic components, such as protein aggregates and damaged organelles, are degraded and recycled to maintain normal cellular homeostasis. In this pathway, cytoplasmic material is delivered to lysosomes, sequestered into autophagosomes, which then fuse with lysosomes, where the cargo is degraded.

[0403] Depending on the context, autophagy and the proteasome share common substrates as well as regulatory factors. The two systems intersect and communicate at multiple points to coordinate and balance their roles in protein homeostasis and organelles (Liu et al., 2020; Ding et al., 2007; Feldman et al., 2019).

[0404] Probably the most important issue that occurs due to reduced proteasome activity is the accumulation of potentially toxic unfolded / misfolded proteins and protein aggregates. Impaired proteasomes will ultimately affect autophagy and cytosolic processes as well as the important functions of all other organelles due to the accumulation of unfolded and damaged proteins (Dikic, 2017; Pohl and Dikic, 2019, Kocaturk and Gozuacik, 2018).

[0405] The present invention encompasses the discovery that NMDA inhibitors, by increasing levels and / or increasing activity, can provide effective treatments, and even preventions, for certain autophagy-related human diseases, such as adult neurodegenerative disorders, including Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, neuronal ceroid lipofuscinosis, fulminant neurodegeneration, dementia with Lewy bodies; pediatric neurodevelopmental disorders, including spinocerebellar ataxia, cortical atrophy and epilepsy, childhood-onset neurodegeneration, BPAN, spastic quadriplegia and brain abnormalities, primary microcephaly, hereditary spastic paraplegia, ataxia with spasticity, Rett syndrome, Joubert syndrome, leukoencephalopathy, juvenile-onset dystonia, CEDNIK syndrome, Pelvic floor disease, West syndrome; hereditary neuropathies, including sensory and autonomic neuropathy type II, peroneal muscle atrophy, IF sensory and autonomic neuropathy, distal hereditary motor neuropathy; ophthalmological diseases, including primary open-angle glaucoma, cataracts; cardiac and skeletal myopathy, including Danon cardiomyopathy, distal myopathy with rimmed vacuoles, dilated cardiomyopathy, sporadic inclusion body myositis, X-linked myopathy with excessive autophagy; inflammatory conditions, including Crohn's disease, ulcerative colitis, childhood asthma; autoimmune diseases, including systemic lupus erythematosus, diabetes, other autoimmune diseases; infectious diseases, including Mycobacterium tuberculosis, Mycobacterium leprae; bone disorders, including osteopetrosis, Paget's disease of bone, Caschin-Baker disease; congenital multisystem disorders, including global dysplasia, Vici syndrome, Zellweger syndrome, glycosylation disorders with autophagy deficiency, Zimmerman-Laband syndrome, Hermansky-Pudlak syndrome, multisystem proteinopathy.

[0406] The present invention encompasses the discovery that NMDA inhibitors activate the proteasome

[0407] The malfunction of the protein homeostasis network (including the accumulation of oxidatively damaged proteins and / or misfolded proteins associated with lysosomal dysfunction) is prevented. The present invention encompasses the discovery that NMDA inhibitors can provide effective treatment or even prevention for lysosomal storage diseases by increasing levels and / or increasing activity on the activation of the proteasome. Lysosomal storage diseases include Gaucher disease, Fabry disease, Niemann-Pick disease, Hunter syndrome, glycogen storage disease II (Pompe disease), Tay-Sachs disease.

[0408] Importantly, in any case, attempts to pharmacologically improve proteostasis must be made at an early stage of the disease, before severe manifestations of cellular dysfunction occur. Indeed, the present invention has demonstrated that early initiation of treatment is more effective in extending life expectancy than late initiation of treatment (Salomon et al., 2012; Halminen et al., 2021).

[0409] Glutamate receptors are key components of the glutamate pathway, functioning as both signal detectors and transmitters. Glutamate receptors are mainly subdivided into two groups: ionotropic glutamate receptors (iGluRs) that constitute ion channels and metabotropic glutamate receptors (mGluRs) that are members of the G protein-coupled receptor (GPCR) superfamily.

[0410] The NMDA receptor belongs to the iGluR family, which includes the kainate receptor, (2-amino-3(3-hydroxy-5-methylisoxazol-4-yl)propanoic acid)AMPA, and (N-methyl-D-aspartate receptor) NMDA receptor.

[0411] Unlike other iGluRs, NMDA receptors are well known for their high Ca2+ ion conductance, voltage-dependent channel blockade, and mandatory binding of two endogenous ligands for receptor activation. Although glutamate synaptic concentrations are tightly regulated, excessive glutamatergic signaling is a key feature of neurodegeneration, leading to a dramatic increase in intracellular Ca2+ ion levels and subsequent neuronal apoptosis.

[0412] Structurally, the NMDA receptor is a heterotetrameric ion channel composed of four residues derived from three related subunits: GluN1 subunit, GluN2 subunit and GluN3 subunit. Activation of the NMDA receptor depends on the combination of two agonists, glutamate and glycine or D-serine. Typically, the NMDA receptor consists of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Specifically, there are eight isoforms of GluN1 (a to h, different splicing variants of a single gene), and four GluN2 (A to D, encoded by four different genes) and two GluN3 (A to B, encoded by two different genes) subunits have been identified. There are multiple ligand binding sites on the NMDA receptor, including the glutamate binding site, the glycine binding site, the ion channel pore and the allosteric binding site on the amino terminal domain (ATD), which regulate receptor activity in a subtype-selective manner. More and more studies have shown that different subtypes of the NMDA receptor produce different functional outputs. Most human-specific GluN splice variants occur in the amino-terminal domain (ATD) and CTD. The corresponding sequences can be easily obtained from existing databases, such as www.ebi.ac.uk EMBL-EBI database or www.i EMIL BIM,Elih.gov The NCBI database under (Cull-Candy et al., 2001; Ishchenko et al., 2021; Paoletti et al., 2013).

[0413] The term "NMDA receptor antagonist or blocker or inhibitor" as used herein relates to a substance capable of negatively regulating the NMDA receptor / NMDA complex. The "negative regulation" includes inhibition and / or blocking. The inhibition and / or blocking may be partial or complete. The antagonists used in the context of the present invention most preferably inhibit / block human NMDA receptors.

[0414] In certain embodiments, an NMDA receptor antagonist relates to a compound that is capable of fully or partially blocking the action and / or function of an NMDA receptor or an NMDA receptor complex in vivo and / or in vitro.

[0415] In certain embodiments, the NMDA receptor antagonist preferentially binds to an extrasynaptic NMDA receptor. In certain embodiments, the extrasynaptic NMDA receptor comprises a NR2B subunit.

[0416] So far, the following three major classes of NMDAR antagonists can be distinguished based on their mechanism of action: competitive antagonists, uncompetitive channel blockers, and noncompetitive allosteric inhibitors. Although ultimately leading to similar results, each antagonist class has a different mechanism of action. Competitive NMDAR antagonists act by occupying the glycine binding site or the glutamate binding site located on the GluN1 subunit or the GluN2 subunit, respectively. This type of antagonist prevents endogenous ligand binding, thereby preventing activation of the receptor. Competitive antagonists and uncompetitive antagonists tend to have poor selectivity for specific GluN1 / GluN2 subunits, while uncompetitive allosteric inhibitors show much higher selectivity. Uncompetitive channel blockers act by blocking the NMDAR ion channel pore.

[0417] This class of uncompetitive ion channel blockers are ketamine, dizocilpine (MK-801), and memantine (Petit-Pedrol et al., 2021; Das 2020; Siala et al., 2020).

[0418] Uncompetitive NMDAR channel blockers are typically positively charged and require depolarization to remove the Mg2+ blockade and expose the binding site. Once bound, these channel blockers can prevent ions from flowing into / out of the channel, resulting in inhibition. Of note, glutamate may still bind to the NMDAR when the channel blocker is in the pore, potentially leading to Ca2+-independent intracellular signaling; although the significance of this phenomenon is not fully understood. Finally, noncompetitive allosteric modulators inhibit the activity of the NMDAR by acting on the N-terminal or "agonist binding" domain of the GluN1 / GluN2 subunit, thereby reducing the activity of the receptor by reducing the binding of endogenous ligands. Noncompetitive NMDAR allosteric inhibitors act on the extracellular domain (e.g., Zn2+, NO), and NMDAR channel blockers block open channels after activation by an agonist.

[0419] All competitive antagonists poorly discriminate between different NMDAR subtypes NR1 / NR2 (A to D), thus leading to general inhibition of NMDARs ( Liu et al., 2020 ; Vieira et al., 2020 ; Groc et al., 2009 ).

[0420] NMDA receptors (NMDARs) are essential for normal physiological processes in the central nervous system, such as development, induction of synaptic plasticity, learning and memory. It has been shown that specific glutamate receptors and glutamate transporters are also expressed in non-neural tissues such as lungs, liver, kidneys, stomach and the immune system, which shows that NMDAR plays an important role in the regulation of physiological functions in various peripheral organs (Du et al., 2016).

[0421] According to some embodiments, the NMDA antagonist is a specific inhibitor of glutamatergic neurotransmission. According to some embodiments, the NMDA antagonist can be a less specific antagonist acting on other neurotransmission pathways. According to some embodiments, the NMDA antagonist can have additional activity, such as serotonergic, cholinergic or dopaminergic antagonist or agonist activity.

[0422] The channel blocker memantine is approved for the treatment of moderate to severe Alzheimer's disease. Memantine's unusual clinical tolerability may well reflect its low affinity for binding to open channels and its relatively rapid unblocking kinetics (Petit-Pedrol et al., 2021; Das 2020; Siala et al., 2020).

[0423] Blockers such as ketamine or (-)MK-801 that remain trapped in the pore during agonist unbinding show a stronger dependence on extracellular pH than other blockers such as (+)MK-801, memantine, or dextromethorphan. Acidic extracellular pH increases the association rate of (-)MK-801 with the intrapore binding site of NMDARs, which appears to be the underlying mechanism for the pH-dependent potency enhancement.

[0424] Another striking observation is that a structurally distinct group of compounds differ in their channel-blocking potency against NMDARs with different NR2 subunits, even at physiological pH. (-)MK-801 and (+)ketamine are more than 10-fold more potent against NR1 / NR2B than NR1 / NR2A receptors, which may be the basis for the development of new truly subunit-selective NMDAR channel blockers. Clinically promising subunit-selective NMDAR channel blockers should exhibit rapid channel unblocking kinetics similar to memantine in addition to pH dependence to prevent the drug from occupying the channel and interfering with normal synaptic transmission. Thus, memantine is very different from (+)MK-801, which binds with higher affinity and has relatively slow unblocking kinetics. Due to these properties, (+)MK-801 has been used as a pharmacological tool to irreversibly block NMDARs for the past 20 years but failed in clinical testing (Petit-Pedrol et al., 2021; Das 2020; Siala et al., 2020).

[0425] Non-limiting examples of NMDA receptor antagonists include memantine (3,5-dimethyladamantan-1-amine), nitromemantine (3-amino-5,7-diethyladamantan-1-yl nitrate), fluoroethylnormemantine (FENM or FNM), ketamine (2-(2-chlorophenyl)-2-(methylamino)-cyclohexanone)), esketamine ((2S)-2-(2-chlorophenyl)-2-(methylamino)-cyclohexanone)), (+)-norketamine ((+)-2-amino-2-(2-chlorophenyl)cyclohexanone hydrochloride), (S)-ketamine, (R,S)- -ketamine, dextromethorphan (DXM), amitriptyline (N,N-dimethyl-3-(2-tricyclo[9.4.0.03,8]pentadecan-1(15),3,5,7,11,13-hexenylidene)propan-1-amine), ifenprodil (a-(4-hydroxyphenyl)-3-methyl-4-benzyl-1-piperidinol (+)-tartrate), orphenadrine β-dimethylaminoethyl-2-methyldiphenyl ether citrate), TCN-201 (3-chloro-4-fluoro-N-[(4-{[2(phenylcarbonyl)hydrazino]carbonyl}phenyl)methyl]benzenesulfonamide), SDZ 220-581 ((S)-a-amino-2-chloro-5-(phosphonomethyl)[1,1?biphenyl]-3-propionic acid hydrochloride), 5,7-dichlorokynurenic acid (5,7-dichloro-4-hydroxyquinoline-2-carboxylic acid monohydrate), MD-Ada (1-N-methylamino-3,5-dimethyladamantane hydrochloride), AP-7 (DL-2-amino-7-phosphonoheptanoic acid), Ro8-4304 (4-{3-[4-(4-fluorophenyl)-3,6-dihydro-2H-pyridin-1-yl)-2-hydroxy-propoxy)-benzamide), L-687,384 (1'-benzylspiro[2,3-dihydro-1H-naphthalene-4,4'-piperidine]), spermine (N,N'-bis(3-aminopropyl)-1,4-diaminobutane), DCOX (6,7-dichloro-2 ,3-quinoxalinedione), troxolidine ((18,2S)-1-(4-hydroxy-phenyl)-2-(4-hydroxy-4-phenylpiperidinyl)-1-propanol), farapane ([[3,4-dihydro-7-(4-morpholinyl)-2,3-dioxo-6(trifluoromethyl)-1(2H)-quinoxaline]methyl]-phosphonic acid hydrate), metafibrate (1-[1-(3-isothiocyanato) phenyl] cyclohexylpiperidinium methanesulfonate), NAAG (N-acetylaspartylglutamic acid, 5-fluoroindole-2-carboxylic acid, (S)-(-)-4-oxo-2-azetidinecarboxylic acid, (S) (-)-4-oxo-2-azetidinecarboxylic acid benzyl ester, (+)-a-amino-3-methoxycarbonyl-5-methylisoxazole-4-propionic acid), CP-101,606 (trexolidine) (1S,=2S)-1-(4-hydroxy-phenyl)-2-(4-hydroxy-4-phenylpiperidinyl)-1-propanol), Neu 2000 (5-aminosalicylic acid), TCS 46b (1,3-dihydro-5-[3-14-(phenylmethyl)-1-2H-benzimidazol-2-one), O-phospho-L-serine (L-SOP) (L-2-amino-3-hydroxypropionic acid-3-phosphate), (+ / -)-huperzine A ((2S)-2-amino-4-phosphonobutyric acid), Gavestinel (1H-indole-2-carboxylic acid, 4,6-dichloro-3-[(1E)-3-oxo-3-(phenylamino)-1-propen-1-yl]-3-(3-anilino-3-oxoprop-1-enyl)-4,6-dichloro-1H-indole-2-carboxylic acid acid), gamma-dgg (D-7-Glu-Gly), N20C hydrochloride (N2-(3,3-diphenylpropyl) glycinamide hydrochloride (1:1)), (1)-CPP ((1)-3-(2-carboxypiperazine-4-yl)propyl-l-phosphonic acid), tissue granule protein, meperidine, methadone, methoxetamine (MXE), phencyclidine (PCP) (1-(1-phenylcyclohexyl) piperidine), nitrous oxide (N2O), D (-)-AP-5 (D (-)-2-amino-5-phosphonopentanoic acid), AP7 (2-amino-7-phosphonoheptanoic acid), CPPene ((3-[(R)-2-carboxypiperazine-4-yl]-prop-2-enyl-1-phosphonic acid), AP-5 (DL-2-amino-5-phosphonopivalic acid), Selfotel, adamantane (adamantan-1-amine), atomoxetine, AZD6765 (as-a-phenyl-2-pyridineethylamine dihydrochloride), agmatine, chloroform, EAA -090 ((P-[2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-2yl)ethyl]-phosphonic acid), D-AP-7 (D(-)-2-amino-7-phosphonoheptanoic acid), dextralorphan, dextromethorphan, dextrorphan ((+)-3-hydroxy-N-methylmorphinan (+)-tartrate), diphenidine, dizocilpine (MK-801) ((5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cycloheptene-5,10-iminomaleate), ethanol, eticyclidine, gacyclidine, ibogaine, magnesium, rolicyclidine, felbamate (2-phenyl-1,3-propanediol dicarbamate), tenocyclidine, methoxydine, tiletamine, neramexane (1,3,3,5,5-pentamethylcyclohexane-l-amine), elirodine (a-(4-chlorophenyl)-4-[(4-fluorophenyl)methyl]-1-piperidinolethanol), dexoxadrol, etoxadrol, Ro 25-6981 ([R-(R*,S*)]-a-(4-hydroxyphenyl)-b-methyl-4(phenylmethyl)-1-piperidinol propanol hydrochloride hydrate), MDL 105,519 ((E)-4,6-dichloro-3-(2-phenyl-2-carboxyvinyl) indole-2-carboxylic acid), remacemide, delucemine, WMS-2539, NEFA, 8A-PDHQ, HU-211, Cerestat (CNS-1102), rhynchophylline, kynurenic acid (4-hydroxyquinoline-2-carboxylic acid), rapastinel (GLYX-13), NRX-1074, 7- Chlorokynurenine, 4-chlorokynurenine (AV-101), TK-40, 1-aminocyclopropanecarboxylic acid (ACPC), L-phenylalanine, xenon, [G1u3,4,7,10,14]cono sleep peptide G (Gly-Glu-Glu-Glu-Leu-Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn), 1-amino-alkylcyclohexane, licostinel (6,7-dichloro-5-nitro-1,4-dihydro-2,3-quinoxalinedione), ACEA 1021, CP-101606, EAB-318 (R-α amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole 2-propionic acid hydrochloride); CP-101606, Ro-25-6981, Co101244, L-701,324, CGP40116, LY235959, LY233053, MRZ2 / 576, LU73068, 4-C1-KYN and their analogs or derivatives, pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers. Also included are ketamine derivatives such as rapatain or Glyx-13. ,

[0426] Non-limiting examples of NIVIDA receptor antagonists also include anti-receptor antibodies, anti-ligand antibodies, inhibitory nucleic acids, and the like.

