Novel Uses of Dextran Sulfate

Abstract

To provide a drug useful for patients suffering from neurological and / or fibrotic conditions. [Solution] Provided is a pharmaceutical composition for the treatment of glutamate excitotoxicity, comprising dextran sulfate or a pharmaceutically acceptable salt thereof, wherein the dextran sulfate or a pharmaceutically acceptable derivative thereof has a number-average molecular weight (Mn) in the range of 1,850 to 3,500 Da as measured by nuclear magnetic resonance (NMR) spectroscopy.

Description

[Technical Field]

[0001] The present embodiments relate generally to neurological and fibrotic conditions, and in particular to the use of dextran sulfate in combating such conditions. [Background technology]

[0002] In neurological diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), as well as injuries to the central nervous system (CNS) or peripheral nervous system (PNS) such as traumatic brain injury (TBI), stroke, and subarachnoid hemorrhage (SAH), loss of differentiation of neurons and glial cells, such as oligodendrocytes and Schwann cells, is one of the first disease stages, followed by cell death. Cellular function is similarly impaired, as evidenced by decreased metabolic function and mitochondrial energy metabolism and elevated oxygen stress. Damaged neurons further release glutamate, which has an excitotoxic effect on nearby neurons, in turn causing further cell damage and death.

[0003] Thus, there are numerous deleterious mechanisms that occur in neurological diseases, disorders and conditions, and therefore there is a general need for drugs that are effective in combating such deleterious mechanisms and thereby can be beneficial to patients suffering from such neurological diseases, disorders and conditions.

[0004] US Patent Application Publication No. 2011 / 0014701 relates to the use of polysulfated polysaccharides to improve the viability of progenitor cells. This US patent application also discloses the use of polysulfated polysaccharides to regulate the differentiation of progenitor cells. Various polysulfated polysaccharides were tested. The polysulfated polysaccharides, dextran polysulfate (M w = 5,000 Da) down-regulates or inhibits differentiation of progenitor cells. Summary of the Invention

[0005] It is a general objective to provide useful drugs to patients suffering from neurological and / or fibrotic conditions.

[0006] This and other objects are achieved by the embodiments defined herein.

[0007] The invention is defined by the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0008] The present embodiments relate to dextran sulfate, or a pharmaceutically acceptable derivative thereof, which has several beneficial effects on patients suffering from neurological and / or fibrotic diseases, disorders or conditions.

[0009] Dextran sulfate or its pharmaceutically acceptable derivatives can, among other things, induce differentiation of glial cells and neurons, reduce oxidative stress in neurons and glial cells, reduce glutamate excitotoxicity, improve mitochondrial metabolic function and energy metabolism in neurons and glial cells, and activate the body's endogenous repair mechanisms.Dextran sulfate or its pharmaceutically acceptable derivatives can also prevent fibrogenesis by suppressing fibrogenic factors such as TGF-β and activating fibrolysis, thereby degrading existing scar tissue and inducing tissue remodeling and tissue healing.Dextran sulfate or its pharmaceutically acceptable derivatives can also be effective in various inflammatory and autoimmune conditions, including neuroinflammatory conditions, by resolving immune or inflammatory responses.