[0427] Several synthetic opioids act as NMDA receptor antagonists, such as pethidine, methadone, meperidine, dextropropoxyphene, tramadol, levorphanol, and ketobemidone.

[0428] Each possibility represents a separate embodiment of the present invention.

[0429] In certain embodiments, the NMDA receptor antagonist is selected from the group consisting of memantine, nitromemantine, FENM, neramexane, ketamine, amantadine, dextromethorphan, L687,384, amitriptyline, 1-benzyl-6-methoxy-6', 7'-dihydrospiro[piperidin-4,4-thieno[3.2-c]pyran), elirodine, ifenprodil, orphenadrine, felbamate and their pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers. Each possibility represents a separate embodiment of the present invention.

[0430] In certain embodiments, the NMDA receptor antagonist is selected from the group consisting of memantine, nitromemantine, neramexane, ketamine and pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers thereof. Each possibility represents a separate embodiment of the present invention.

[0431] According to some embodiments of the invention, the NMDA receptor antagonist is selected from the group consisting of memantine, elirodine and ifenprodil or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0432] In certain embodiments, the NMDA receptor antagonist is memantine (3,5-dimethyl-1-adamantanamine hydrochloride) or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof.

[0433] Memantine is described in detail herein, and more details can be found in U.S. Patent No. US2017 / 0273917 A1; U.S. Patent No. 4,122,193; U.S. Patent No. 4,273,774 or U.S. Patent No. 5,061,703. Likewise, as noted herein above, all compounds used according to the present invention can also be used in the form of pharmaceutically acceptable salts, such as memantine hydrochloride, etc.

[0434] Memantine is the first representative of a new class of Alzheimer's disease drugs - a medium-affinity NMDA receptor antagonist. Memantine was developed by Merz Pharmaceuticals in Germany and is approved in Europe and the United States for the treatment of moderate to severe Alzheimer's disease. According to a specific embodiment, the NMDA receptor antagonist specifically binds to the NMDA receptor without cross-reactivity with other receptors.

[0435] Several substances are selective for the NR2B NMDA receptor subtype. The NMDAR2B subunit has received increasing attention as a therapeutic target for a variety of CNS pathologies, including acute and chronic pain, stroke and head trauma, drug-induced movement disorders, and dementia. The apparently excellent preclinical and clinical data may reflect the subtype selectivity, unique mode of action, and cellular localization of the NR2B receptor in the CNS (Pender et al., 2020; Vieira et al., 2020). The NMDANR2B subunit receptor-specific antagonist CP-101606 increased the rate of functional recovery and protected against ischemic brain injury in a dose-dependent manner. It has been concluded that the NMDANR2B receptor subunit represents a potential target for reducing not only functional deficits but also neuronal death in the cortex and several midbrain regions produced by cerebral ischemia (Liu et al., 2020). Elirodi also antagonizes the NR2B subunit. Eliprodil has been shown to protect against NMDA receptor-mediated excitotoxicity during ethanol withdrawal. Col 01244, another new potent and selective NR1 / 2B NMDA receptor antagonist, has been found to have promise in anti-epileptic medicine (Ahmed et al., 2020; U.S. Patent No. US2017 / 0273917 Al).

[0436] Many compounds that act as NMDA receptor antagonists are known to have further biological activities.Thus, according to other specific embodiments, the NMDA receptor antagonist may modulate the activity of other receptors.

[0437] As used herein, the term "modulate" refers to changing the activity by inhibiting (ie, antagonist) or by activating (ie, agonist) the activity and / or expression of a receptor. According to a specific embodiment, modulating the activity and / or expression is inhibiting the activity and / or expression.

[0438] According to a specific embodiment, regulating activity and / or expression is activating activity and / or expression. Therefore, in certain embodiments, the NMDA receptor antagonist also has serotonergic activity. As used herein, the term "serotonergic activity" refers to the ability of NMDA receptor antagonists to also regulate the activity of 5-HT receptor family receptors. As used herein, the term "5-HT receptor family" refers to a group of proteins that act as receptors for serotonin. The group contains a subgroup of proteins encoded by genes, and these genes show greater than 72% or higher homology to each other in the amino acid sequence derived from it in the assumed transmembrane region (a linear continuous fragment of hydrophobic amino acids bounded by charged amino acids or polar amino acids, whose length is sufficient to form a secondary protein structure across lipid bilayers). So far, four human 5-HT receptor subfamilies are known: 5-HT1, 5-HT2, 5-HT3, 5-HT4.

[0439] In certain embodiments, the NMDA receptor antagonist is also a 5-HT2 receptor antagonist.

[0440] In certain embodiments, the NMDA receptor antagonist having 5-HT2 receptor antagonist activity is ketamine or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof. Each possibility represents a separate embodiment of the present invention.

[0441] In certain embodiments, the NMDA receptor antagonist also has cholinergic activity. As used herein, the term "cholinergic activity" refers to the ability of the NMDA receptor antagonist to also regulate the activity of nicotinic acetylcholine receptor (nACHR) family receptors. In certain embodiments, the NMDA receptor antagonist is also a nicotinic acetylcholine receptor (nACHR) antagonist.

[0442] As used herein, the term "nicotinic acetylcholine receptor (nACHR) family" refers to a group of proteins that act as receptors for acetylcholine, which signal muscle contraction when ligand binds. In certain embodiments, the NMDA receptor antagonist with nACHR antagonist activity is selected from the group consisting of amantadine and dextromethorphan and their pharmaceutically acceptable salts, hydrates, and pharmaceutically active enantiomers. Each possibility represents a separate embodiment of the present invention.

[0443] In certain embodiments, the NMDA receptor antagonist also has dopaminergic activity. As used herein, the term "dopaminergic activity" refers to the ability of an NMDA receptor antagonist to also modulate the activity of receptors of the dopamine receptor family.

[0444] As used herein, the term "dopamine receptor family" refers to a group of proteins that act as receptors for dopamine. The group contains subsets of proteins encoded by genes that exhibit greater than 65% homology to each other in the amino acid sequences deduced therefrom within the putative transmembrane region. To date, three human dopamine receptor subfamilies are known: dopamine D receptors, dopamine D2 receptors, and dopamine D3 receptors.

[0445] In certain embodiments, the NMDA receptor antagonist is also a dopamine D2 receptor agonist. In certain embodiments, the NMDA receptor antagonist with dopamine D2 receptor agonist activity is ketamine or a pharmaceutically acceptable salt, hydrate or pharmaceutically active enantiomer thereof. Each possibility represents a separate embodiment of the present invention.

[0446] In certain embodiments, the NMDA receptor antagonist with sigma-1 receptor agonist activity is selected from the group consisting of L-687,384, amitriptyline and 1-benzyl-1-6-methoxy-6,7'-dihydrospiro? piperidine-4,4'-thieno[3.2-c]pyran] and their pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers. Each possibility represents a separate embodiment of the present invention.

[0447] As used herein, the term "sigma-1 receptor" refers to the expression product of the SIGMAR1 gene encoding a chaperone protein at the endoplasmic reticulum (ER) that regulates calcium signaling through the IP3 receptor.

[0448] A subject in need of such treatment or prevention is administered a therapeutically effective amount of the NMDA receptor antagonist memantine, less than 10 mg / kg.

[0449] In certain embodiments, the effective amount of the agent of the present invention is a dosage of about 0.01 mg to about 10 mg (mg / kg) per kilogram of subject body weight, i.e., about 0.01 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, the effective amount of the compound of the present invention ranges from 0.001 mg body weight / kg to about 9 mg / kg body weight, 0.001 mg / kg body weight to about 8 mg / kg body weight, about 0.01 mg / kg body weight to about 7 mg / kg body weight, about 0.01 mg / kg body weight to about 2 mg / kg body weight, about 0.01 mg / kg body weight to about 6 mg / kg body weight, about 0.05 mg / kg body weight to about 5 mg / kg body weight, about 0.05 mg / kg body weight to about 4 mg / kg body weight, about 0.05 mg / kg body weight to about 3 mg / kg body weight. In some aspects, the effective amount of the compounds of the present invention is about 0.01mg / kg body weight to about 1.5mg / kg body weight, about 0.01mg / kg body weight to about 1.3mg / kg body weight, about 0.01mg / kg body weight to about 1.2mg / kg body weight, about 0.01mg / kg body weight to about 1.1mg / kg body weight, about 0.01mg / kg body weight to about 1.0mg / kg body weight, about 0.01mg / kg body weight to about 0.9mg / kg body weight or about 0.05mg / kg body weight to about 0.8mg / kg body weight. In some aspects, the dosage is about 0.05mg / kg to about 0.5mg / kg. In some aspects, the dosage is less than about 0.5mg / kg body weight, less than about 0.4mg / kg body weight or less than about 0.3mg / kg body weight. In some aspects, the effective amount of the compounds of the present invention is about 0.01mg / kg body weight to about 1.5mg / kg body weight. In some aspects, the effective amount of the compounds of this invention is a dosage within the range of about 0.01mg / kg body weight to about 1mg / kg body weight. In some aspects, the effective amount of the compounds of this invention is a dosage within the range of about 0.01mg / kg body weight to about 0.75mg / kg body weight. In some aspects, the effective amount of the compounds of this invention is a dosage within the range of about 0.75mg / kg body weight to about 1.5mg / kg body weight. In some aspects, the effective amount of the compounds of this invention is a dosage within the range of about 0.5mg / kg body weight to about 1.2mg / kg body weight. In some aspects, the effective amount of the compounds of this invention is a dosage within the range of about 0.05mg / kg to about 0.5mg / kg. In some aspects, the effective amount of the compounds of this invention is a dosage within the range of about 0.2mg / kg body weight or about 0.4mg / kg body weight.In some aspects, the dosage of the compound of the invention is about 0.01 mg / kg body weight to about 1 mg / kg body weight, about 0.1 mg / kg body weight to about 0.5 mg / kg body weight, about 0.8 mg / kg body weight to about 1.2 mg / kg body weight, about 0.7 mg / kg body weight to about 1.1 mg / kg body weight, about 0.05 mg / kg body weight to about 0.7 mg / kg body weight, about 0.01 mg / kg body weight, about 0.05 mg / kg body weight, about 0.1 mg / kg body weight, about 0.2 mg / kg body weight, about 0.3 mg / kg body weight, about 0.4 mg / kg g body weight, about 0.5 mg / kg body weight, about 0.6 mg / kg body weight, about 0.7 mg / kg body weight, about 0.8 mg / kg body weight, about 0.9 mg / kg body weight, about 1.0 mg / kg body weight, about 1.1 mg / kg body weight, about 1.2 mg / kg body weight, about 1.3 mg / kg body weight, about 1.4 mg / kg body weight, about 1.5 mg / kg body weight, about 1.6 mg / kg body weight, about 1.7 mg / kg body weight, about 1.8 mg / kg body weight, about 1.9 mg / kg body weight, about 2.0 mg / kg body weight or about 3 mg / kg body weight.

[0450] In certain embodiments, the dosage of the compound of the invention per administration is from about 1 mg to about 250 mg, from about 10 mg to about 300 mg, from about 10 mg to about 250 mg, from about 10 mg to about 200 mg, from about 15 mg to about 175 mg, from about 20 mg to about 175 mg, from about 8 mg to about 32 mg, from about 50 mg to about 75 mg, from about 25 mg to about 150 mg, from about 25 mg to about 125 mg, from about 25 mg to about 100 mg, from about 50 mg to about 100 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100 mg, or from about 75 mg to about 2 In some aspects, the dosage of the compounds of this invention is about 50 mg. In some aspects, the dosage of the compounds of this invention is about 75 mg. In some aspects, the total dosage of the compounds of this invention is about 100 mg.

[0451] In certain embodiments, the therapeutically effective dose of NMDAR blockers can be adjusted according to the condition of the disease / disorder to be treated or preventively treated, the age, weight, general health, sex and diet of the subject, dosing interval, route of administration, excretion rate and the combination of drugs. The initial dose of the medicament of the present invention can be large, followed by one or more smaller maintenance doses. Other ranges are also possible, depending on the subject's response to treatment. The initial dose can be the same as the dose subsequently administered, or lower or higher than the dose subsequently administered.

[0452] The present invention's agent / composition can be administered daily, weekly, biweekly, or several times a day. The duration and frequency of treatment can depend on the subject's response to treatment.

[0453] In certain embodiments, 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses or more of an agent / composition of the invention may be administered to a subject.

[0454] The number and frequency of dosage can be determined based on the subject's response to the administration of the composition, for example, if one or more of the patient's symptoms improve and / or if the subject tolerates the administration of the composition without adverse reactions. In certain embodiments, the agent / composition of the present invention is administered at least once a day, at least twice a day, at least three times a day, or more.

[0455] In certain embodiments, the agent / composition of the present invention is administered to a subject prior to a stressor. The accumulation of potentially toxic unfolded / misfolded proteins and protein aggregates is referred to as a stressor. In addition, proteasome damage is also referred to as a stressor, which may or may not be dependent on the accumulation of potentially toxic unfolded / misfolded proteins and protein aggregates. Therefore, in certain embodiments, the agent / composition of the present invention is administered to a subject who is 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or older.

[0456] In certain embodiments, the agents / compositions of the present invention are administered to a subject who is a human less than 25 years old.

[0457] In certain embodiments, agents / compositions of the invention are administered to a subject for life.

[0458] The subject of the present invention can be a mammal, and in a specific embodiment is a human being, which can be an infant, a child, an adult or an elderly person. The terms "subject" and "individual" are used interchangeably and refer to a mammal. For example, in the context of the present invention, a mammal is a human, a non-human primate, a domestic animal such as a dog, a cat, a sheep, a cow, a goat, a pig, a horse, etc.

[0459] The agents or compositions of the present invention may be administered to a subject alone, or may be administered to a subject in combination with one or more other therapeutic regimens / agents.

[0460] In certain embodiments, combination therapy means administering the agents simultaneously in the same dosage form, administering the agents simultaneously in different dosage forms, or administering the agents separately.