[0010] Some embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0011] [Figure 1]Figure 1 shows the propidium iodide (PI) content of mouse cortical neurons. Cells were stained with PI, which binds to DNA. Based on DNA content, cells can be classified into different phases of the cell cycle. Because DNA content changes during the cell cycle, PI staining can indicate cell cycle progression. The data showed that most cells remained in the G1 phase of the cell cycle (dotted arrow), but low molecular weight dextran sulfate (LMW-DS) appeared to increase the number of cells in the G2 / M phase (solid arrow). [Figure 2] Figure 2 shows the PI content of human motor neurons. The data showed that most cells remained in the G1 phase of the cell cycle (dotted arrows), but LMW-DS appeared to increase the number of cells in the G2 / M phase (solid arrows). [Figure 3] Figure 3 shows the PI content of human Schwann cells. The data showed that most cells remained in the G1 phase of the cell cycle (dotted arrows), but LMW-DS appeared to increase the number of cells in the G2 / M phase (solid arrows). [Figure 4] FIG. 4 shows representative photographs of βIII-tubulin expression in mouse cortical neurons. [Figure 5A] Figures 5A and 5B show the effect of LMW-DS on βIII-tubulin expression in mouse cortical neurons. Graphs show the overall intensity (Figure 5A) and average size (Figure 5B) of positive cells. [Figure 5B] Figures 5A and 5B show the effect of LMW-DS on βIII-tubulin expression in mouse cortical neurons. Graphs show the overall intensity (Figure 5A) and average size (Figure 5B) of positive cells. [Figure 6A] 6A and 6B show the effect of LMW-DS on βIII-tubulin expression in human motor neurons. The graphs show the overall intensity (FIG. 6A) and average size (FIG. 6B) of positive cells. [Figure 6B] 6A and 6B show the effect of LMW-DS on βIII-tubulin expression in human motor neurons. The graphs show the overall intensity (FIG. 6A) and average size (FIG. 6B) of positive cells. [Figure 7] FIG. 7 is a representative photograph of βIII-tubulin expression in human motor neurons. [Figure 8A] 8A and 8B show the effect of LMW-DS on myelin basic protein (MBP) expression in human Schwann cells. The graphs show the overall intensity (FIG. 8A) and average size (FIG. 8B) of positive cells. [Figure 8B] 8A and 8B show the effect of LMW-DS on myelin basic protein (MBP) expression in human Schwann cells. The graphs show the overall intensity (FIG. 8A) and average size (FIG. 8B) of positive cells. [Figure 9] FIG. 9 is a representative photograph of MBP expression in human Schwann cells. [Figure 10] FIG. 10 shows the mean experimental autoimmune encephalomyelitis (EAE) severity scores after EAE induction in mice for the negative control (vehicle), the positive control cyclosporin A (cyclo), and LMW-DS. [Figure 11] 11 shows the mean EAE severity scores after EAE induction in mice for negative control (vehicle) and HGF. Arrows indicate the start of treatment. [Figure 12] FIG. 12 shows changes in brain glutamate levels. [Figure 13A] 13A to 13D are diagrams showing the change in the levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a function of mitochondrial phosphorylation capacity. [Figure 13B] 13A to 13D are diagrams showing the change in the levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a function of mitochondrial phosphorylation capacity. [Figure 13C] 13A to 13D are diagrams showing the change in the levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a function of mitochondrial phosphorylation capacity. [Figure 13D]13A to 13D are diagrams showing the change in the levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a function of mitochondrial phosphorylation capacity. [Figure 14A] 14A-14D show the change in levels of oxidized and reduced nicotine coenzyme. [Figure 14B] 14A-14D show the change in levels of oxidized and reduced nicotine coenzyme. [Figure 14C] 14A-14D show the change in levels of oxidized and reduced nicotine coenzyme. [Figure 14D] 14A-14D show the change in levels of oxidized and reduced nicotine coenzyme. [Figure 15A] 15A to 15C are diagrams showing the change in the levels of biomarkers indicative of oxidative stress. [Figure 15B] 15A to 15C are diagrams showing the change in the levels of biomarkers indicative of oxidative stress. [Figure 15C] 15A to 15C are diagrams showing the change in the levels of biomarkers indicative of oxidative stress. [Figure 16] FIG. 16 shows altered levels of nitrate as a measure of NO-mediated nitrosative stress. [Figure 17A] 17A-17C show the changing levels of N-acetylaspartate (NAA) and its substrates. [Figure 17B] 17A-17C show the changing levels of N-acetylaspartate (NAA) and its substrates. [Figure 17C] 17A-17C show the changing levels of N-acetylaspartate (NAA) and its substrates. [Figure 18] FIG. 18 shows a schematic representation of the effect of oxidative stress on mitochondrial dysfunction. [Figure 19] FIG. 19 shows a schematic representation of molecules involved in the glutamate signaling pathway. [Figure 20]FIG. 20 shows the change in laminin immunoreactivity in the angle of subjects with primary open-angle glaucoma (POAG) treated with saline control or LMW-DS. [Figure 21] FIG. 21 shows changes in fibronectin immunoreactivity in the angle of subjects with POAG treated with saline control or LMW-DS. [Figure 22] Figure 22 shows amyloid beta monomer and oligomer preparations. Preparations of oligomers (lanes 1, 2, 5-7) or monomers (lanes 3 and 4) of amyloid beta (1-42)A or amyloid beta biotin (B). 50 pmoles (lane 5), 100 pmoles (lanes 1, 3, and 6), or 200 pmoles (lanes 2, 4, and 7) of each peptide preparation were loaded onto the gel. Proteins from the ongoing Western blot were immunolabeled with anti-amyloid beta. Expected oligomers and molecular weight markers are shown. [Figure 23] FIG. 23 shows dextran sulfate sodium salt (DSSS) and LMW-DS competition for protein-protein interaction between amyloid-β and PrP C . [Figure 24] Figure 24 shows the concentration of NAA measured in deproteinized brain homogenates of rats sacrificed 2 days after TBI with or without a single administration of increasing doses of LWM-DS (1, 5, and 15 mg / kg body weight) administered 30 minutes after trauma induction. Controls are sham-operated animals. Values ​​are the means of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from 2 days after sTBI, p<0.01. [Figure 25] Figure 25 shows the concentration of ATP measured in deproteinized brain homogenates of rats sacrificed 7 days after sTBI with or without administration of increasing doses of LWM-DS (single doses of 1, 5, and 15 mg / kg body weight and repeated doses of 15 mg / kg body weight). Controls are sham-operated animals. Values ​​are the mean of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from 2 days after sTBI, p<0.01. [Figure 26] Figure 26 shows the concentration of ascorbic acid measured in deproteinized brain homogenates of rats sacrificed 7 days after sTBI with or without administration of increasing doses of LWM-DS (single doses of 1, 5, and 15 mg / kg body weight and repeated doses of 15 mg / kg body weight). Controls were sham-operated animals. Values ​​are the mean of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from 2 days after sTBI, p<0.01. [Figure 27] Figure 27 shows glutathione (GSH) concentrations measured in deproteinized brain homogenates of rats sacrificed 7 days after sTBI with or without administration of increasing doses of LWM-DS (single doses of 1, 5, and 15 mg / kg body weight and repeated doses of 15 mg / kg body weight). Controls were sham-operated animals. Values ​​are the means of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from 2 days after sTBI, p<0.01. [Figure 28] Figure 28 shows the concentration of NAA measured in deproteinized brain homogenates of rats sacrificed 7 days after sTBI with or without administration of increasing doses of LWM-DS (single doses of 1, 5, and 15 mg / kg body weight and repeated doses of 15 mg / kg body weight). Controls were sham-operated animals. Values ​​are the means of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from 2 days after sTBI, p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present embodiments relate generally to neurological and fibrotic conditions, and in particular to the use of dextran sulfate in combating such conditions.

[0013] Neuropathy is any disorder of the body's nervous system, i.e., the brain, spine, and nerves that connect them. Structural, biochemical, or electrical abnormalities in the brain, spinal cord, or other nerves can result in a range of symptoms. Although the brain and spinal cord are surrounded by tough membranes, confined within the bones of the skull and vertebrae, and chemically isolated by the blood-brain barrier, they are highly susceptible to compromise. Although nerves are often thought of as located deep beneath the skin, they can still be exposed to injury. Individual neurons, as well as the neural networks and nerves they form, are susceptible to electrochemical and structural destruction. While nerve regeneration can occur in the peripheral nervous system, thus overcoming or functioning around injury to some extent, it is thought to be rare in the brain and spinal cord.

[0014] The specific causes of neurological problems vary but can include genetic disorders, congenital abnormalities or disorders, infections, lifestyle habits, environmental health issues including nutritional deficiencies, and brain, spinal cord, or nerve injuries. Problems can also begin in other body systems that interact with the nervous system. For example, cerebrovascular disorders involve brain damage due to problems with the blood vessels, i.e., the cardiovascular system, that supply the brain; autoimmune disorders involve damage caused by the body's own immune system; and lysosomal storage disorders, such as Niemann-Pick disease, can lead to neurological exacerbations.

[0015] A neurodegenerative disease, disorder or condition is a disease, disorder or condition that causes progressive loss of structure and / or function of neurons, including death of neurons.

[0016] Non-limiting examples of such neurodegenerative diseases, disorders, or conditions include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS).

[0017] AD is characterized by the loss of neurons and synapses in the cerebral cortex and subcortical regions. Classic neuropathological findings of AD include amyloid plaques, neurofibrillary tangles, and synaptic and neuronal cell death. White matter disease (WMD) is frequently observed in AD on neuropathological examination. It is defined as near-total tissue loss with loss of myelin, axons, and oligodendrocytes, and an increase in astrocytes.

[0018] PD is a neurodegenerative disease of the CNS. The motor symptoms of PD result from the death of dopamine-producing cells in the substantia nigra. In affected nerves, the myelin sheath around axons begins to erode. Neuroinflammation is a pathological hallmark of PD and is characterized by activated microglia and infiltrating T cells at the site of neuronal damage.

[0019] HD is a neurodegenerative disease that affects muscle coordination and leads to cognitive decline and psychiatric problems. The disease is caused by an autosomal dominant mutation in a gene called huntingtin. Part of this gene is a repeating segment called a trinucleotide repeat, which varies in length between individuals. When the length of this repeat reaches a certain threshold, it produces an altered form of the protein. The protein encoded by the huntingtin gene (Htt) interacts with over 100 other proteins and has multiple biological functions. Mutant forms of Htt are particularly toxic to certain cell types in the brain. HD is characterized by damage to the myelin sheath on nerves. Increased activated T cells in the peripheral blood have been identified in HD patients.

[0020] ALS, also known as Lou Gehrig's disease, is a debilitating disease with diverse etiologies characterized by rapidly progressive weakness, muscle atrophy and fasciculation, muscle spasticity, dysarthria, dysphagia, and respiratory distress. ALS is the most common motor neuron disease (ALS, hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), and pseudobulbar palsy). The cardinal feature of ALS pathology is the loss of motor neurons in the anterior horn of the spinal cord and motor nuclei of the brainstem. This results in secondary atrophy of the corresponding muscles (muscle atrophy). Neuroinflammation is a pathological hallmark of ALS and is characterized by activated microglia and infiltrating T cells at sites of neuronal damage. "Lateral sclerosis" refers to corticospinal tract degeneration (the lateral part of the spinal cord). Myelin loss actually occurs in the corticospinal tract. The sclerosis in ALS involves the lateral columns, or corticospinal tracts, and is a secondary phenomenon.

[0021] The neurological disease, disorder, or condition may be a demyelinating disease, disorder, or condition. A demyelinating disease, disorder, or condition is a disease of the nervous system in which the myelin sheath of neurons is damaged. Such damage reduces signal conduction in the affected nerves, thereby resulting in sensory, motor, cognitive, and other functional deficits, depending on the nerve involved in the damage.

[0022] Non-limiting examples of such demyelinating diseases, disorders, or conditions include multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), central nervous system (CNS) neuropathy, central pontine myelinolysis (CPM), myelopathy, leukoencephalopathy, and leukodystrophies (all affecting the CNS), as well as Guillain-Barré syndrome (GBS), peripheral neuropathy, and Charcot-Marie-Tooth (CMT) disease (all affecting the peripheral nervous system (PNS)).

[0023] MS is an inflammatory disease that damages the fatty myelin sheath surrounding axons in the brain and spinal cord, leading to demyelination and scarring and a wide range of signs and symptoms. MS involves T cells that direct an immune response against the white matter of the brain and spinal cord. MS is a disease of myelin, not primarily a disease of nerve cells. Because myelin occurs throughout the nervous system, lesions can and usually occur in multiple locations. However, the disease only affects central nervous system myelin, not peripheral nerve myelin. Therefore, the condition is a CNS-specific disorder.

[0024] ADEM is an immune-mediated disease of the brain. It can occur after viral, bacterial, or parasitic infection or occur spontaneously. ADEM attacks nerves in the CNS, damaging their myelin insulation, which ultimately destroys white matter. It is similar to MS because it involves autoimmune demyelination and is considered part of the MS spectrum. ADEM produces multiple inflammatory lesions in the brain and spinal cord, particularly in the white matter. ADEM requires cytokines secreted by myelin-reactive T cells.

[0025] Neuropathies, including CNS neuropathy and peripheral neuropathy, are damage to or diseases affecting a group of affected nerves. Depending on the type of nerve affected, they can impair sensation, movement, glandular or organ function, or other health conditions. Common causes include systemic diseases such as diabetes or leprosy; vitamin deficiencies; medications, such as chemotherapy agents and commonly prescribed antibiotics; trauma; ischemia; radiation therapy; excessive alcohol use; immune system disorders; celiac disease; or viral infections. Neuropathies can be acute. Acute neuropathy requires urgent diagnosis. Motor nerves, which control muscles, sensory nerves, or autonomic nerves, which control autonomic functions such as heart rate, body temperature, and breathing, can be affected. Two or more nerves can be affected simultaneously.

[0026] CPM is a neurological disorder caused by severe damage to the myelin sheath of nerve cells in the brainstem, more precisely in the area called the pons, primarily of iatrogenic etiology. It is characterized by acute paralysis, dysphagia and dysarthria, and other neurological symptoms.

[0027] Myelopathy refers to any neurological disorder associated with the spinal cord. When due to trauma, it is commonly known as spinal cord injury (SCI); when inflammatory, it is commonly known as myelitis; and when vascular in nature, it is known as vascular myelopathy. The most common form of myelopathy in humans, cervical spondylotic myelopathy (CSM), is caused by arthritic changes of the cervical spine (spondylosis), which leads to narrowing of the spinal canal (spinal stenosis) and ultimately compression of the spinal cord.