[0461] In certain embodiments, the second medicament / treatment regimen is used as an adjuvant therapy for medicament or composition of the present invention. In certain embodiments, the treatment is included in the stage of treating with the second medicament / treatment regimen after stopping treatment with medicament or composition of the present invention. In certain embodiments, the treatment includes treating with medicament or composition of the present invention and treating overlapping stages with the second medicament / treatment regimen. Combination therapy can be used in sequence or can be used simultaneously. In either case, these medicines and / or therapies are referred to as "co-administered".

[0462] In certain embodiments, the subject is treated with the present invention medicament or composition and the second medicament at the same time (or concomitantly). In certain embodiments, the subject is initially treated with the present invention medicament or composition, then stops treating with the present invention compound or composition, and starts treating with the second medicament. In certain embodiments, the present invention medicament or composition is used as initial treatment, for example, by administering one dose, two doses, or three doses, and the second medicament is administered to prolong the effect of the present invention compound or composition, or alternatively, to enhance the effect of the present invention medicament or composition. It will be appreciated by those of ordinary skill in the art that other variations of the proposed scheme are possible, for example, treating the subject with the present invention medicament or composition, then treating the subject with the second medicament as the adjuvant therapy for the treatment of the present invention medicament or composition for a period of time, and then stopping treating with the present invention medicament or composition. The present invention medicament and other pharmaceutically active medicaments can be administered together or separately, and when administered separately, administration can be performed simultaneously or sequentially in any order. The amount of the present invention medicament and other pharmaceutically active medicaments and the relative timing of administration will be selected to achieve the desired combined therapeutic effect.

[0463] In certain embodiments, a composition provided herein and a second agent are administered to a subject sequentially within a time interval such that a composition provided herein can act together with the other agent to provide a greater benefit than when they are otherwise administered.

[0464] The present invention provides the effects of NMDA receptor antagonists on activating the proteasome 20S subunit, including chymotrypsin-like activity, trypsin-like activity and caspase-like activity.

[0465] As used herein, the term "activator" refers to an agent that increases the level and / or activity of a target entity compared to the level and / or activity of the target entity under comparable conditions in the absence of the activator. For example, compared to the level and / or activity of the target entity under comparable conditions in the absence of the activator, the activator may increase the level and / or activity of the target entity by at least about 5%, including at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more. In some embodiments, the activator increases the level and / or activity of its target entity to a point within a predetermined range of a reference level and / or activity. In some embodiments, the reference level and / or activity is the level and / or activity observed with a wild-type version of the target entity in its natural context. In some embodiments, the activator binds directly to its target. In some embodiments, the activator binds indirectly (i.e., by binding to a physically different entity that binds to the target). In some embodiments, an activator does not physically interact with its target, either directly or indirectly, but increases the level and / or activity of the target through other actions (e.g., binding to a regulatory site in a nucleic acid that increases target expression; activating or inhibiting an enzyme that modifies the target and changes its activity, etc.). In some embodiments, an activator stabilizes and / or prolongs the half-life of its target entity. In some embodiments, an activator stabilizes its target entity in a specific three-dimensional conformation. In some embodiments, an activator competes with an inhibitor for binding to its target entity. In some embodiments, an activator prevents or reduces the aggregation of a target entity. In some embodiments, an activator stabilizes the interaction of its target entity with another entity (e.g., a substrate protein, RNA or DNA, a small molecule, a peptide, or a carbohydrate). In some embodiments, an activator binds to a target entity, and the activator increases the interaction of these entities compared to the interaction of the target entity with another entity under comparable conditions in the absence of the activator. In some embodiments, an activator-mediated increase in the interaction of a target entity with another entity increases the level and / or activity of the target entity compared to the level and / or activity of the target entity under comparable conditions in the absence of the activator. In some embodiments, an activator binds to a target entity and the activator reduces the interaction of the entities compared to the interaction of the target entity with another entity under comparable conditions in the absence of the activator. In some embodiments, an activator-mediated decrease in the interaction of a target entity with another entity increases the level and / or activity of the target entity compared to the level and / or activity of the target entity under comparable conditions in the absence of the activator.

[0466] In general, an activator can be or comprise a compound of any chemical class (e.g., a small molecule, a metal, a nucleic acid, a polypeptide, a lipid, and / or a carbohydrate). In some embodiments, the activator is or comprises an antibody or an antibody mimetic. In some embodiments, the activator is or comprises a nucleic acid agent (e.g., an antisense oligonucleotide, siRNA, shRNA, etc.) or a mimetic thereof. In some embodiments, the activator is or comprises a small molecule. In some embodiments, the activator is or comprises a naturally occurring compound (e.g., a small molecule). In some embodiments, the activator has a chemical structure that is artificially produced and / or modified. In general, an activator increases the level or activity of one or more target entities present and / or produced in a cell or organism. In some embodiments, the target entity is or comprises a polypeptide. In some embodiments, the target entity is or comprises a nucleic acid (e.g., a nucleic acid that encodes or regulates [e.g., by changing the expression and / or activity of a polypeptide] a polypeptide). In some embodiments, the target entity is or comprises a carbohydrate. In some embodiments, the target entity is or comprises a lipid. In some embodiments, the target entity is or comprises an enzyme. In some embodiments, the target entity is or comprises a polypeptide involved in cellular trafficking.

[0467] Prevention and / or inhibition of the biological function of the NMDA receptor can be achieved at the protein level, but can also be achieved at the genome and / or transcript level using various molecules that interfere with the transcription and / or translation of the receptor.

[0468] Thus, non-limiting examples of antagonists that may be used according to some embodiments of the present invention include small molecules, antibodies, inhibitory peptides, enzymes that cleave polypeptides, aptamers, homologous recombination agents, site-specific endonucleases, and RNA silencing agents.

[0469] According to a specific embodiment, the antagonist is an antibody. According to a specific embodiment, the antagonist antibody specifically binds to at least one epitope of the NMDA receptor. According to a specific embodiment, the antibody can cross the blood-brain barrier.

[0470] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants are usually composed of chemically active surface groups of molecules such as amino acids or carbohydrate side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0471] The term "antibody" as used in the present invention includes complete molecules and functional fragments thereof that are capable of binding to an epitope of an antigen, such as Fab, F(ab')2, Fv, scFv, dsFv or single domain molecules such as VH and VL. Antibodies can be monospecific (capable of recognizing one epitope or protein), bispecific (capable of binding to two epitopes or proteins) or multispecific (capable of recognizing multiple epitopes or proteins).

[0472] Another form of antibody fragment is a peptide encoding a single complementarity determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest.

[0473] It should be understood that for human therapy or diagnosis, humanized antibodies are preferably used. Non-human (e.g., mouse) antibodies in humanized form are chimeric molecules of immunoglobulins, immunoglobulin chains or their fragments, which contain minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (receptor antibodies), wherein the residues from the complementary determining regions (CDRs) of the receptor are replaced by residues from the CDRs of non-human species (donor antibodies) (such as mice, rats or rabbits) with desired specificity, affinity and ability. In some cases, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are neither present in the receptor antibody nor in the CDR or framework sequences of the input. Typically, the humanized antibody will include substantially all at least one, usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin consensus sequences. Optimally, the humanized antibody also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0474] Another agent that can be used as an antagonist in some embodiments of the present invention is an aptamer. As used herein, the term "aptamer" refers to a double-stranded or single-stranded RNA molecule that binds to a specific molecular target (such as a protein). Various methods known in the art can be used to design protein-specific aptamers.

[0475] Downregulation of nucleic acid levels is generally achieved using nucleic acid agents having a nucleic acid backbone, DNA, RNA, their mimetics, or a combination thereof. Nucleic acid agents can be encoded by a DNA molecule or provided to the cell itself.

[0476] Therefore, the antagonists of some embodiments of the present invention can be RNA silencing agents. As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms mediated by RNA molecules [e.g., RNA interference (RNAi), transcriptional gene silencing, post-transcriptional gene silencing, suppression, co-suppression and translation repression], which lead to the inhibition or "silencing" of the expression of the corresponding protein-coding gene. RNA silencing is observed in many types of organisms, including plants, animals and fungi.

[0477] As used herein, the term "RNA silencing agent" refers to an RNA that can specifically inhibit or "silence" the expression of a target gene (e.g., NMDAR gene). In certain embodiments, RNA silencing agents can prevent the complete processing (e.g., complete translation and / or expression) of mRNA molecules by a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, such as RNA duplexes comprising paired chains, and precursor RNAs that can produce such small non-coding RNAs.

[0478] Exemplary RNA silencing agents include dsRNA, such as siRNA, miRNA, and shRNA.

[0479] In one embodiment, the RNA silencing agent is capable of inducing RNA interference.

[0480] In another embodiment, the RNA silencing agent can mediate translation repression. According to one embodiment of the present invention, the RNA silencing agent is specific to the target RNA (e.g., NAMDR), and does not cross-repress or silence other targets or splice variants, and it exhibits an overall homology of 99% or less with the target gene, for example, with the target gene having an overall homology lower than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%; As determined by PCR, Western blotting, immunohistochemistry and / or flow cytometry.

[0481] RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNA (siRNA).

[0482] The following is a detailed description of RNA silencing agents that can be used according to specific embodiments of the present invention.

[0483] DsRNA, siRNA, and shRNA - The presence of long dsRNA in cells stimulates the activity of an RNase III enzyme called Dicer.

[0484] Dicer

[0485] The process of processing dsRNA into short pieces of dsRNA, which are called short interfering RNA (siRNA). The short interfering RNA obtained by Dicer activity is usually about 21 to about 23 nucleotides in length, and the short interfering RNA contains a duplex of about 19 base pairs. The RNAi reaction also features a nuclease complex commonly referred to as the RNA-induced silencing complex (RISC), which mediates the cleavage of single-stranded RNA with sequence complementarity to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex.

[0486] Thus, some embodiments of the invention contemplate the use of dsRNA to downregulate protein expression from mRNA.

[0487] According to some embodiments of the invention, the dsRNA is provided in a cell in which the interferon pathway is not activated.

[0488] According to one embodiment of the present invention, the long dsRNA is specifically designed not to induce the interferon pathway and the PKR pathway for down-regulating gene expression.

[0489] Another method to escape the interferon pathway and PKR pathway in mammalian systems is to introduce small inhibitory RNA (siRNA) by transfection or endogenous expression. The term "siRNA" refers to a small inhibitory RNA duplex (usually between 18 and 30 base pairs) that induces RNA interference (RNAi) pathways. Typically, siRNAS are chemically synthesized as 21 polymers with a central 19bp duplex region and symmetrical 2-base 3'-overhangs at the ends or at different positions.

[0490] The strands of a double-stranded interfering RNA (eg, siRNA) may be linked to form a hairpin or stem-loop structure (eg, shRNA). Thus, as described above, the RNA silencing agent of some embodiments of the present invention may also be a short hairpin RNA (shRNA).

[0491] As used herein, the term "shRNA" refers to an RNA agent with a stem-loop structure, the agent comprising a first district and a second district of complementary sequences, the degree of complementarity and orientation of the two districts being sufficient to allow base pairing to occur between the two districts, the first district and the second district being connected by a loop region, the loop being produced due to the lack of base pairing between nucleotides (or nucleotide analogs) in the loop region. The number of nucleotides in the loop is a number between 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11, and includes the above numbers. Some nucleotides in the nucleotides in the loop can participate in base pair interactions with other nucleotides in the loop. The effects of these molecules on the target can be simply tracked by protein expression of the target (i.e., NMDAR receptor herein).

[0492] It should be understood that, as described above, the RNA silencing agents of some embodiments of the present invention are not necessarily limited to those molecules containing only RNA, but also encompass chemically modified nucleotides and non-nucleotides and methods of preparation.

[0493] According to another embodiment, the RNA silencing agent may be a miRNA.

[0494] The terms "microRNA," "miRNA," and "miR" are synonymous and refer to a collection of noncoding single-stranded RNA molecules of approximately 19 to 28 nucleotides in length that regulate gene expression. miRNAs are present in a wide range of organisms and have been shown to play roles in development, homeostasis, and disease etiology.

[0495] Although miRNAs initially exist as double-stranded species, miRNAs are ultimately incorporated as single-stranded RNAs into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC), where they are removed and degraded.

[0496] Antisense is a single-stranded RNA designed to prevent or inhibit the expression of a gene by specifically hybridizing to its mRNA. Downregulation of receptors can be achieved using antisense polynucleotides encoding receptors or receptor subunits.

[0497] The prior art teaches a number of delivery strategies that can be used to effectively deliver oligonucleotides to a variety of cell types [see, Molecular Cloning: A Laboratory Manual, 3rd edition, Sambrook and Russel eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2001; Brown TA (Genomes. 2nd edition, Oxford, Wiley-Liss: 2002; Auld et al., -The assay Guidance Manual; https: / / www.ncbi.nlm.nih.gov / books / )]

[0498] Nucleic acid agents can also act at the DNA level, as summarized below.

[0499] Inhibition of the biological function of the NMDA receptor can also be achieved by inactivating the gene by introducing targeted mutations involving loss-of-function changes (eg, point mutations, deletions, and insertions) in the gene structure.

[0500] As used herein, the phrase "loss of function change" refers to any mutation in the DNA sequence of a gene that results in downregulation of the expression level and / or activity of the expression product (i.e., mRNA transcript and / or translated protein). Non-limiting examples of such loss of function changes include missense mutations, i.e., mutations that change an amino acid residue in a protein with another amino acid residue, thereby eliminating the effect of the protein; nonsense mutations, i.e., mutations that introduce a stop codon in a protein, such as an early stop codon, which results in a shorter protein lacking enzymatic activity; frameshift mutations, i.e., mutations of deletions or insertions of proteins, usually nucleic acids, that change the reading frame of the protein and can result in early termination (e.g., truncated proteins lacking enzymatic activity) by introducing a stop codon into the reading frame, or in longer amino acid sequences that affect the secondary or tertiary structure of the protein (e.g., read-through proteins), and result in a non-functional protein lacking the enzymatic activity of a non-mutated polypeptide; read-through mutations that eliminate enzyme activity, which are caused by frameshift mutations or modified stop codon mutations (i.e., when the stop codon is mutated to amino acid codon); promoter mutations, i.e. mutations in the promoter sequence, usually 5' to the transcription start site of the gene, which result in downregulation of a specific gene product; regulatory mutations, i.e. mutations in upstream or downstream regions or within the gene, which affect the expression of the gene product; deletion mutations, i.e. mutations that delete the coding nucleic acid in the gene sequence and can result in frameshift mutations or whole-frame mutations (within the coding sequence, the deletion of one or more amino acid codons); insertion mutations, i.e., mutations that insert coding or non-coding nucleic acids into the gene sequence and can result in frameshift mutations or whole-frame insertions of one or more amino acid codons; inversions, i.e. mutations that result in the reversal of the coding or non-coding sequence; splicing mutations, i.e. mutations that result in abnormal or poor splicing; and duplication mutations, i.e., mutations that result in duplication of coding or non-coding sequences, which can be whole-frame or can cause frameshifts.

[0501] Methods for introducing nucleic acid changes into a gene of interest are well known in the art and include site-specific recombinases (e.g., Cre recombinase and Flp recombinase), PB transposases (e.g., Sleeping Beauty, piggyBac, To12, or Frog Prince), genome editing by engineered nucleases (e.g., mega nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas systems), and genome editing using a recombinant adeno-associated virus (rAAV) platform.

[0502] The NMDA antagonists and / or agents of some embodiments of the present invention may be administered to an organism as such or in the form of a pharmaceutical composition mixed with a suitable carrier or excipient.

[0503] As used herein, the term "pharmaceutical composition" refers to any composition comprising at least one pharmaceutically active ingredient and at least one pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. In this article, the term "active ingredient" refers to an NMDA receptor antagonist that is responsible for a biological effect.

[0504] As used herein, the terms "pharmaceutically acceptable carrier" and "physiologically acceptable carrier" are interchangeable and refer to any type of non-toxic solid,

[0505] Semisolid or liquid fillers, carriers diluents or excipients which do not cause significant irritation to the organism and do not abrogate the biological activity and properties of the administered compound.