[0028] Leukoencephalopathy is a broad term for leukodystrophy-like disorders. It applies to all cerebral white matter disorders, regardless of whether the molecular cause is known. Specific examples of leukoencephalopathy include progressive multifocal leukoencephalopathy, toxic leukoencephalopathy, vanishing white matter disease, leukoencephalopathy with neuroaxonal spheroids, reversible posterior leukoencephalopathy syndrome, and macrocephalic leukodystrophy with subcortical cysts.

[0029] Leukodystrophies are a group of disorders characterized by degeneration of white matter in the brain. Leukodystrophies are caused by incomplete growth or development of the myelin sheath, a fatty covering that acts as insulation around nerve fibers. When damage occurs to the white matter, an immune response can lead to inflammation in the CNS, along with loss of myelin. Leukodystrophies are characterized by specific symptoms, including decreased motor function, muscle stiffness, and ultimately vision and hearing loss. Specific types of leukodystrophies include adrenomyeloneuropathy, Alexander disease, cerebrotendinous xanthomatosis, hereditary CNS demyelinating disorders, Krabbe disease, metachromatic leukodystrophy, Pelizaeus-Merzbach disease, Canavan disease, vanishing white matter disease, adrenoleukodystrophy, and Refsum disease.

[0030] GBS, also known as Landry's palsy or Guillan-Barré-Strohl syndrome, is an acute polyneuropathy affecting the PNS. In GBS, immune cells attack the myelin sheath (the fatty substance that covers nerve fibers). Ascending paralysis is a common symptom. GBS is thought to be an immune-mediated disease involving an abnormal T-cell response triggered by infection. Cellular and humoral immune mechanisms are likely involved in its development. Most patients report an infectious illness several weeks before the onset of GBS. Many identified infectious agents induce the production of antibodies that cross-react with specific gangliosides and glycolipids, such as GM1 and GD1b, which are thought to be distributed throughout the myelin of the peripheral nervous system.

[0031] CMT is one of a group of diverse hereditary motor and sensory neuropathies, inherited disorders of the peripheral nervous system characterized by progressive loss of muscle tissue and touch throughout various parts of the body. CMT was previously classified as a subtype of muscular dystrophy.

[0032] In neurological disorders, the loss of differentiated neurons and glial cells, such as oligodendrocytes and Schwann cells, is one of the first steps in disease progression, and the disorder is generally followed by the cell death of such neurons and glial cells.

[0033] Therefore, drugs that can promote the differentiation of neurons and glial cells would be beneficial to patients suffering from neurological diseases, disorders, or conditions. Such differentiation-inducing drugs may be neuroprotective and, for example, may be useful in the treatment of neurological diseases, disorders, or conditions.

[0034] The experimental data presented herein demonstrate that dextran sulfate of the present invention can induce the differentiation of neurons and glial cells. This effect is observed on both cortical neurons and motor neurons, and on neurons of both mouse and human origin. Correspondingly, dextran sulfate can induce the differentiation of Schwann cells, which constitute a type of glial cell.

[0035] Dextran sulfate of embodiments further demonstrated favorable effects in an in vivo model of inflammatory demyelinating disease of the CNS, which is currently the most widely accepted animal model of MS and ADEM.

[0036] These results regarding the induction of neuronal and glial cell differentiation by dextran sulfate of the embodiment are consistent with those of dextran sulfate (M w This was highly surprising in light of U.S. Patent Application Publication No. 2011 / 0014701, which states that dextran sulfate (=5,000 Da) does not induce differentiation of progenitor cells, but rather downregulates or suppresses them. Thus, it appears that the cell differentiation ability of the embodiments of dextran sulfate may be cell type specific, potentially limited to neurons and glial cells. Prior art data indicates that dextran sulfate may in fact have the opposite effect on other cell types as demonstrated by progenitor cells in the aforementioned U.S. patent application.

[0037] Neurons, also called nerve cells, are electrically excitable cells that process and transmit information through electrical and chemical signals. These signals between neurons occur via specialized connections with other cells at synapses. Neurons can connect with each other to form neural networks. Neurons are core components of the brain and spinal cord in the CNS and of the ganglia in the PNS. Specialized types of neurons include sensory neurons, which respond to touch, sound, light, and all other stimuli that affect cells in the sensory organs, which then send signals to the spinal cord and brain; motor neurons, which receive signals from the brain and spinal cord and produce muscle contractions and affect glandular output; and interneurons, which connect neurons to other neurons within the same brain or spinal cord region in neural networks.

[0038] A typical neuron consists of a cell body (neuronal cell body), dendrites, and an axon. The term neurite is used to describe either the dendrite or axon, specifically in their undifferentiated stages. Dendrites are thin structures arising from the cell body, often extending hundreds of micrometers and branching multiple times to give rise to a complex "dendritic tree." Axons, also called nerve fibers when myelinated, are specialized cell extensions that arise from the cell body at sites called axon hillocks and extend for some distance. Nerve fibers are often bundled into fascicles; in the PNS, bundles of fibers constitute nerves. At most synapses, signals are sent from the axon of one neuron to the dendrites of another.

[0039] Neurons do not undergo cell division. In most cases, neurons are generated by specialized types of stem cells. Astrocytes are astroglial cells that have also been observed to transform into neurons due to the pluripotency characteristic of stem cells. In humans, neurogenesis is largely halted during adulthood, but there is strong evidence of the generation of substantial numbers of new neurons in two brain regions: the hippocampus and the olfactory bulb.

[0040] Dextran sulfate in embodiments can induce increased neuronal expression in β-tubulin, particularly βIII-tubulin.

[0041] βIII-tubulin, also known as class III β-tubulin, is a microtubule component expressed exclusively in neurons. The microtubule cytoskeleton is essential for neuronal development and survival. Microtubules are constructed from tubulin heterodimers, which contain different tubulin isotypes. Microtubules are polarized; in neurons, their "minus ends" typically point toward the centrosome in the cell body, while their "plus ends" protrude toward the axon tip. Microtubule polarity plays an important function in both differentiating and adult neurons. During differentiation, tubulin increases in cells and assembles microtubules that extend or retract growing axons in response to guidance cues, allowing differentiating neurons to maintain directional growth toward postsynaptic targets. Their activity is essential for cell migration, axon development, and guidance, as well as for the function and viability of adult neurons (Bioscience Reports (2010), 30:319-330).