[0506] Suitable routes of administration may include, for example, oral delivery, rectal delivery, transmucosal delivery, especially nasal, enteral or parenteral delivery, including intramuscular and subcutaneous delivery.

[0507] In certain embodiments, the above-mentioned NMDA receptor antagonist and / or medicament are formulated for oral delivery. In certain embodiments, the above-mentioned NMDA receptor antagonist and / or medicament are formulated for injection. In certain embodiments, the NMDA receptor antagonist and / or medicament are formulated for sustained release.

[0508] Sustained release allows a specific drug to be delivered at a program-controlled rate, which results in drug delivery that lasts for a long period of time. Sustained release can maintain the concentration of the drug in the blood or target tissue at a constant level by adjusting the drug release rate. When a drug is dissolved in an aqueous body fluid, it can be easily transported to the target receptor with the body fluid. Some studies have shown that one way to achieve sustained drug release is to prevent the drug molecules from completely entering the aqueous environment for a manageable period of time. This inhibition can be identified by adjusting the degradation rate of the carrier, or by adjusting the diffusion rate of the drug molecules on the insoluble polymer matrix or shell.

[0509] Conventional methods for delivering drugs to the central nervous system (CNS) include, but are not limited to, neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion); molecular manipulation of agents (e.g., generating chimeric fusion proteins comprising a combination of a transport peptide with affinity for endothelial cell surface molecules and an agent that cannot itself cross the BBB), attempting to utilize one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of agents (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and transient disruption of BBB integrity caused by hyperosmotic disruption (caused by injection of mannitol solution into the carotid artery or use of bioactive agents such as angiotensin peptides). Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example by injecting the pharmaceutical composition directly into a tissue region of the patient.

[0510] The pharmaceutical composition of the NMDAR blocker can be prepared by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, embedding or lyophilizing methods.

[0511] Thus, the pharmaceutical compositions used according to some embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and adjuvants that facilitate processing of the active ingredients into pharmaceutically usable preparations. Appropriate formulations depend on the chosen route of administration.

[0512] For injection, the active ingredients of the pharmaceutical composition can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0513] For oral administration, pharmaceutical compositions can be easily prepared by combining active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers allow pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by patients. Pharmacological preparations for oral use can be prepared using solid excipients, optionally grinding the resulting mixture, and if necessary, after adding suitable adjuvants, processing the granular mixture to obtain tablets or dragee cores. Suitable excipients, in particular fillers such as sugars, include lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers, such as polyvinyl pyrrolidone (PVP). If necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or its salt, such as sodium alginate, can be added.

[0514] Orally usable pharmaceutical compositions include push-fit capsules made of gelatin and soft sealed capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). Push-fit capsules may contain active ingredients mixed with fillers (such as lactose), binders (such as starch), lubricants (such as talc or magnesium stearate) and optional stabilizers. In soft capsules, these active ingredients may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin or liquid polyethylene glycol. In addition, stabilizers may be added. The dosage of all formulations for oral administration should be suitable for the selected route of administration.

[0515] For oral administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0516] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example by bolus injection or continuous infusion. Formulations for injection can be in unit dosage form, for example in ampoules or multidose containers, optionally with preservatives added. The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain preparatants, such as suspending agents, stabilizers and / or dispersants.

[0517] Pharmaceutical compositions for parenteral administration include aqueous solutions of active preparations in water-soluble form. Additionally, the suspension of active ingredient can be prepared into suitable oil-based injection suspensions or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (such as sesame oil), synthetic fatty acid esters (such as ethyl oleate, triglycerides), or liposomes. Aqueous injection suspensions can contain materials that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or medicaments, which increase the solubility of the active ingredient to allow the preparation of highly concentrated solutions.

[0518] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water based solution) before use.

[0519] The pharmaceutical compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, eg, conventional suppository bases such as cocoa butter or other glycerides.

[0520] Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions wherein the active ingredient is included in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means the amount of an active ingredient (e.g., NMDA receptor antagonist) that effectively prevents, alleviates or improves the symptoms of a disorder (e.g., aging, e.g., aging-related diseases) or prolongs the life span of a subject.

[0521] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0522] For any preparation used in the methods of the present invention, the therapeutically effective amount or dosage can be initially estimated based on in vitro and cell culture assays. For example, a dosage can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine an effective dosage in humans.

[0523] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, cell culture or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage can vary depending on the dosage form employed and the route of administration used.

[0524] Dosage and interval can be adjusted, respectively, to provide a level of active ingredient sufficient to induce or inhibit a biological effect (minimum effective concentration, MEC). The MEC for each preparation is different but can be estimated from in vitro data. The dosage necessary to reach the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0525] Pharmaceutically active prodrugs of NMDA receptor antagonists can be used. As used herein, the term "prodrug" refers to (i) an inactive form of a drug that exerts its effects after being converted into an available or active form during metabolic processes in the body, or (ii) a substance that produces a pharmacologically active metabolite, although it itself is inactive (i.e., an inactive precursor).

[0526] Depending on the severity and responsiveness of the condition to be treated, dosing may be single or multiple administrations, with courses lasting from days to weeks or lifelong.

[0527] The amount of composition to be administered will, of course, be dependent upon the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like.

[0528] The terms "comprises / comprising", "includes / including", "having" and their variations mean "including but not limited to".

[0529] The term "consisting of" means "including and limited to."

[0530] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but the additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method or structure.

[0531] As used herein, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0532] The present invention and its embodiments have been described in detail. However, the scope of the present invention is not intended to be limited to any specific embodiment of any process, manufacture, composition of matter, compound, means, method and / or step described in the specification. Various modifications, substitutions and changes may be made to the disclosed materials without departing from the essence and / or basic features of the present invention. Therefore, those of ordinary skill in the art will easily understand from the disclosure that, according to such relevant embodiments of the present invention, subsequent modifications, substitutions and / or changes may be utilized to perform substantially the same functions as the embodiments described herein or achieve substantially the same results as the embodiments described herein. Therefore, the following claims are intended to include modifications, substitutions and changes to the processes, manufactures, compositions of matter, compounds, means, methods and / or steps disclosed herein within their scope.

[0533] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to those manners, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine or readily developable from known manners, means, techniques and procedures.

[0534] It should be understood that, for the sake of clarity, certain features of the invention described in the context of each independent embodiment may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, various features of the invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination, or, where appropriate, in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment does not function without those elements.

[0535] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. However, they should in no way be construed as limiting the broad scope of the invention.

[0536] The geroscience hypothesis posits that fundamental aging mechanisms are the “root causes” contributing to the increasing burden of diseases and conditions that occur with aging and that account for the majority of morbidity, mortality, and health costs in both developed and developing countries. These fundamental aging processes include: (1) macromolecular dysfunction (e.g., protein misfolding and aggregation, decreased proteasome activity, DNA damage, telomere uncapping, increased advanced glycation end products [AGEs], lipotoxicity, and accumulation of bioactive lipids) and organelle dysfunction (nuclear membrane alterations associated with lamin B deficiency, mitochondrial dysfunction resulting in decreased fatty acid metabolism, increased glucose utilization, depletion of NAD+, and increased production of reactive oxygen species [ROS], etc.); 2) chronic low-grade “sterile” (absent bacteria, fungi, etc.) inflammation often accompanied by fibrosis; 3) stem cell, progenitor, and immune cell dysfunction (including altered proliferation capacity and impaired differentiation, failure to develop into functional mature cells, decreased “anti-aging” factors [e.g., α-Klotho] that lead to stem cell and progenitor cell dysfunction); and 4) cellular senescence.

[0537] The disease monism of fundamental aging processes assumes that these fundamental aging processes are likely to be interconnected. Therefore, targeting any one fundamental aging process genetically or pharmacologically (e.g., reduced proteasome activity, protein misfolding-aggregation, depletion of NAD+, cellular senescence) should affect many or perhaps all of the remaining processes. Indeed, consistent with the disease monism, senescent cells contribute to inflammation, fibrosis, DNA damage, development of protein aggregates, autophagy failure, lipotoxicity, mitochondrial dysfunction, depletion of NAD+, ROS generation, and stem cell, progenitor, and immune cell dysfunction.

[0538] Therefore, in another embodiment, the present invention provides a drug for improving the onset and / or progression of aging and aging-related diseases or even treating them by targeting more than one fundamental aging process, including protein misfolding and aggregation, reduced proteasome activity, increased oxidized proteins, AGEs, lipotoxicity and accumulation of bioactive lipids, and organelle dysfunction (mitochondrial and autophagic lysosomal dysfunction), stem and progenitor cell dysfunction, depletion of NAD+, and cellular senescence, comprising the following steps: administering a therapeutically effective amount of an NMDAR antagonist or a pharmaceutically acceptable salt or prodrug thereof.

[0539] Example

[0540] NMDA inhibitors degrade the cellular proteins p21, p27, and p53.

[0541] In the present invention, it has been found that NMDA receptor antagonists (ketamine and memantine) lead to a decrease in p21, p27, and p53 proteins in HepG2, T98G, and SH-SY5Y cell lines ( Figure 1A ). These cellular proteins (including p21, p27, p53) are known to be classified as amyloidogenic IDPs, and up to 41% of the eukaryotic proteome is predicted to contain IDRs. Degradation is concentration-dependent and begins within minutes, as demonstrated by western blotting in cells incubated with an NMDAR antagonist for 10 minutes ( Figure 1B The decrease in p21 levels in the presence of cycloheximide indicates that p21 protein is degraded rather than protein synthesis is inhibited ( Figure 1C to Figure 1D ).

[0542] It is known in our laboratory and described previously that some NMDR blockers, including ketamine, inhibit AktlSer473 phosphorylation, leading to Aktl inactivation ( Figure 2A ). Therefore, to understand whether inhibition of Aktl Ser473 phosphorylation would affect p21 degradation, the allosteric phospho-AKT S473 inhibitor MK-2206 and a specific inhibitor of PI3K, an upstream activator of phospho-AKT S473, were tested. Figure 2B The results showed that although MK-2206 and wortmannin reduced pAkt1 phosphorylation at Ser 473 much more than ketamine did, they did not affect the amount of p21 protein. Therefore, the effect of ketamine on p21 degradation is independent of the PI3K / Aktl pathway.

[0543] Proteasome inhibitors prevent the degradation of p21, p27, and p53 proteins caused by NMDAR blockade

[0544] Further experiments showed that the presence of the proteasome inhibitors Mg132, lactacystin, or both prevented or reduced the degradation of p21, p27, and p53 caused by NMDAR blockade (data for lactacystin not shown) ( FIG. 3A to FIG. 3C These results suggest that the degradation of p21, p27, and p53 involves the proteasome system; however, proteasome inhibitors had no effect on the reduction in Aktl phosphorylation that occurred in the presence of NMDAR blockers ( Figure 3D ). Therefore, the effects of NMDAR blockade on Aktl Ser473 phosphorylation and p21 protein amount occur via different mechanisms and independently of each other.

[0545] NMDAR inhibitors also degrade phosphorylated tau (p-tau) and serine racemase (SR), and their functions are inhibited by proteasome inhibitors.

[0546] Tau is the main axonal microtubule-associated protein. Abnormal phosphorylation of tau interrupts its binding to microtubules and leads to the destruction of MTN. Intracellular aggregates composed mainly of phosphorylated tau (p-tau) are one of the hallmarks of Alzheimer's disease (AD). P-tau accumulation is a prominent feature in AD, which can hinder the normal function of MTN and lead to a series of catastrophic cascades.

[0547] Serine racemase (SR) converts free form L-serine to D-serine (DS) in the mammalian brain. DS acts as a co-agonist of NMDAR. Overactivation of NMDA receptors leads to many neurological disorders such as stroke, amyotrophic lateral sclerosis and AD. NMDAR inhibitors (ketamine and memantine) degrade phosphorylated tau (p-tau) and serine racemase (SR), and their function is inhibited by proteasome inhibitors ( Figure 4A , Figure 4B ). Figure 4A and Figure 4B Only results for HepG cells and ketamine are shown. Similar results were obtained with memantine in T98G and SH-SY5Y cell lines.

[0548] NMDAR blockade enhances proteasome activity

[0549] Therefore, the activity of NMDA receptor antagonists on the proteasome was tested next.

[0550] A common method for measuring proteasome activity is to use fluorescent substrates. In this type of experiment, a small peptide is linked to a 7-amino-4-methylcoumarin (AMC) group. When a protease such as the proteasome cleaves the group, the AMC group becomes fluorescent and the signal can be measured over time. These types of experiments are almost always performed with cell lysates or purified proteasomes. Therefore, data from these types of experiments can be independent of more complex environments (such as whole cells or in vivo situations).

[0551] Indeed, we demonstrated in a series of experiments using cell lysates that NMDA receptor antagonists had no effect on any of the three isoforms of proteasome activity, including trypsin-like activity, chymotrypsin-like activity, and caspase-like activity ( FIG. 5A to FIG. 5C ).

[0552] We developed a cell-permeable form of the fluorescent substrate (the most effective concentration was determined by adjusting the digoxigenin-Tween ratio) to overcome these limitations and termed it the "intracellular fluorescent proteasome assay". Indeed, using the cell-permeable form of the fluorescent substrate, we demonstrated that NMDAR blockade statistically significantly increased the proteasome activity of trypsin-like activity, chymotrypsin-like activity, and caspase-like activity in HepG cells ( Fig. 6A , Figure 6B and Figure 6C ). In addition, it was determined that NMDAR blockers increased the activities of trypsin-like enzymes, chymotrypsin-like enzymes, and caspase-like enzymes, depending on the amount of drug ( Fig.6D ) (Only results from the HepG-chymotrypsin-ketamine experiment are shown).

[0553] Further experiments using the cell-based proteasome luciferase system (Promega), a method applied to living cells, also showed that NMDA receptor antagonists increased the proteasome activity of chymotrypsin, similar to the results of the intracellular fluorescent proteasome assay in HepG2 cells ( Fig. 7A ). In addition, when Promega's luciferase system was used with cell lysates, no effect on chymotrypsin activity was observed, which was identical to the results of the lysate-based fluorescence assay ( Figure 7B Further experiments using the cell-based proteasome luciferase system (Promega), a method applied to living cells, also showed that the NIVIDA receptor antagonist increased the proteasome activity of trypsin and chymotrypsin, similar to the results of the intracellular fluorescent proteasome assay ( Figure 7C , Fig.7D ).

[0554] NMDAR blockade increased trypsin-, chymotrypsin-, and caspase-like activities in T98G glioblastoma and Hep3B hepatocellular carcinoma cell lines, in addition to HepG2. FIG. 8A to FIG. 8C ) (Memantine and Hep3B results not shown).

[0555] In further experiments, in addition to ketamine, IU1 and other NMDAR blockers previously defined as proteasome activators (including memantine and MK801) were tested and their effects on the proteasome were compared. Mg132 was used as a proteasome inhibitor control. The results showed that NMDAR blockers had no significant effect on HepG2 ( 9A to 9C ) and T98G cell lines ( FIG. 10A to FIG. 10C). IU1 was used as a proteasome activator and increased all three forms of proteasome activity at different levels in HepG2, similar to ketamine ( 9A to 9C ).

[0556] The experiment also showed that Mg132 inhibited only chymotrypsin activity, but not trypsin-like activity and caspase-like activity. In contrast to chymotrypsin activity, Mg132 did not inhibit the trypsin activity of the proteasome in HepG2, T98G, and Hep3B cell lines ( 9A to 9C , FIG. 10A to FIG. 10C ).

[0557] Effects of NMDAR blockade are independent of autophagy

[0558] Autophagy is another intracellular degradation system that obtains its degradation capacity from lysosomes. The most well-studied form of autophagy is macroautophagy, which delivers cytoplasmic material to lysosomes via double-membrane autophagosomes. Autophagy is a multistep process that includes not only autophagosome formation but also, most importantly, flux through the entire system, including degradation following fusion with lysosomes. Other forms of autophagy, namely chaperone-mediated autophagy and microautophagy, occur directly on lysosomes.