[0042] The increased expression of βIII-tubulin in neurons indicates that dextran sulfate of the embodiments functions as a differentiation factor for these cells.

[0043] In certain embodiments, the neuron is selected from the group consisting of a cortical neuron and a motor neuron.

[0044] Motor neurons are nerve cells whose cell bodies are located in the spinal cord and whose axons project outside the spinal cord to directly or indirectly control effector organs, primarily muscles and glands. The axons of motor neurons are efferent nerve fibers that carry effector signals from the spinal cord and produce the effect.

[0045] Motor neuron diseases (MNDs) are neurological disorders that selectively affect motor neurons. These MNDs include ALS, HSP, PLS, PMA, PBP, pseudobulbar palsy, spinal muscular atrophy (SMA), and post-polio syndrome (PPS). They are neurodegenerative in nature and cause progressive disability and ultimately death.

[0046] HSP, also known as hereditary spastic paraplegia, familial spastic paraplegia, French settlement disease, or Strampel-Lorraine disease, is a group of genetic disorders characterized primarily by progressive gait disturbance. The disease manifests as progressive stiffness (spasticity) and shortening of the legs. This symptom results from dysfunction of long axons in the spinal cord. The affected cells are primary motor neurons; therefore, the disease is an upper motor neuron disorder. HSP is caused by impaired transport of proteins, structural proteins, protein-maintaining cells, lipids, and other substances through the cell.

[0047] PLS is a rare neuromuscular disorder characterized by progressive weakness in voluntary muscles. PLS affects only upper motor neurons.

[0048] PMA, also known as Duchenne-Allan muscular atrophy, is a rare subtype of MND that affects only the lower motor neurons.

[0049] PBP is a disease that attacks the nerves of the bulbar muscles. These disorders are characterized by degeneration of motor neurons in the cerebral cortex, spinal cord, brainstem, and pyramidal tract, particularly the glossopharyngeal (IX), vagus (X), and hypoglossal (XII) nerves.

[0050] Pseudobulbar palsy is a condition characterized by the inability to control facial movements, such as chewing and speaking, and is caused by a variety of neurological disorders. Patients who experience difficulty chewing and swallowing also have increased reflexes and spasticity in the tongue and bulbar region, slurred speech, and sometimes uncontrolled emotional outbursts. This condition is usually caused by damage to, and bilateral degeneration of, neurons in the brainstem, particularly the corticobulbar tract (the upper motor neuron pathway to the cranial nerve motor nuclei).

[0051] SMA, also known as autosomal recessive proximal spinal muscular atrophy and 5q spinal muscular atrophy, is a rare neuromuscular disorder characterized by motor neuron loss and progressive muscle wasting, often leading to early death. The disorder is caused by a genetic defect in the SMN1 gene, which encodes SMN, a protein that is widely expressed in all eukaryotic cells and required for motor neuron survival. Lower levels of this protein result in loss of neuronal function in the anterior horn of the spinal cord and subsequent tissue-wide skeletal muscle atrophy.

[0052] PPS, also known as post-polio syndrome or polio sequelae, is a condition that affects approximately 25-40% of people who survive a first acute attack from a previous poliovirus (a viral infection of the nervous system) infection. Symptoms include acute or increasing muscle weakness, muscle pain, and fatigue. The same symptoms may develop years after infection with non-paralytic polio (NPP). The exact mechanism that causes PPS is unknown. It shares many characteristics with chronic fatigue syndrome, but unlike that disorder, it tends to be progressive and can cause muscle strength loss.

[0053] Cortical neurons are cells in the cerebral cortex of the brain. Most of the complex brain activities that enable thought, perception, and voluntary movement are associated with the activity of cortical neurons.

[0054] Cortical neuron loss occurs in several neurodegenerative diseases, including AD.

[0055] Glial cells, sometimes called neuroglia, are non-neuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons in the CNS and PNS. Glial cells have four important functions: surrounding neurons and holding them in place; supplying neurons with nutrients and oxygen; insulating neurons from each other and destroying pathogens; and removing dead neurons.

[0056] There are many types of glial cells present in the CNS or PNS. Glial cell types present in the CNS include astrocytes, oligodendrocytes, ependymal cells, radial glia, and microglia. Glial cell types present in the PNS include Schwann cells, satellite cells, and enteric glial cells.

[0057] Astrocytes, also known as astroglia, are the most abundant type of macroglial cell in the CNS. Astrocytes possess numerous processes that anchor neurons to their blood supply. They regulate the external chemical environment of neurons by removing excess ions and recycling neurotransmitters released during synaptic transmission. Astrocytes can regulate vasoconstriction and vasodilation by producing substances such as arachidonic acid, whose metabolites are vasoactive.

[0058] Oligodendrocytes are cells in the CNS that coat axons with their cell membrane, forming a specialized membrane differentiation called myelin, producing the so-called myelin sheath, which provides insulation to axons, allowing them to transmit electrical signals more efficiently.

[0059] Ependymal cells, also known as ependymal cells, line the spinal cord and ventricular system. These cells are involved in the production and secretion of cerebrospinal fluid (CSF), their cilia help circulate CSF, and constitute the blood-CSF barrier. They are also thought to function as neural stem cells.

[0060] Radial glial cells arise from neuroepithelial cells after the initiation of neurogenesis. Their differentiation potential is more restricted than that of neuroepithelial cells. In the developing nervous system, radial glia function as both neural progenitor cells and a scaffold on which newborn neurons migrate. In the mature brain, the cerebellum and retina possess characteristic radial glial cells. In the cerebellum, these are Bergmann glia, which regulate synaptic plasticity. In the retina, radial Müller cells are the primary glial cells and are involved in bidirectional communication with neurons.