[0559] Therefore, the aim was to investigate whether the degradation of proteins, including p21 and p53, is involved in the effects of NMDAR blockers on the autophagic-lysosomal pathway.

[0560] The most direct method for monitoring autophagic activity experimentally is to detect microtubule-associated protein light chain 3 (LC3) protein processing.LC3 is the only known protein specifically associated with all types of autophagic membranes, including phagosomes, autophagosomes and autolysosomes (hybrid organelles formed by the fusion of autophagosomes and lysosomes).LC3 is specifically associated with autophagosomes.After autophagy induction, cytoplasmic LC3 (LC3-I) is conjugated with phosphatidylethanolamine (PE) to form lipidated LC3 (LC3-II).LC3-II is bound to the extended separation membrane and remains in combination with complete autophagosomes.Therefore, the amount of LC3-II is closely related to the number of autophagosomes, and LC3-II is widely used as a marker of autophagosomes.Autophagy is a multi-step process, which not only includes the formation of autophagosomes, but also the most important thing is to include the flux through the entire system (including the degradation of LC3-II after fusion with lysosomes).Therefore, the expected result is that LC3-II protein is degraded to autophagy completely. Fig.11AIt was shown that in HepG2 cells stably expressing LC3, the amount of LC3-II increased in the presence of ketamine. This result could indicate an increase in autophagosome formation due to autophagy induction. However, the total amount of LC3 (LC3-I plus LC3II) remained unchanged. Therefore, NMDAR blockade does not lead to lysosomal degradation.

[0561] In addition to LC3, the level of p62 (also known as SQSTM1 / sequestome 1) can also be used to monitor autophagic flux. Although the proteasome pathway is also involved in p62 degradation, the main degradation mechanism of p62 is considered to be autophagy. Therefore, it is generally believed that the amount of p62 is negatively correlated with autophagic activity. The accumulation of p62 has been used as a marker for autophagy inhibition, and similarly, a decrease in p62 levels indicates autophagy activation.

[0562] A key point is that autophagy is a highly dynamic multi-step process that can be regulated in several steps. The amount of autophagosomes detected at any particular time point is a function of the balance between their rate of generation and the rate of degradation by fusion with lysosomes. Therefore, changes in the amount of p62 may reflect an increase in autophagosome formation due to autophagy induction, or a blockage of downstream steps of autophagy (such as a reduction in ineffective fusion or autophagosome degradation). Therefore, detecting only p62 levels at a specific time point is not enough to fully assess autophagic flux, which refers to the complete process of autophagy, including the sequestration of cargo within the autophagosome, the delivery of cargo to lysosomes, and the subsequent release of decomposition products. Therefore, p62 turnover needs to be studied by immunoblotting in the presence and absence of lysosomal degradation. Therefore, experiments were performed using a combination of leupeptin and NH4Cl to block lysosomal degradation. In the results, although lysosomal protease inhibitors were found to increase the amount of p62, it was determined that ketamine had no effect on the amount of p62 ( Fig. 11B ).

[0563] Another basic protocol for autophagy detection is to monitor the number of autophagosomes by visualizing LC3 dots using a fluorescence microscope. After induction of autophagy, LC3 binds to the autophagosome membrane. This change in the subcellular distribution of LC3 can be observed by indirect immunofluorescence or by detecting signals from fluorescent proteins labeled with LC3 (e.g., green fluorescent protein GFP-LC3). For this purpose, the LC3 gene construct is stably inserted into the cells, and after achieving its continuous expression, the changes in the punctate area in the presence of NMDAR blockers are studied using LC3-specific antibodies. The results showed that there was no change in the density of the punctate area in the presence of NMAR blockers compared to the control ( Fig. 11C ).

[0564] To more directly understand the possible relationship between p21 and p53 degradation and autophagy in the presence of NMDAR blockers, NMDAR blockers were tested in the presence of lysosomal inhibitors. The results showed that although proteasome inhibitors prevented p21 and p53 degradation due to NMDAR blockers as shown previously, lysosomal activity blockers did not inhibit p21 and p53 degradation in the presence of ketamine ( Fig.11D ).

[0565] All the above results related to autophagy clearly demonstrated that the autophagy pathway was not involved in NMDAR-induced degradation of p21 and p53.

[0566] The ubiquitin pathway is not involved in the effects of NMDAR blockade on proteasome activity

[0567] In the present invention, it has been found that NMDAR blockers can improve proteasome activity and can increase the degradation of p21, p27 and p53 proteins. The degradation process of ubiquitin-dependent 26S proteasome mediates the degradation of damaged and short-lived regulatory proteins. This active ATP-dependent pathway involves a cascade of three different types of enzymes. Ubiquitin is first activated by E1 ubiquitin activating enzyme in an ATP-dependent reaction, and the ubiquitin is attached to the E1 ubiquitin activating enzyme through a thioester bond. Subsequently, the activated ubiquitin is transferred to the cysteine ​​active site of the E2 ubiquitin-conjugating enzyme. Ubiquitin-protein ligase (E3) catalyzes ubiquitin transfer to the protein destined to be degraded by the 26S proteasome together with E2.

[0568] Therefore, the aim was to investigate whether the effect of NMDAR blockers is related to these steps. Furthermore, in the present invention, it was demonstrated by using a series of experiments that the effect of NMDAR blockers is independent of the ubiquitin-ATP-pathway.

[0569] This finding is supported by the following experiments; First, a model was developed to discover one or more inhibitors of ubiquitin-dependent degradation in the Hep3B cell line. For this purpose, Hep3B cells expressing the Ub-YFP genome were prepared. Ub-YFP is a system for detecting ubiquitin-dependent proteasome activity (26S). It is also known that Ub-YFP is not affected by the ubiquitin-independent proteasome system. In further experiments, we tested molecules involved in different parts of the ubiquitination process, including PYR41 (ubiquitin-activating enzyme (El) inhibitor), NCC697923 (UBE2N E2 ubiquitin conjugating enzyme inhibitor), SMER3 (ubiquitin ligase E3 inhibitor), SZL-P1-411 (Skp2 E3 ligase inhibitor), SKPIN Ct (Skp2 E3 ligase inhibitor), P005091 (USP7 inhibitor), VialinA (USP4 and USP5 inhibitor), SpautinA (USP10 and USP13 inhibitor), ML 323 (USP1-UAF1 inhibitor), IU1 (USP14 inhibitor) and Mg132 (proteasome inhibitor) for inhibiting Ub-YFP degradation. The experiments showed that PYR41 inhibited Ub-YFP degradation and YFP expression appeared under fluorescence microscopy. MG132 was used as an inhibitor of proteasome activity ( Fig. 12A ). In the next experiment, the amount of YFP protein was investigated by Western blotting in Hep3B cells in the presence of ketamine, Pyr41, and ketamine and Pyr41 together. Fig. 12B It was shown that although Pyr41 increased the amount of YFP protein in cells, ketamine did not reduce the amount of YFP protein.

[0570] It is known that the degradation of p21 protein occurs in the ubiquitin-proteasome system (UPS) (26S) and the ubiquitin-independent proteasome system (UIPS) (20S). In further experiments, the effect of PYR-41 on endogenous p21 and the activity of NMDAR blockers in HepG2 hepatocellular carcinoma cells (Hep3B, as we know, does not contain p21) in the presence of PYR-41 were tested. The experiments showed that PYR-41 increased the amount of p21 protein in control cells and cells with PYR-41, and that NMDAR blockers reduced the amount of p21 protein in a similar manner ( Fig.13A , Fig. 13B ).

[0571] In further studies, we prepared a p21-GFP construct and provided expression of the p21-GFP fusion protein in Hep3B in the presence of doxicillin. Similar to the results with endogenous p21 in HepG cells, this experiment also showed that PYR-41 increased the amount of p21 and GFP protein in control cells and cells with PYR-41, and that NMDAR antagonists reduced the amount of p21 and GFP in Hep3B cells in a similar manner ( FIG. 14A to FIG. 14C ).

[0572] Therefore, until this stage, the results showed that NMDAR blockade degraded p21 protein without involving ubiquitination-dependent processes.

[0573] NMDA receptor antagonists induce an increase in proteasome activity by increasing the activity of the 20S subunit of the proteasome.

[0574] The human 26S proteasome is composed of a barrel-shaped 20S core particle (CP) capped by one or two 19S regulatory particles (RP). 19S RP is responsible for the opening of the 20S gate, substrate recognition and binding, unfolding, and guiding ubiquitinated substrates into the 20S CP. 19S RP consists of two subcomplexes, that is, the base and peripheral lid that directly interact with 20S. The base consists of hexameric AAA-ATPase subunits (Rpt1 to Rpt6) and tetrameric non-ATPase subunits (Rpn1, Rpn2, Rpn10 and Rpn13). Rpn13, as an ubiquitin receptor, directs polyubiquitinated proteins to the proteasome. The lid is composed of nine non-ATPase subunits (Rpn3, Rpn5-Rpn9, Rpn11, Rpn12 and Sem1). The lid, particularly the Rpn11 subunit, acts as a deubiquitinating enzyme. Rpn subunits also create docking sites for other proteins, including the proteasome-associated deubiquitinases USP14 and UCH37.

[0575] The present invention further proves through a series of experiments that NMDA receptor antagonists increase the activity of proteasome through 20S subunit, while the 19S subunit of proteasome has no effect.

[0576] For this purpose, the first studies performed were to investigate the effect of NMDAR blockade on protein degradation and the chymotrypsin activity of the proteasome when the key ubiquitin receptor subunit of the 19S regulatory particle, Rpn13, was inhibited.

[0577] RA190 has recently been described as a highly selective inhibitor of Rpn13. First, the effect of NMDAR blockade on p21 degradation in the presence and absence of RA190 was investigated by Western blotting. The findings showed an increase in the amount of p21 in the presence of RA190, most likely due to inhibition of UPS-dependent degradation ( Fig.15A ). However, ketamine was just as effective in degrading p21 in the presence of RA190 as in control cells without RA190. In further experiments, it was determined that NMDAR blockade was just as effective in increasing the chymotrypsin activity of the proteasome in the presence of RA190 as in the absence of RA190. The results are shown as a line graph for measurements over an hour and as a bar graph for measurements at 15 minutes ( Fig. 15B , Fig. 15C ). Only ketamine results are shown in these experiments. In the p21 degradation and chymotrypsin activity tests, the memantine results were very similar to the ketamine results. The UPS includes a large family of deubiquitinating enzymes (DUBs). DUBs mediate the deubiquitination of the proteolytic process of the UPS. Although the exact role of proteasomal DUBs is not completely clear, they regulate the degradation of ubiquitinated proteins by removing ubiquitinated chains from substrates. DUBs belong to the protease superfamily. The human genome encodes at least 98 DUBs, which belong to 6 subfamilies based on sequence and structural similarity. Including ubiquitin carboxyl-terminal hydrolases (UCH), ubiquitin-specific proteases (USPs), and JAMM / MPN domain-associated metallopeptidases (JAMMs).

[0578] There are three important DUBs (JAMM family member POH1 (also known as RPN11 / pdal / S13 / mpr1), USP family member USP14 and UCH family member UCHL5 (also known as UCH37)) associated with the 19S regulatory particle of the proteasome. POH1 is a Zn-dependent metalloprotease, while USP14 and UCHL5 are cysteine ​​proteases.

[0579] In further experiments, gliotoxin was used to elucidate the effects of NMDAR blockade on the proteasome. It has been previously described that gliotoxin inhibits the 19S proteasome regulator in vitro and in cells by targeting its essential deubiquitinase subunit Rpn11 (also known as PSMD14). PSMD14 is a component of the 19S regulatory particle of the 26S proteasome. In addition, gliotoxin has also been shown to be a non-competitive inhibitor of the chymotrypsin activity of the 20S proteasome at high doses. The findings showed that the amount of p21 increased with increasing dose and time in the presence of gliotoxin, indicating that UPS-dependent degradation was inhibited ( Fig.16A However, NMDAR blockers degraded p21 in the presence of 5 μM and 10 μM gliotoxin, which was the same as the control group without gliotoxin ( Fig.16A). As mentioned above, gliotoxin is a non-competitive inhibitor of chymotrypsin activity of 20S, and increasing doses of gliotoxin reduced p21 degradation by inhibiting the action of ketamine. These results may indicate that the effect of NMDAR blockade on p21 degradation occurs through the chymotrypsin activity of 20S proteasome subunits rather than trypsin-like and caspase-like actions. In the next study, the effect of gliotoxin at low (5 μM) and high (25 μM) concentrations on the chymotrypsin activity of the proteasome was investigated in the presence or absence of NMDAR blockers. Fig. 16B and Fig. 16C The results showed that the chymotrypsin activity of the proteasome was not statistically changed compared to the low-dose gliotoxin control, although it showed a trend toward improvement. Importantly, NMDR blockers increased the chymotrypsin activity of the proteasome in the presence of gliotoxin as much or more than in the absence of gliotoxin ( Fig. 16B , Fig. 16C Similar to the inhibition of p21 degradation, high doses of gliotoxin (25 μM) inhibited the chymotrypsin activity of the proteasome in the presence of NMDR blockers ( Fig.16D , Fig.16E )(Only ketamine results are shown). Surprisingly, at high doses of gliotoxin, p53 also underwent the same degradation as the control, unlike p21 ( Fig.16F ). This may indicate that p53 degradation occurs independently of chymotrypsin activity. To ensure the results, immunoblotting was performed using p53 and p21 antibodies together on the same membrane.

[0580] Capzimin is another inhibitor of PSMD14. Capzimin was tested in a similar manner to gliotoxin. Fig.17A As shown, capsulomine increases the amount of p21, and NMDAR blockade in the presence of capsulomine leads to the degradation of p21 ( Fig.17A As previously mentioned, PSMD14 is part of the 19S proteasome subunit and is required for ubiquitination-dependent protein degradation. In further experiments, it was determined that NMDAR blockade in the presence of capsids was as effective as in the absence of capsids in increasing the chymotrypsin activity of the proteasome ( Fig. 17B , Fig. 17C ).

[0581] To further confirm and make it clear that the effects of NMDAR blockade are independent of PSMD14 and, therefore, the 19S subunit of the proteasome system, PSMD14 RNA expression was silenced using a lentiviral siRNA system containing DNA encoding specific DNA for PSMD14 ( Fig.18AThe results showed that NMDAR blockade reduced p21 protein in PSMD14-silenced cells to the same extent as in control cells ( Fig.18B In further experiments, it was determined that NMDAR blockade increased the chymotrypsin activity of the proteasome more in PSMD14-silenced cells than in cells where the PSMD14 protein was not silenced ( Fig.18C , Fig.18D ).

[0582] As mentioned above, one of the other important DUBs associated with the 19S regulatory particle of the proteasome is USP14.

[0583] It has been previously described that IU1 inhibits USP14 and increases proteasome activity. Although not completely confirmed, it is possible that these two functions of IU1 are independent of each other. In the present invention, it was determined that the presence of IU1 in cells significantly increased the amount of p21, while NMDAR blockers effectively reduced the amount of p21 in the presence of IU1 ( Fig.19 In the present invention, it has been previously determined that IU1 increases the trypsin-like activity, chymotrypsin-like activity, and caspase-like activity of the proteasome ( 9A to 9C ).

[0584] To further confirm and more definitively demonstrate that NMDAR inhibitors act in an independent manner as ubiquitin and 19S subunits, USP14 gene expression was silenced using a lentiviral siRNA system containing DNA encoding specific for USP14 ( Fig. 20A In addition, the expression of USP14 in cells was increased using a doxicillin-inducible retroviral system ( Fig. 20B The results showed that silencing of USP14 led to a decrease in the amount of p21 ( Fig. 20B On the other hand, the amount of p53 increased in USP14-silenced cells ( Fig. 20B ). However, as a result of a study conducted to investigate the effect of NMDAR blockers on p53 protein in USP14-silenced cells, it was determined that the reduction of p53 was similar to the effect of NMDAR blockers in control cells ( Fig. 20B ).

[0585] Importantly, although chymotrypsin activity was increased in USP14-silenced cells relative to control cells, addition of NMDAR blockade resulted in a significant increase in chymotrypsin activity in USP14-silenced cells compared to control cells ( Fig. 20C , Fig.20D ). These results are similar to those described above, indicating that NMDAR blockers act independently of 19S regulatory particles.

[0586] N-ethylmaleimide, described as a general inhibitor of deubiquitinating enzymes including USP14 and UCHL15, was used to further demonstrate that NMDAR blockade activates the proteasome independently of 19S regulatory particles and ubiquitination processes. Fig.21A The results showed that inhibition of all DUBs increased the amount of p21, which could indicate a reduction in UPS-dependent p21 degradation. Furthermore, NMDAR blockade reduced p21 to the same extent as control cells, suggesting that NMDAR blockade acts in a DUB-independent manner ( Fig. 21C and Figure 21D ).

[0587] All the above experiments indicate that the activity of NMDAR blockers is neither through the ubiquitination nor the deubiquitination process of the UPS.

[0588] More interestingly, when 19S regulatory particles (including Rpn13, PSMD14, and UCHL15) were inhibited by chemical or silencing methods, the effect of NMDAR blockade on the chymotrypsin activity of the 20S proteasome was enhanced more than that of the normally functioning 19S regulatory particles.

[0589] Up to this stage of the present invention, it has been shown that NMDAR blockers increase the trypsin-like activity, chymotrypsin-like activity and caspase-like activity of the proteasome, this increase is prevented by lactacystin and Mg132 as proteasome inhibitors, and that the activation of the proteasome and protein degradation by NMDAR blockers occurs independently of the UPS.

[0590] The next experiment was designed to show that the activity of the NMDAR blocker was directly correlated with 20S proteasome activity.

[0591] As mentioned above, the 26S proteasome consists of a barrel-shaped 20S core particle (CP) capped by one or two 19S regulatory particles. The 20S CP is a threonine protease composed of four stacked rings. The two inner β-rings contain three catalytic subunits (β5, β2, and β1), which exhibit chymotrypsin-like (CT-L), trypsin-like (Tryp-L), and caspase-like (Casp-L) activities, respectively. Unlike the 26S proteasome, which mainly degrades polyubiquitinated proteins, the 20S proteasome directly degrades misfolded proteins, oxidatively damaged proteins, as well as IDPs and IDR-containing proteins, and does not require the unfoldase activity of the 19S base.

[0592] Many other non-ATPase particles, such as the 11S complex (PA28α, β, and γ) and PA200, reversibly bind to and regulate 20S. PA28γ (REGgamma), a member of the 11S proteasome activators, has been shown to bind to and activate the 20S proteasome to promote proteasome-dependent degradation of important regulatory proteins, especially p21, which is known to be degraded by both UPS and UIPS pathways, and the UIPS degradation of p21 is regulated by PA28γ. In the next experiment, PA28γ (REGgamma) siRNA sequences cloned using the Lenti siRNA vector were used to silence PA28γ in HepG cells. Fig.22A and Fig. 22B The results showed that in PA28γ-silenced cells, p21 degradation caused by NMDAR blockade occurred much less than in control cells. In addition, although proteasome activity was reduced in PA28γ-silenced cells, the effect of NMDAR blockade on proteasome activity was also reduced in PA28γ-silenced cells compared with control cells ( Fig. 22C , Fig.22D ). This finding suggests that NMDAR blockers have activity beyond the 20S and its regulatory particles.

[0593] NMDAR blockers are more effective in degrading p21 protein in the presence of H2O2

[0594] Previously different researchers described that oxidatively modified proteins are known to be more susceptible to proteolytic degradation by the proteasome than native proteins. In addition, it was described that the 20S proteasome is more resistant to oxidative stress than the 26S proteasome, since the 20S complex remains active even when treated with moderate to high concentrations of H2O2, while the 26S proteasome is more fragile, probably because the 19S particles were observed to separate from the 20S core in the presence of H2O2 (Ishii et al., 2005). Therefore, the aim of the next study was to reveal the effect of NMDAR inhibitors on oxidatively damaged proteins. For this purpose, different concentrations of H2O2 were tested on HepG2 cells into which the p21-GFP genome was inserted, in order to find out whether H2O2 has an effect on GFP expression and, therefore, on p21. It was determined that H2O2 significantly increased the GFP signal during 12 hours and at a dose of 0.2 mM ( Fig.23A ). Similarly, at the same time and dose, H2O2 significantly increased the endogenous p21 in cells ( Fig. 23B ). The figure also shows that, crucially, NMDAR blockade more effectively reduces endogenous p21 in the presence of H2O2.

[0595] This result may indicate that NMDAR blockade is more effective against p21 that is oxidatively modified in the presence of H2O2 than against p21 in control cells. However, this result may also indicate that the separation of 19S particles from the 26S proteasome complex in the presence of H2O2 allows NMDAR blockade to enhance 20S proteasome activity, similar to the results of the above-mentioned 19S particle inhibitors.

[0596] Effects of NMDAR blockade on aging and aging-related phenotypes

[0597] The role of oxidative stress in age-related organ changes seems to be generally considered as an increase in ROS accompanies aging, leading to functional changes, increased morbidity, and shortened lifespan. Following oxidative damage, proteins unfold and expose hydrophobic regions, which makes these proteins prone to aggregation.

[0598] Therefore, the clearance of misfolded proteins is critical for cell survival because misfolding alters the three-dimensional structure of proteins, impairs their biological activity and increases their propensity to form toxic aggregates.

[0599] Modulation of protein concentrations by regulating the proteolytic machinery has long been demonstrated to be a promising setting for developing therapeutic strategies against different aging and aging-related human diseases, such as neurodegeneration, cancer and autoimmunity, aging and aging-related diseases.

[0600] The proteasome is the first defense mechanism of the cell against toxic stress induced by accumulated proteins due to oxidative damage.

[0601] Because the ability to establish homeostatic adaptive responses declines with age, leading to a decrease in overall proteostasis, measuring the adaptive response to oxidative stress and the elimination of damaged proteins by the 20S proteasome could potentially provide useful biomarkers and prognostic data to simplify treatment of aging patients.

[0602] Therefore, in the next study of the present invention, the effect of NMDA blockers activating the 20S proteasome on cellular and organismal aging was investigated.

[0603] Effects of NMDAR blockade on senescence and aging-related phenotypes in primary fibroblasts

[0604] First, primary human fibroblasts were divided into two groups at passage 4: a control group and an NMDAR-added group, and then cultured under the same conditions until passage 10.

[0605] Cellular senescence is defined as the complete and irreversible loss of replicative capacity in primary somatic cells (Hayflick 1961). Therefore, the MTT assay was used to measure cellular metabolic activity as an indicator of cell viability and proliferation in cells established at passage 8. It was found that proliferation was higher in cells treated with ketamine or memantine compared to controls ( Fig.24A ).

[0606] Another major characteristic of senescent cells is the increase in senescence-associated β-galactosidase (SAβ-gal) activity.

[0607] Therefore, cell senescence was investigated by SAβ-gal staining in cells at passage 10. It was found that the SAβ-gal staining of control cells was 2.6 times higher than that of cells treated with ketamine, and 1.8 times higher than that of cells treated with memantine, with p = 0.001 and 0.02, respectively ( Fig. 24B ).

[0608] In another experiment, the amount of intracellular lipid droplets as an indicator of lipid metabolism was studied in senescent cells by performing Nile red staining in late passage cells. Nile red is a lipophilic stain that stains intracellular lipid droplets and proteins. Lipid droplets are highly dynamic organelles involved in the storage and metabolism of neutral lipids.

[0609] Fig.24C and Fig.24D As shown, cells containing ketamine or memantine were found to produce more fluorescence than cells without drug (P=0.0001). These results may indicate that senescent cells not only lose their proliferation capacity, but also their fat metabolism becomes insufficient.

[0610] Effects of NMDAR blockade on aging and aging-related phenotypes in Caenorhabditis elegans

[0611] Caenorhabditis elegans is a free-living multicellular organism. At the tissue, cell and molecular levels, the obvious age-dependent physiological changes similar to those of humans make Caenorhabditis elegans a valuable model for research in the field of aging and lifespan research (Ambros, 2006). Therefore, how NMDAR blockers affect the lifespan of Caenorhabditis elegans was studied. In Caenorhabditis elegans related studies, the use of drugs has been applied in different ways. The drug is applied to LB medium containing bacterial OP50 and then spread on nematode growth medium (NGM) plates (LB medium method), or applied to NGM containing live bacteria (NGM live bacteria method) or dead bacteria (NGM dead bacteria method), or the drug solution is directly spotted on the surface of the plate (spotting dead bacteria method), or worms are cultured in liquid culture medium (liquid culture method) (Ambros, 2006). The liquid culture method achieves the best absorption efficiency in worms. The drug concentration in worms is comparable to that in mice, providing a bridge for dose conversion from worms to mammals. Therefore, in the present invention, all experiments related to C. elegans were done in liquid culture medium. In order to understand the effectiveness of the drug with the onset time, memantine was added to the culture medium on the 1st, 7th and 14th day during 24 days, and the number of live worms was determined on the 20th day.

[0612] The results showed that when memantine was added starting at day 7 or day 1, the survival of C. elegans in the presence of memantine was almost 3 times higher than that of the control at day 20 ( Fig.25A When memantine was added starting at day 14, survival of C. elegans in the presence of memantine was 67% higher than the control at day 20 ( Fig.25B elegans, memantine was added starting at day 7.

[0613] In C. elegans, aging-related characteristics can lead to less active, uncoordinated movements, sluggishness, and accumulation of autofluorescent deposits in cells. Therefore, on day 18, C. elegans grown on standard nematode growth medium (NGM) plates were shaken at 200 rpm at 20°C and their movements were recorded for 5 minutes. All C. elegans in the wells were tracked and counted for 5 minutes using three different eyes. In the results, the number of immobile C. elegans obs...

Claims

1. The method of the present invention is based on a model in which an N-methyl-D-aspartate receptor (NMDAR) complex antagonist increases proteasome activity.

2. The method of claim 1, wherein the NMDAR antagonist directly activates the 20S proteasome.

3. The method of claim 1, wherein the NMDAR antagonist indirectly activates the 20S proteasome.

4. The method of claim 1, wherein the NMDAR antagonist activates the effects of the proteasome 20S subunit, including chymotrypsin-like activity, trypsin-like activity, and caspase-like activity.

5. The method of claim 1, wherein the NMDAR antagonist activates the proteasome 20S subunit primarily independent of UPS (ubiquitin-ATP-dependent proteolysis).

6. The method of claims 1-5, wherein the use of the NMDAR antagonist provides a new therapeutic approach involving activation of the proteasome 20S as a target.

7. The method of claims 1-5, wherein the use of the NMDAR antagonist provides a composition and method for treating diseases caused by oxidative damage and / or protein misfolding disorders by increasing the proteasome activity.

8. The method of claims 1-5, wherein the use of the NMDAR antagonist provides compositions and methods for treating malfunctions of the protein homeostasis network, which interfere with key signaling pathways and are frequently associated with a variety of human diseases.

9. The method of claims 1-5, wherein the use of the NMDAR antagonist provides a composition and method for treating oxidative damage and / or protein misfolding diseases associated with amyloid-promoting intrinsic disordered proteins, wherein the amyloid-promoting intrinsic disordered proteins are selected from the proteins and related diseases shown in Table 1.

10. The method of claims 1-5, wherein the use of the NMDAR antagonist provides a composition and method for treating when age-dependent proteasome trypsin-like activity, chymotrypsin-like activity, and caspase-like activity are reduced.

11. The method of claims 1-10, wherein the use of the NMDAR antagonist provides a composition and method that effectively improves the onset and / or progression of aging and age-related diseases by increasing the proteasome activity and regulating the protein homeostasis network.

12. The method of claims 1-10, wherein the use of the NMDAR antagonist provides the following compositions and methods, which are not only effective in treating diseases, but more importantly, effectively preventing the causes of diseases associated with malfunction of the protein homeostasis network, including the accumulation of oxidatively damaged proteins and / or misfolded proteins.

13. The method of claim 11, wherein the aging-related disease is selected from, but not limited to, congestive heart failure, myocardial infarction, dementia, stroke, most cancers, diabetes and metabolic diseases, renal dysfunction, chronic lung disease, osteoporosis, arthritis, blindness and frailty, sarcopenia, falls, incontinence and mild cognitive impairment, among many others.

14. The method of claims 1-10, wherein the use of the NMDAR antagonist provides a composition and method that is effective in protecting, alleviating or treating the symptoms of aging.

15. The method of claim 14, wherein the use of the NMDAR antagonist provides a composition and method that effectively reduces or treats the symptoms of aging, including reducing or alleviating: susceptibility to infection, risk of heat stroke or hypothermia; Thinning of the spine bones, fractures, joint changes (ranging from mild stiffness to severe arthritis); loss of muscle mass and strength (sarcopenia), stooped posture, slowed and limited movement; decreased hand strength and dexterity, including "hand and finger strength and the ability to control submaximal pinch force and maintain a stable, precise pinching position, manual speed, and hand feel"; weakness, a syndrome of decreased strength, physical activity, physical performance, and energy; decreased maximum oxygen uptake and maximum heart rate; Constipation, urinary incontinence; slowed thinking, memory and reasoning, decreased reflexes and coordination, and difficulty with balance; Decreased visual acuity, decreased peripheral vision, hearing loss; wrinkling and sagging of the skin, whitening or graying of hair, weight loss, partly due to loss of muscle tissue; loss of strength in the eye's ciliary muscles, resulting in difficulty focusing on near objects, or presbyopia; menopause, which usually occurs between the ages of 44 and 58; loss of elasticity in the arteries and the resulting hardening of the blood vessels, atherosclerosis, leading to cardiovascular disease (e.g., stroke and heart attack), the most common cause of death worldwide.

16. The method of claim 14, wherein the use of the NMDAR antagonist provides a composition and method that is effective in preventing age-related symptoms of major body systems, including: Cells, Organs, and Tissues: Cells become less able to divide, telomeres shorten, oxidation products accumulate, connective tissue between cells becomes stiff, and the maximum functional capacity of many organs decreases; Heart and Blood Vessels: The walls of the heart become thicker, the heart muscle becomes less efficient (it needs to work harder to pump the same amount of blood), the aorta (the main artery in the body) becomes thicker, harder, and less flexible, and many arteries in the body, including those that supply blood to the heart and brain, slowly develop atherosclerosis, although some people never become seriously ill; Vital Signs: The body has a harder time controlling its temperature, and the heart rate takes longer to return to normal after exercise; Bones, Muscles, and Joints: Bones become thinner and less strong, joints become stiffer and less flexible, cartilage and bone in the joints begin to weaken, and muscle tissue becomes less thick and less strong; Digestive System: Food passes through the body more slowly. Movement through the digestive system becomes slower, and the stomach, liver, pancreas, and small intestine produce smaller amounts of digestive juices; Brain and nervous system: The number of nerve cells in the brain and spinal cord decreases, the number of connections between nerve cells decreases, abnormal structures called amyloid, plaques and tangles may form in the brain, cognitive impairment (such as memory and / or disorientation) or global functioning (overall functioning, including daily living activities) impairment and / or slowing or reversing the progressive worsening of global or cognitive impairment, age-related mood anxiety; Eyes and ears: The retina becomes thinner, the iris becomes harder, the lens becomes less clear, the walls of the ear canal become thinner, and the eardrum becomes thicker; Skin, nails, and hair: The skin becomes thinner and less elastic, sweat glands produce less sweat, nails grow more slowly, and hair becomes gray and some no longer grows.

17. The method of claims 1-10, wherein use of the NMDAR antagonist provides compositions and methods for preventing or treating aging associated with proteotoxic disorders, which are frequently associated with a variety of human diseases.