[0061] Microglia are a type of neuroglial cell located throughout the brain and spinal cord. As resident macrophage cells, they function as the first and primary form of active immune defense in the CNS. Microglia are important cells in maintaining the whole brain; they constantly clean the CNS, removing plaques, damaged or unnecessary neuronal synapses, and infectious agents.

[0062] Schwann cells are functionally similar to oligodendrocytes, but are present in the PNS rather than the CNS. Thus, Schwann cells enable myelination of axons in the PNS. They also possess phagocytic activity, removing cellular debris and allowing regeneration of PNS neurons.

[0063] Satellite glial cells are small cells that surround neurons in sensory, sympathetic, and parasympathetic ganglia. These cells help regulate the external chemical environment. They are highly sensitive to injury and inflammation and appear to contribute to pathological conditions such as chronic pain.

[0064] Enteric glial cells are found in the intrinsic ganglia of the digestive system. They have many roles in the intestinal system, some of which are thought to be related to homeostasis and myodigestive processes.

[0065] Dextran sulfate in embodiments further increases myelin basic protein (MBP) expression in glial cells.

[0066] MBP is a key protein in the myelination process of nervous system neurons and is a major component of the myelin sheath of oligodendrocytes and Schwann cells. MBP maintains the proper structure of myelin and interacts with lipids in the myelin membrane. Interest in MBP has focused on its role in demyelinating diseases, particularly MS.

[0067] Axonal myelination is an essential process for normal functioning of the vertebrate CNS. In the PNS, myelin is formed by differentiation of the Schwann cell plasma membrane. Loss of axonal contact following nerve injury leads to downregulation of myelin gene expression (Progress in Neurobiology (2000), 61:267-304). Schwann cell differentiation and increased MBP in injured peripheral nerves are essential for regeneration after injury (Frontiers in Neuroscience (2015), 9:Article 298, 1-13).

[0068] Increased expression of MBP in glial cells indicates that dextran sulfate of the present invention functions as a differentiation factor for these cells (Physiological Reviews (2001), 81(2):871-927, Journal of Neurochemistry (2013), 125(3):334-361).

[0069] In certain embodiments, the glial cells are myelinating cells, i.e., cells that form a myelin sheath wrapped around one or more axons of adjacent neurons. Thus, in certain embodiments, the glial cells are selected from the group consisting of Schwann cells and oligodendrocytes.

[0070] Dextran sulfate of the embodiments not only induces differentiation of cells in the CNS and PNS, but is also beneficial for neurological diseases, disorders, and conditions. Experimental data presented herein demonstrate that dextran sulfate of the embodiments has a favorable effect in combating metabolic changes observed in neurological diseases, disorders, and conditions, such as traumatic brain injury (TBI). Accordingly, many neurological diseases, disorders, and conditions are characterized by the regulation of various metabolites related to cellular energy status and mitochondrial function. Furthermore, modulation of amino acid metabolism is observed in many neurological diseases, disorders, and conditions. These metabolic changes are early cellular signals that influence enzyme activity and changes in gene and protein expression indicative of pathological tissue responses. Dextran sulfate of the embodiments functions to favorably regulate cellular metabolism in injured tissue, thereby inhibiting or at least suppressing any subsequent modulation of enzyme activity and gene and protein expression that contributes to adverse outcomes.

[0071] More specifically, dextran sulfate of the embodiment can reduce the level of glutamate excitotoxicity and restore the harmful changes in metabolic homeostasis, thereby effectively protecting mitochondrial function and providing neuroprotective effects. Dextran sulfate of the embodiment had a positive effect on various compounds related to energy metabolism and mitochondrial function. Of particular interest are the adenine nucleotide concentration and ATP / ADP ratio as measures of mitochondrial phosphorylation capacity.

[0072] Dextran sulfate of the present embodiment also led to a significant reduction in oxidative stress. In particular, the levels of ascorbic acid, the major water-soluble brain antioxidant, and glutathione (GSH), the major intracellular sulfhydryl group (SH) donor, were significantly improved. In addition, the levels of malondialdehyde (MDA), which is used as an end product of polyunsaturated fatty acids in membrane phospholipids and therefore as a marker of reactive oxygen species (ROS)-mediated lipid peroxidation, showed a significant decrease after dextran sulfate administration. After dextran sulfate treatment, all of the above oxidative stress markers showed an improvement in the restoration of antioxidant status.

[0073] Dextran sulfate administration also significantly reduced nitrate levels in both acute and chronic neurological diseases, disorders, and conditions. Thus, dextran sulfate of the embodiments has a favorable effect on NO-mediated nitrosative stress.

[0074] N-acetylaspartate (NAA) is a brain-specific metabolite and a useful biochemical marker for monitoring deterioration or recovery after neurological diseases, disorders, and conditions such as traumatic brain injury (TBI). NAA is synthesized in neurons from aspartate and acetyl-CoA by aspartate N-acetyltransferase. Dextran sulfate of the embodiment showed significant improvement in NAA levels.

[0075] The experimental data presented herein demonstrate that dextran sulfate embodiments can protect against cell loss caused by oxidative stress and / or glutamate excitotoxicity in diseased and injured nervous systems. By protecting cellular metabolism, dextran sulfate embodiments may be useful protective therapeutic agents in many degenerative conditions in which cells are progressively lost due to ischemic, oxidative, or traumatic injury, such as stroke, ALS, MND, MS, dementia, TBI, SCI, and retinal injury. These neurological diseases, disorders, and conditions share a common link in terms of the death and impairment of neuronal function of neurons that occurs in all conditions. The causes of this neuronal death are similar. Of particular relevance is toxicity caused by high levels of the neurotransmitter glutamate released from dying neurons. Dextran sulfate embodiments induce the clearance of released glutamate in glial cells, thereby preventing the accumulation of toxic amounts of glutamate in the interneuronal space. This may be useful in all neurodegenerative diseases, disorders, and conditions, both acute and chronic, in which neurons are in a dying state.

[0076] Excitotoxicity is a pathological process in which neurons are damaged or killed by excessive stimulation by neurotransmitters, particularly glutamate. This occurs when receptors for the excitatory neurotransmitter glutamate, such as N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, are overactivated by a glutamatergic storm or when neurons are damaged or die and release their glutamate content.

[0077] Excitotoxicity may also be involved in SCI, stroke, TBI, hearing loss (due to noise overexposure or ototoxicity), and neurodegenerative diseases of the CNS such as MS, ALS, PD, alcoholism or withdrawal and especially very rapid benzodiazepine withdrawal, as well as HS. Another common condition that results in excessive glutamate concentrations around neurons is hypoglycemia.

[0078] Under normal conditions, glutamate concentrations can increase to 1 mM in the synaptic cleft, which rapidly declines over the course of several milliseconds. If glutamate concentrations around the synaptic cleft cannot be reduced or reach high levels, neurons will kill themselves through a process called apoptosis. This pathological phenomenon can also occur after brain injuries such as TBI and SCI. Within minutes of injury, damaged neurons in the lesion site release glutamate into the extracellular space, where it stimulates presynaptic glutamate receptors, enhancing further glutamate release. Brain trauma or stroke can cause ischemia, in which blood flow is reduced to inadequate levels. Following ischemia, glutamate accumulates in the extracellular fluid, causing cell death, which is exacerbated by oxygen and glucose deprivation. The biochemical cascade resulting from ischemia and associated excitotoxicity is called the ischemic cascade. Due to events resulting from ischemia and glutamate receptor activation, deep chemical coma may be induced in patients with brain injury to reduce the cerebral metabolic rate, oxygen and glucose requirements, and to conserve energy used in actively removing glutamate.

[0079] Furthermore, increased extracellular glutamate levels increase Ca levels in the myelin sheath and on oligodendrocytes. 2+ This leads to activation of permeable N-methyl-D-aspartate (NMDA) receptors, which increases Ca 2+ This leaves oligodendrocytes susceptible to influx and subsequent excitotoxicity. One adverse consequence of excess calcium in the cytosol is the initiation of apoptosis through cleaved caspase processing. Another adverse consequence of excess calcium in the cytosol is the opening of the mitochondrial permeability transition pore (a pore in the mitochondrial membrane that opens when the organelle absorbs excess calcium). The opening of the pore causes mitochondria to swell, releasing reactive oxygen species and various proteins, which can lead to apoptosis. The pore can also cause mitochondria to release even more calcium. Furthermore, adenosine triphosphate (ATP) production is halted, and ATP synthase may actually begin hydrolyzing ATP rather than producing it.

[0080] Insufficient ATP production resulting from brain trauma can eliminate the electrochemical gradients of certain ions. Glutamate transporters are required to maintain these ion gradients in order to remove glutamate from the extracellular space. Disappearance of the ion gradient not only halts glutamate uptake but also reverses transporter activity. Na transport on neurons and astrocytes + Glutamate transporters reverse their glutamate transport and begin secreting glutamate at concentrations capable of inducing excitotoxicity, leading to glutamate accumulation and further deleterious activation of glutamate receptors.

[0081] At the molecular level, calcium influx is not the only factor involved in excitotoxicity-induced apoptosis. Recently, it has been shown that extrasynaptic NMDA receptor activation, induced by both glutamate exposure or hypoxic / ischemic conditions, activates cAMP response element-binding (CREB) protein blockade, which in turn leads to loss of mitochondrial membrane potential and apoptosis.

[0082] Thus, activation of glutamate transporters in glial cells by dextran sulfate embodiments to prevent or at least reduce the accumulation of toxic levels of glutamate effectively protects surrounding neurons from glutamate excitotoxicity, and as a result, dextran sulfate embodiments protect neurons from damage and cell death that would otherwise occur as a result of this glutamate excitotoxicity.

[0083] In addition, when any tissue, including the CNS and PNS, and the brain, is particularly sensitive to changes in oxygen / energy supply, the energy supply to cells is impaired when damaged or diseased.As a result, the cells in tissues such as the CNS, PNS, or brain cannot function efficiently.Therefore, the reduction of oxidative stress by dextran sulfate in embodiments, that is, the protection of mitochondrial energy supply, allows surviving cells to function more efficiently, and also protects damaged neurons from apoptosis death.

[0084] Thus, dextran sulfate of the present embodiment was effective in restoring severely imbalanced mitochondrial energy metabolism in subjects with brain injury, such as severe traumatic brain injury (sTBI), with favorable effects on the concentrations of purine triphosphate and pyrimidine nucleotides. In particular, ATP levels were 16% lower than those in healthy control subjects, while a 35% decrease was observed in untreated sTBI subjects. Of note, NAA concentrations in dextran sulfate-treated sTBI subjects were 16% lower than those in healthy control subjects, while sTBI subjects exhibited 48% lower levels of this compound. This finding again strongly confirms the strict link between NAA homeostasis and proper mitochondrial energy metabolism and emphasizes the importance of pharmacological interventions that can favorably affect mitochondrial function.

[0085] The general restoration of brain metabolism produced by dextran sulfate administration was also accompanied by the metabolism of nicotine coenzymes and free CoA-SH and CoA-SH derivatives, indicating that dextran sulfate-treated subjects, despite suffering from sTBI, had subnormal coenzymes that compensated for the correct redox reactions and allowed the TCA cycle to function properly.

[0086] Furthermore, the above-mentioned improvement in brain metabolism was responsible for another significant effect of dextran sulfate, namely the abolition of glutamate excitotoxicity. Furthermore, dextran sulfate affected sulfur-containing amino acids. This effect may be related to the dextran sulfate molecule containing an S atom. The increased bioavailability of this atom resulted in a net increase in the biosynthesis of these amino acids. One of these, MET, is crucial for methylation reactions and the so-called methyl cycle.

[0087] Additional positive effects documented were increased antioxidants and decreased biochemical signatures of oxidative / nitrosative stress in sTBI subjects receiving dextran sulfate. It is pertinent that the effects of dextran sulfate were more evident 7 days after sTBI than 2 days after. This strongly suggests that the overall recovery of cerebral metabolism with dextran sulfate administration was not a transient phenomenon.