18. The method of claims 1-10, wherein use of the NMDAR antagonist provides compositions and methods for treating aging associated with malfunction of the proteostasis network, which perturbs key signaling pathways and is frequently associated with a variety of human diseases.

19. The method of claims 1-10, wherein use of the NMDAR antagonist provides compositions and methods for treating aging associated with malfunction of the proteostasis network, including accumulation of oxidatively damaged proteins and / or misfolded proteins.

20. The method of claims 1-10, wherein use of the NMDAR antagonist provides compositions and methods for preventing malfunction of the proteostasis network, including accumulation of oxidatively damaged proteins and / or misfolded proteins, thereby extending lifespan.

21. The method of claims 1-10, wherein use of the NMDAR antagonist provides compositions and methods for detoxifying cells having oxidatively damaged proteins by directly targeting the oxidatively damaged proteins to detoxify the cells.

22. The method of claims 1-21, wherein use of the NMDAR antagonist provides compositions and methods for increasing motor activity in a subject, thereby extending healthspan.

23. The method of claims 1-22, wherein use of the NMDAR antagonist provides a drug that extends both healthspan and lifespan.

24. The method of claims 1-10, 21, 22, wherein the use of the NMDAR antagonist provides a composition and method for maintaining and increasing muscle mass, muscle strength and muscle function by reducing the amount of oxidized protein.

25. The method of claim 24, wherein use of the NMDAR antagonist provides compositions and methods for maintaining and increasing muscle mass, muscle strength, and muscle function thereby providing regulation of glucose and fat oxidation.

26. The method of claims 24 and 25, wherein the use of the NMDAR antagonist provides compositions and methods for maintaining and increasing muscle mass, muscle strength and muscle function, which prevent and / or treat metabolic syndrome, which includes (i) mitochondrial dysfunction, which causes various SkM pathologies such as sarcopenia and muscular dystrophy; (ii): Inflammatory myopathies caused by inflammation and oxidative stress, such as dermatomyositis, polymyositis, necrotizing autoimmune myositis, and sporadic inclusion body myositis; and (iii) chronic diseases that cause SkM impairment, such as type 2 diabetes, obesity, chronic kidney disease, and chronic obstructive pulmonary disease, syndromes that often lead to a decline in the patient's quality of life over time and increase the patient's mortality rate.

27. The method of claims 1-10, wherein the use of the NMDAR antagonist provides a composition and method for preventing and / or treating heart disease by maintaining and / or improving myocardial function, and the composition and method are effective in improving the onset and / or progression of cardiac dysfunction following ischemia / reperfusion injury and aging-induced cardiomyopathy due to oxidative stress, which is the main cause of cardiac dysfunction following ischemia / reperfusion injury and aging-induced cardiomyopathy.

28. The method of claims 1-10, wherein the use of the NMDAR antagonist provides compositions and methods for preventing and / or treating adipogenesis and lipid accumulation during aging by maintaining and / or improving the function of the lipid-protein homeostasis network.

29. The method of claims 1-10, wherein the use of the NMDAR antagonist provides compositions and methods for effectively treating and even preventing obesity and obesity-related disorders by activating the proteasome to regulate malfunction of the protein homeostasis network, including the accumulation of oxidatively damaged proteins and / or misfolded proteins, including cardiovascular disease, insulin resistance, diabetes, dyslipidemia.

30. The method of claims 1-10, wherein the use of the NMDAR antagonist provides a composition and method for effectively treating or even preventing neurodegenerative diseases by activating the proteasome to regulate the dysfunction of the protein homeostasis network, including the accumulation of oxidatively damaged proteins and / or misfolded proteins, wherein the neurodegenerative disease is selected from neurodegeneration caused by pathological insults including anti-cancer therapy, cerebral ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, retinal ganglion cell loss, long-term sequelae of concussion, reduction of the neurotoxic effects of astrocytes in Alzheimer's disease, epileptic seizures, and the like.

31. The method of claims 20 and 21, wherein the use of the NMDAR antagonist provides a composition and method for detoxifying cells having oxidatively damaged proteins by directly targeting oxidatively damaged proteins to detoxify the cells, thereby preventing and / or treating skin aging symptoms including skin wrinkles, skin sagging, age spots and wrinkling on the skin, whitening or graying of hair, and hair loss.

32. The method according to claims 21 and 31, wherein the use of the NMDAR antagonist provides the following compositions and methods for detoxifying the cells having oxidatively damaged proteins and lipids by inducing the activity of the 20S proteasome complex, and thus can be used in cosmetics.

33. The method of claims 1-10, wherein the use of the NMDAR antagonist provides a composition and method for preventing and / or treating ocular pathologies, including age-related macular degeneration, cataracts, glaucoma, and diabetic retinopathy associated with oxidative stress, which is one of the most influential factors in ocular diseases and directly affects the process of autophagic activity, which may occur due to reduced proteasome activity, is the accumulation of potentially toxic unfolded / misfolded proteins and protein aggregates.

34. The method of claims 1-5, wherein the use of the NMDAR antagonist demonstrates that increased proteasome activity in the presence of the NMDAR blocker reduces serine racemase, which plays a role in neurodegenerative diseases caused by any neural damage, thereby reducing the conversion of L-serine to D-serine.

35. The method of claim 34, wherein the NMDAR blocker reduces serine racemase, thereby reducing the conversion of L-serine to D-serine, wherein the serine racemase plays a role in neurodegenerative proteinopathies associated with increased D-serine, the neurodegenerative proteinopathies comprising a disorder selected from cerebral ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, hyperactivity syndrome, retinal ganglion cell loss, long-term sequelae of concussion, epileptic seizures, retinal ganglion cell loss in diabetic patients, chronic social defeat stress and other neurodegenerative diseases.

36. The method according to claims 34 and 35, wherein the NMDAR blocker reduces serine racemase which prevents or treats many neuronal diseases by reducing astrocyte toxicity in different ways.

37. The method of claim 36, wherein the neuronal disease associated with astrocyte toxicity is selected from the group consisting of microvascular damage in diabetic retinopathy, synaptic damage after traumatic brain injury, neuronal overactivation, choroidal neovascularization, pathological symptoms of Alzheimer's disease, and neurodegeneration.

38. The method of claim 34, wherein the NMDAR blocker reduces serine racemase, thereby preventing and / or treating schizophrenia and other psychological disorders, since the serine racemase plays a role in the pathogenesis of these diseases.

39. The method of claim 1-10, wherein the use of the NMDAR antagonist provides a composition and method for preventing and / or treating a patient's renal disease caused by an increase in oxidatively damaged proteins and / or misfolded proteins due to aging, hyperlipidemia, hypertension, smoking, diabetes, obesity and other factors, or the composition and method are used to treat disease onset in a patient in need or reduce the likelihood of disease onset, comprising administering an effective amount of an NMDAR blocker to the subject.

40. The method of claim 39, wherein the renal diseases are acute and chronic renal diseases caused by different causes.

41. The method of claims 1-10, wherein use of the NMDAR antagonist provides compositions and methods for preventing and / or treating cell proliferative diseases including cancer.

42. The method of claim 41, wherein the cell proliferative disease and / or cancer is associated with an increased unfolded protein response (UPR) in the patient caused by protein unfolding / misfolding stress.

43. The method of claims 1-10, 41, 42, wherein the use of the NMDAR antagonist provides a composition and method for preventing, reducing or even treating the cell proliferative disease by reducing or preventing the accumulation of the unfolded protein and the subsequent UPR, wherein the UPR alters the sensitivity of cancer cells to anti-tumor drugs.

44. The method of claim 41, wherein the cell proliferative disease encompasses a variety of conditions characterized by aberrant cell growth, preferably abnormally increased cell proliferation, such as proteinopathic cell proliferative diseases, disorders and / or conditions including, but not limited to, cancer, immune-mediated responses and diseases (e.g., transplant rejection, graft-versus-host disease, immune response to gene therapy, autoimmune diseases, pathogen-induced immune disorders, etc.), certain circulatory diseases, and certain neurodegenerative diseases.

45. The method according to claim 41, 42, wherein the cancer disease is selected from but not limited to carcinoma, leukemia and lymphoma, such as cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, lymphoma associated with human T-cell virus (HTLV), such as adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma, acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, mesothelioma, adult Common solid tumors, such as head and neck cancer (e.g., oral cancer, laryngeal cancer and esophageal cancer), genitourinary cancer (e.g., prostate cancer, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, testicular cancer, rectal cancer and colon cancer), lung cancer, breast cancer, liver cancer, colon cancer, small intestine cancer, pancreatic cancer, melanoma and other skin cancers, gastric cancer, tumors of the central nervous system (CNS), neuroectodermal cancer, spinal tumors, gliomas, meningiomas, pituitary adenomas, hepatoma and thyroid cancer and / or childhood solid tumors, such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors and soft tissue sarcomas.

46. ​​The term "cancer" according to claims 41 and 45 also includes cancer metastasis.

47. The method of claim 42, wherein the use of the NMDAR antagonist provides a composition and method for increasing the sensitivity of cancer cells to anti-tumor drugs by reducing the UPR.

48. The method of claims 1-10, wherein use of the NMDAR antagonist provides compositions and methods for preventing and / or treating a disease associated with endoplasmic reticulum (ER) stress in a patient, or treating or reducing the likelihood of disease onset in a patient.

49. The method of claim 48, wherein the ER stress-related disease is selected from, but not limited to, metabolic diseases, including insulin resistance, atherosclerosis, diabetes, obesity, alcoholic and non-alcoholic fatty liver disease, hyperlipidemia; cancers, including leukemia, multiple myeloma, breast cancer, prostate tumors; immune system-related diseases, including viral infection, bacterial infection, vitiligo, rheumatoid arthritis, type 1 diabetes; and many nervous system diseases are known to be associated with ER stress, including cerebral ischemia, sleep apnea, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, prion disease, Parkinson's disease and Huntington's disease and neuroserpin inclusion body encephalopathy.

50. The method of claims 1-10, wherein the use of the NMDAR antagonist provides a composition and method for preventing and / or treating a disease associated with autophagy dysfunction associated with a malfunction of the protein homeostasis network including accumulation of oxidatively damaged proteins and / or misfolded proteins.

51. The method of claim 50, wherein the disease associated with autophagy dysfunction is selected from, but not limited to, adult neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, neuronal ceroid lipofuscinosis, fulminant neurodegeneration, and Lewy body dementia; pediatric neurodevelopmental disorders, including spinocerebellar ataxia, cortical atrophy and epilepsy, childhood-onset neurodegeneration, BPAN, spastic quadriplegia and brain abnormalities, primary microcephaly, hereditary spastic paraplegia, ataxia with spasticity, Rett syndrome, Joubert syndrome, leukoencephalopathy, juvenile-onset dystonia, CEDNIK syndrome, Pelvic floor disease, and West syndrome; hereditary neuropathy, including sensory and autonomic neuropathy type II, Charcot-Marie-Tooth disease, sensory and autonomic neuropathy type IF, and teleneuropathy. hereditary motor neuropathies; ophthalmological diseases, including primary open-angle glaucoma and cataracts; cardiac and skeletal myopathies, including Danon cardiomyopathy, distal myopathy with rimmed vacuoles, dilated cardiomyopathy, sporadic inclusion body myositis, and X-linked myopathy with excessive autophagy; inflammatory conditions, including Crohn's disease, ulcerative colitis, and childhood asthma; autoimmune diseases, including systemic lupus erythematosus, diabetes, and other autoimmune diseases; infectious diseases, including Mycobacterium tuberculosis and Mycobacterium leprae; bone disorders, including osteopetrosis, Paget's disease of bone, and Caschin-Baker disease; congenital multisystem disorders, including global dysplasia, Vici syndrome, Zellweger syndrome, glycosylation disorders with autophagy deficiency, Zimmerman-Laband syndrome, Hermansky-Pudlak syndrome, and multisystem proteinopathy.

52. The method of claims 1-10, wherein the use of the NMDAR antagonist provides compositions and methods for preventing and / or treating diseases associated with lysosomal dysfunction associated with malfunction of the protein homeostasis network including accumulation of oxidatively damaged proteins and / or misfolded proteins.

53. The method of claim 52, wherein the lysosomal dysfunction-related disease is selected from but not limited to Gaucher disease, Fabry disease, Niemann-Pick disease, Hunter syndrome, glycogen storage disease type II (Pompe disease), Tay-Sachs disease.

54. The method of claims 1-10, wherein the use of the NMDAR antagonist provides a composition and method for preventing and / or treating a disease associated with certain dysfunctional mitochondrial diseases in a patient, wherein the mitochondrial disease is directly or indirectly associated with a malfunction of the protein homeostasis network, including accumulation of oxidatively damaged proteins and / or misfolded proteins.

55. The method of claim 54, wherein the disease associated with mitochondrial dysfunction is selected from, but not limited to, mitochondrial myopathy, diabetes and deafness, Leber hereditary optic neuropathy, Leigh's syndrome, subacute sclerosing encephalopathy, neuropathy, ataxia, retinitis pigmentosa, ptosis, progressive symptoms of dementia, myoneurogenic gastrointestinal encephalopathy, MERRF syndrome (myoclonic epilepsy with ragged red fibers), MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes), mitochondrial DNA depletion syndrome, Huntington's disease, cancer, Alzheimer's disease, Parkinson's disease, bipolar disorder, schizophrenia, aging and senescence, anxiety disorders, Alper syndrome, Lowe syndrome, Luft syndrome, Menke curly hair syndrome, Zellweger syndrome, mitochondrial myopathy, and proximal limb chondrodysplasia punctata.

56. The method of claims 1-10, wherein use of the NMDAR antagonist provides compositions and methods for preventing and / or treating diseases associated with immunoproteasome deficiency.

57. A method according to claims 1-10, wherein the use of the NMDAR antagonist reduces carbonylated and carbamylated proteins in the patient that are responsible for structural changes that lead to exposure of abnormally denatured areas on the surface of proteins, thereby generating new antigens that lead to proteinopathic inflammatory diseases, disorders and / or conditions and autoimmunity, wherein the proteinopathic inflammatory diseases, disorders and / or conditions are directly or indirectly related to malfunction of the protein homeostasis network, including the accumulation of oxidatively damaged proteins and / or misfolded proteins.

58. The method of claim 57, wherein the immunoproteasome deficiency-related disease associated with carbonylated and carbamylated proteins is selected from, but not limited to, altered cytokine patterns such as diabetes, Sjögren's syndrome, Nakajo-Nishimura syndrome (NNS), joint contractures, muscle atrophy, microcytic anemia and panniculitis-induced lipodystrophy (JMP) syndrome, Japanese autoinflammatory syndrome with lipodystrophy (JASL) and chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), now classified as a series of diseases called proteasome-associated autoinflammatory syndrome (PRAAS).

59. The method of claims 1-9, 57, 58, wherein the use of the NMDAR antagonist provides a composition and method for preventing and / or treating a patient with proteinopathic inflammatory diseases, disorders and / or conditions and autoimmune diseases, wherein the proteinopathic inflammatory diseases, disorders and / or conditions are directly or indirectly associated with a malfunction of the protein homeostasis network, including accumulation of oxidatively damaged proteins and / or misfolded proteins.

60. according to the method for claim 59, wherein said proteinopathy inflammatory disease, disorder and / or patient's condition is selected from but not limited to inflammatory pelvic disease, urethritis, skin sunburn, sinusitis, pneumonia, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis, dermatitis, gingivitis, appendicitis, pancreatitis, cholecystitis, irritable bowel syndrome, ulcerative colitis, glomerulonephritis, dermatomyositis, scleroderma, vasculitis; Allergic disease comprises asthma such as bronchial asthma, over-exposure to wind and cold, and the like. Allergic asthma, intrinsic asthma, exogenous asthma and dust asthma, especially chronic or refractory asthma (e.g., late-stage asthma, airway hyperresponsiveness) and bronchitis, chronic obstructive pulmonary disease, multiple sclerosis, rheumatoid arthritis; Gastrointestinal disorders, including but not limited to celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, pancreatitis, Crohn's disease, ulcerative colitis, food-related allergies with effects away from the intestine, e.g., migraine, rhinitis and eczema; as well as Conditions characterized by inflammation of the nasal mucosa, including acute rhinitis, allergic rhinitis, atrophic rhinitis, and chronic rhinitis, including caseous rhinitis, hypertrophic rhinitis, purulent rhinitis, sicca rhinitis, and rhinitis medicamentosa; membranous rhinitis, including croupous rhinitis, fibrinous rhinitis, pseudomembranous rhinitis, and adenotic rhinitis; seasonal rhinitis, including neurogenic rhinitis (hay fever) and vasomotor rhinitis, sarcoidosis, farmer's lung and related diseases, fibrotic lung and idiopathic interstitial pneumonia, acute pancreatitis, chronic pancreatitis, and adult respiratory distress syndrome and / or acute inflammatory reactions (such as acute respiratory distress syndrome and ischemia / reperfusion injury).

61. The method of claim 60, wherein the other autoimmune-related diseases, disorders and / or conditions include rejection after transplantation of synthetic or organic transplant materials, cells, organs or tissues to replace all or part of the function of tissues, such as heart, kidney, liver, bone marrow, skin, cornea, blood vessels, lung, pancreas, intestine, limb, muscle, neural tissue, duodenum, small intestine, pancreatic islet cells, including xenografts, etc.; treatment of graft-versus-host disease, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, thyroiditis, Hashimoto's thyroiditis, etc. Adenitis, multiple sclerosis, myasthenia gravis, type I diabetic uveitis, juvenile onset or recent onset diabetes, uveitis, Graves' disease, psoriasis, atopic dermatitis, autoantibody-mediated diseases, aplastic anemia, Evans syndrome, autoimmune hemolytic anemia, etc.; and further treatment of infectious diseases that cause abnormal immune responses and / or activation, such as traumatic or pathogen-induced immune disorders, including, for example, immune disorders caused by hepatitis B infection and hepatitis C infection, HIV, Staphylococcus aureus infection, viral encephalitis, sepsis, parasitic diseases (e.g., leprosy) in which the damage is induced by an inflammatory response. Other immune-mediated responses and diseases involve graft-versus-host disease (especially allogeneic graft-versus-host disease), rheumatoid arthritis, systemic lupus erythematosus, psoriasis, atopic dermatitis, Crohn's disease, ulcerative colitis, and / or multiple sclerosis.

62. The method of claims 1-10, wherein use of the NMDAR antagonist demonstrates that increased proteasome activity in the presence of the NMDAR blocker reduces accumulation of pTau protein in cells causing tauopathy-related diseases, including neuronal cells and / or non-neuronal cells.

63. The method of claim 62, wherein the neurodegenerative tauopathies include sporadic and inherited diseases characterized by filamentous tau deposition in the brain and spinal cord, such as amyotrophic lateral sclerosis (ALS), Parkinson's syndrome, argyrophilic grain dementia, diffuse neurofibrillary tangles with calcifications, frontotemporal dementia associated with chromosome 17, corticobasal degeneration, Pick's disease, progressive supranuclear palsy, progressive subcortical gliosis and tangle dementia, argyrophilic grain dementia, pugilistic dementia, diffuse neurofibrillary tangles with calcifications, Down's syndrome, frontotemporal dementia associated with chromosome 17, and Parkinson's syndrome. syndrome (FTDP-17), Giestmann-Strauss disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob encephalopathy (CJD), multiple system atrophy, Niemann-Pick disease (NPC), Pick's disease, prion protein cerebral amyloid angiopathy, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-predominant Alzheimer's disease, corticobasal degeneration (CBD), myotonic dystrophy, non-guanamian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, and tangle dementia.

64. The method of claim 62, wherein the non-neuronal tauopathy comprises diseases associated with disrupted mitochondrial dynamics and impaired mitophagy and autophagy resulting from the appearance of pTau and other cleavage products.

65. The method of claim 64, wherein the disease associated with non-neuronal tauopathy comprises disrupted mitochondrial dynamics and impaired mitophagy as described in claims 53 and 54.

66. The method of claims 1-10, wherein use of the NMDAR antagonist demonstrates that increased proteasome activity in the presence of the NMDAR blocker induces actin turnover that prevents neurodegenerative diseases by enhancing axonal regeneration.

67. The method of claim 66, wherein the use of the NMDAR antagonist provides compositions and methods by increasing axonal regeneration, which prevent and / or treat neurodegeneration caused by pathological injuries including anti-cancer therapy, cerebral ischemia, traumatic brain injury, peripheral neuropathy, cerebral ischemia, choroidal neovascularization, retinal ganglion cell loss, long-term sequelae of concussion, reduction of the neurotoxic effects of astrocytes in Alzheimer's disease, epileptic seizures, and the like.

68. The method of claim 34, wherein the NMDAR blocker reduces serine racemase, thereby reducing the conversion of L-serine to D-serine that plays a role in colorectal cancer by reducing serine racemase, which enhances the growth of the colorectal cancer by generating pyruvate from serine.

69. The method of claims 1-5, wherein the use of the NMDAR antagonist, particularly ketamine, provides compositions and methods that are effective in protecting, alleviating or treating depression, acute psychosis, anxiety, insomnia, psychosis and migraine, especially in situations where an urgent and rapid effect is desired.

70. The method of claims 1-5, wherein the use of the NMDAR antagonist provides compositions and methods for effectively protecting, alleviating or treating diseases in which the proteasome activity is reduced and which are defective, including viral infection, delayed recovery, and autoimmunity following infection.

71. The method of claim 70, wherein the viral disease is selected from but not limited to HCV, HIV and other viral infections.

72. The method of claims 1-9, wherein the use of the NMDAR antagonist provides compositions and methods that effectively maintain and increase stemness and lifespan characteristics, namely the potential for stem cell self-renewal, multi-lineage differentiation, and lineage reprogramming.

73. The method of claim 72, wherein the use of the NMDAR antagonist provides compositions and methods that maintain and increase stemness characteristics and prevent loss of muscle mass and strength, irreversible muscle loss, and muscle diseases that severely affect the quality of life of the elderly by increasing proteasome activity.

74. The method of claim 73, wherein the effects of the present invention are also not limited to muscle tissue, the present invention also provides compositions and methods for maintaining and increasing stemness characteristics and preventing aging and related diseases by ensuring the continuation of the regenerative properties of all tissues including neurons.

75. The method of claims 1-11, 15, 16, wherein the use of the NMDAR antagonist provides a composition and method for effectively protecting, alleviating or treating the cognitive impairment and dementia in normal aging and cognitive impairment and dementia caused by any cause.

76. The method of claims 1-11, 34, 48, 50, 54, 57, 62, 63, 66, 67, 72, 73 and 74, wherein use of the NMDAR antagonist provides compositions and methods for neuroprotection in neurological diseases.

77. The method of claims 1-11, wherein the use of the NMDAR antagonist provides compositions and methods for preventing and / or treating acute and chronic renal insufficiency caused by aging, hyperlipidemia, hypertension, smoking, diabetes, obesity and other factors that lead to oxidative damage to proteins.

78. The method of claims 1-11, wherein use of the NMDAR antagonist provides compositions and methods for increasing telomerase activity due to increased proteasome activity.

79. The method of claim 78, wherein the increased telomerase activity due to increased proteasome activity increases lifespan.

80. According to the method of claim 79, the present invention further provides the following NMDA receptor antagonist, which delays cell senescence by increasing telomerase activity, thereby extending lifespan.

81. The method of claims 1-11, wherein the use of the NMDAR antagonist provides a composition and method that increases proteasome activity and causes a decrease in p21, p53, pAkt1, and ppRb, which are the main target molecules for senescent cell-eliminating drugs used for the ablation of senescent cells.

82. The method of claim 81, wherein the NMDAR antagonist provides compositions and methods for ablation of senescent cells, which has been postulated to be a promising therapeutic approach for targeting the aging phenotype, thereby preventing, delaying or ameliorating aging-related diseases.

83. The method of claims 81 and 82, wherein the NMDAR antagonist provides compositions and methods for ablation of senescent cells for treating aging, which is caused by cellular senescence and is associated with an increased risk of developing multiple chronic diseases, geriatric syndromes, impaired physical resilience, and mortality.

84. The method of claims 81, 82, and 83, wherein the aging and related diseases (benefits of senescent cell removal drugs) include but are not limited to diabetes / obesity, metabolic diseases, cardiac dysfunction, congestive heart failure, myocardial infarction, vascular hyporesponsiveness / calcification, AV fistula, frailty, age-related muscle loss (sarcopenia), arthritis, osteoporosis, falls, chemotherapy complications, radiation complications, cancer, bone marrow transplant complications, organ transplant complications, myeloma / MGUS (monoclonal gammopathy of undetermined significance), age-related Related cognitive impairment, other dementias, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia, obesity-related neuropsychiatric dysfunction, renal dysfunction, urinary incontinence, osteoporosis, osteoarthritis, age-related disc disease, idiopathic pulmonary fibrosis, hyperoxia-induced lung injury, chronic obstructive pulmonary disease, tobacco, hepatic steatosis, cirrhosis, primary biliary cirrhosis, progeria, preeclampsia, macular degeneration, glaucoma, cataracts, blindness, prostatic hypertrophy, incontinence, psoriasis, healthy life expectancy, life expectancy and many other diseases.

85. The method according to claims 1-84, wherein the present invention provides a drug that improves the onset and / or progression of aging and aging-related diseases or even treats aging and aging-related diseases by targeting more than one "basic aging process", wherein the "basic aging process" includes: 1) macromolecular dysfunction (e.g., reduced proteasome activity, protein misfolding and aggregation, telomere uncapping, increased advanced oxidation-glycation end products, lipotoxicity, DNA damage, and accumulation of bioactive lipids) and organelle dysfunction (nuclear membrane alterations associated with lamin B deficiency, mitochondrial dysfunction leading to reduced fatty acid metabolism, increased glucose utilization, depletion of NAD+, and increased ROS generation, etc.), 2) stem cell, progenitor cell, and immune cell dysfunction (including altered proliferation capacity and differentiation disorders, failure to develop into functional mature cells, and decreased "anti-aging" factors [e.g., α-Klotho], leading to stem cell and progenitor cell dysfunction), 3) cellular senescence, and the associated 4) chronic low-grade "sterile" (no bacteria, fungi, etc.) inflammation.

86. A disease monism of basic aging processes, wherein the unified theory of basic aging processes assumes that targeting any one basic aging process (as described above) by gene or drug will affect many or possibly all of the remaining processes, and therefore, according to the method of claims 1-85, the present invention provides a drug (NMDAR blocker) for improving the onset and / or progression of aging and aging-related diseases or even treating aging and aging-related diseases, wherein the drug targets more than one basic aging process (as described above).

87. The method of any one of claims 1-86, wherein the NMDAR blocker is selected from the group consisting of memantine, nitromemantine, neramexane, ketamine, amantadine, dextromethorphan, L-687,384, amitriptyline, 1-benzyl-6-methoxy-6,7'-dihydrospiro[piperidin-4,4'-thieno[3.2-c]pyran], ifenprodil, orphenadrine, kynurenic acid, felbamate, D(-)-AP-5, (+)-CPP, EAA-090, TCN-201, AP-5, AZD6765, SDZ 220-581, (+)-norketamine, eliprodil, dextrorphan, 5,7-dichlorokynurenic acid monohydrate, [G1u3,4,7,10,14]-cono sleep peptide G, D-AP-7, MD-Ada, AP-7, Ro 8-4304, spermine, Ro25-6981, DCOX, trexolotide, MDL105,519, fanapanel, metaphit, Ro25-6981, NAAG, 5-fluoroindole-2-carboxylic acid, (S)-(-)-4-oxo-2-azetidinecarboxylic acid, (S) (-)-4-oxo-2-azetidinecarboxylic acid benzyl ester and (+)-a-amino-3-methoxycarbonyl-5-methylisoxazole-4-propionic acid and their pharmaceutically acceptable salts, hydrates and pharmaceutically active enantiomers, each possibility representing a separate embodiment of the present invention.

88. according to the method described in any one of claims 1-86, wherein the NMDAR blocker is selected from the compound that antagonizes or inhibits the effect of NMDA receptor, and the NMDA receptor antagonist can be a competitive antagonist, an uncompetitive antagonist, a non-competitive antagonist and / or a glycine antagonist.

89. A method according to claims 1-86, wherein the NMDAR blocker can be or include a compound of any chemical class (e.g., small molecules, metals, nucleic acids, polypeptides, lipids and / or carbohydrates), an antibody or antibody mimetic, a nucleic acid agent (e.g., antisense oligonucleotides, siRNA, shRNA, etc.) or its mimetic, a naturally occurring compound (e.g., small molecules), an artificially produced and / or modified chemical structure.

90. A method according to claims 1-86, wherein the NMDAR blocker can be or include a compound that increases the level or activity of one or more target entities present in and / or produced by a cell or organism, wherein the target is the trypsin-like activity, chymotrypsin-like activity, and caspase-like activity of the proteasome, but the target entity can also be or include a polypeptide, a nucleic acid (e.g., a nucleic acid encoding or regulating [e.g., by altering the expression and / or activity of the polypeptide] a polypeptide), a carbohydrate, a lipid, an enzyme, or a polypeptide involved in cellular trafficking.

91. The method of claims 1-86, wherein the NMDAR blocker can be or include a compound, non-limiting examples of antagonists that can be used according to some embodiments of the present invention include small molecules, antibodies, inhibitory peptides, enzymes that cleave polypeptides, aptamer homologous recombination agents, site-specific endonucleases, and RNA silencing agents.

92. The method of claim 1-86, wherein the use of the NMDAR antagonist provides compositions and methods, wherein the therapeutically effective dose of the NMDAR blocker can be adjusted according to the condition of the disease / disorder to be treated or prophylactically treated, the age and associated symptoms, body weight, general health, sex and diet of the subject, dosing interval, route of administration, excretion rate, and combination of drugs.

93. The method of claim 91, 92, wherein the therapeutically effective dose of the NMDAR blocker and the effective dose of all possible unrestricted compounds used as NMDAR blockers or inhibitors can be adjusted according to the condition of the disease / disorder to be treated or preventively treated, the age and related symptoms, body weight, general health, sex and diet of the subject, dosing interval, route of administration, excretion rate and combination of drugs.

94. The method of claims 1-86, wherein the memantine is administered at a dose of about 5 mg / day to about 100 mg / day.

95. The method of claims 1-86, wherein the dosage is about 10 mg / day to about 40 mg / day.

96. The method of claims 1-86, wherein the NMDAR blocker is administered to the subject for life.

97. The method of claims 1-86, wherein the NMDAR blocker is administered to the subject such that pharmacological improvement of protein homeostasis is necessary at an early stage of the disease before severe cellular dysfunction manifests.

98. according to the method described in claim 1-86, wherein the NMDAR blocker is administered to the subject, and the subject can be a mammal, and in a specific embodiment is a human, can be an infant, a child, an adult or an elderly person, in addition, the terms "subject" and "individual" are used interchangeably and refer to mammals, such as mammals in the context of the present invention are humans, non-human primates, domestic animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc.

99. The method of claim 1-86, wherein the NMDAR blocker is administered to the subject by a suitable route, which may, for example, include oral delivery, rectal delivery, transmucosal delivery, especially nasal delivery, intestinal delivery or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections, as well as intrathecal injections, direct intraventricular injections, intracardiac injections, such as into the right or left ventricle, into the common coronary artery, intravenous injections, intraperitoneal injections, intranasal injections or intraocular injections.

100. The method of claims 1-86, wherein the NMDAR antagonist and / or the agent of some embodiments of the present invention can be administered to an organism by itself or in the form of a pharmaceutical composition mixed with a suitable carrier or excipient.

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