[0088] Dextran sulfate in embodiments further comprises oligomeric amyloid beta and PrP. C and have competing affinity in protein-protein interactions between them, which may have beneficial effects in subjects suffering from AD, prion disease, or amyloidosis.

[0089] Gene expression data presented herein indicates that dextran sulfate of the embodiments has a role in Schwann cells, neurons, and human umbilical vein endothelial cells (HUVECs) with respect to protecting against apoptosis; inducing angiogenesis (in HUVECs); increasing cell migration and movement; increasing cell viability and survival; and inducing cell differentiation.

[0090] Results from a HUVEC cell model indicate that dextran sulfate of the embodiments can protect against cell injury and promote the development of new blood vessels in injured or diseased tissue, such as after stroke or other ischemic conditions.

[0091] Analysis of key molecular pathways demonstrated that dextran sulfate reduced the effects of oxidative stress on mitochondria and increased the uptake of harmful glutamate in Schwann cells. Consequently, gene expression data confirmed the results observed in animal models of TBI. Of particular interest was the finding that dextran sulfate of embodiments inhibited complex III. Inhibition of complex III subsequently leads to reduced mitochondrial oxidative stress. Furthermore, dextran sulfate of embodiments also induced the expression of the following protein complexes: calmodulin (CALM) (a multifunctional intermediate calcium-binding messenger protein); G beta-gamma complex (Gβγ) (a tightly coupled dimeric G protein complex consisting of one Gβ and one Gγ subunit); metabotropic glutamate receptor 7 (GRM7); and protein interacting with C kinase 1 (PICK1). As shown schematically in Figure 19, this protein complex subsequently suppresses glutamate release from presynaptic neurons.

[0092] The results with Schwann cells indicate that dextran sulfate of the embodiments can protect against cell loss caused by oxidative stress and / or glutamate excitotoxicity in the diseased and injured nervous system, which is applicable, for example, to neurodegenerative diseases and TBI.

[0093] Results from neurons demonstrate that dextran sulfate of embodiments can prevent and suppress apoptosis, its negative effects on amyloid-beta and Lewy body pathology, and mitochondrial fragmentation and dysfunction, and subsequent damage, as well as inhibiting inhibitory fatty acid oxidation. Dextran sulfate of embodiments also improved mitochondrial function and reduced mitochondrial levels of H2O2 and reactive oxygen species.

[0094] Analysis of upstream regulators of genes regulated by dextran sulfate demonstrated that dextran sulfate of the embodiments enhanced the effects of existing growth factors on cells. As shown in Tables 12-14, dextran sulfate of the embodiments was able to modulate the effects of several growth factors by increasing their activation or reducing their inhibition. This means that dextran sulfate of the embodiments may be used for diseases, disorders, and conditions in which increasing the activity or reducing the inhibition of these growth factors may be beneficial to patients. Non-limiting examples of such diseases, disorders, and conditions include ALS; stroke; SCI; depression and other psychiatric disorders, such as mood disorders and bipolar disorder; and metabolic disorders.

[0095] One hypothesis is that dextran sulfate binds to growth factor molecules and promotes their binding to their receptors. This hypothesis is also supported by the observation that dextran sulfate-induced differential gene expression in HUVECs (normal control medium already containing heparin) was relatively less than in Schwann cells (normal control medium not containing heparin). This mechanism of action also explains why dextran sulfate is primarily effective in the acute phase of TBI, when growth factors are present, but less effective at later stages, when early repair attempts have already been curtailed.

[0096] Thus, at least some of the therapeutic effects of dextran sulfate of the embodiments may depend on pre-existing repair mechanisms that are amplified by dextran sulfate. In such cases, it is generally recommended that dextran sulfate be administered at an early stage of any neurodegenerative disease, disorder, or condition, when the tissue has sufficient repair potential.

[0097] By protecting cellular metabolism, dextran sulfate of the embodiments may be a useful protective therapeutic agent for many degenerative conditions in which cells are progressively lost due to ischemic, oxidative, or traumatic injury. Non-limiting examples of such degenerative conditions include stroke, ALS, MS, dementia, TBI, SCI, retinal injury, AD, etc. Dextran sulfate of the embodiments may support damaged tissues and restore some lost function while enhancing remaining endogenous repair mechanisms.

[0098] Thus, gene expression data confirms the potential therapeutic utility of dextran sulfate embodiments in the injured CNS and PNS by promoting revascularization, reducing secondary tissue damage, and promoting repair for neurodegenerative diseases, disorders, and conditions, where dextran sulfate can promote neuronal survival, differentiation, and ultimately repair.

[0099] Another interesting effect of dextran sulfate of the embodiments is its effect on cell adhesion. Cell adhesion was primarily affected in neurons and Schwann cells, with dextran sulfate of the embodiments promoting cell detachment and migration. The effect on cell adhesion is primarily due to the expression of metalloproteinase-type enzymes. This finding also explains the anti-scarring effect of dextran sulfate of the embodiments. This result suggests that the anti-scarring effect is mediated by dextran sulfate of the embodiments by activating degradative enzymes that support tissue remodeling and block fibrogenic (scar-forming) signals in damaged tissue.

[0100] Metalloproteinase-type enzymes activated by dextran sulfate of the embodiments act specifically by breaking down fibrous molecules that form scars. See Tables 10-11. These enzymes are released by cells migrating into damaged tissue. Thus, dextran sulfate of the embodiments makes these cells more mobile and reduces their adhesiveness, allowing them to migrate better for repair, release scar-degrading enzymes, and remodel tissue.

[0101] Thus, the anti-scarring properties of dextran sulfate in embodiments suggest its potential use in the treatment of fibroproliferative (scar-forming) conditions, including, for example, glaucoma, proliferative vitreoretinopathy, cerebrospinal trauma, cerebral subarachnoid hemorrhage, invasive surgery, postoperative adhesions, rotator cuff injuries, burns, reconstructive surgery, and ulcerative conditions (diabetes). Other fibrotic diseases and conditions include pulmonary fibrosis, such as pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, progressive massive fibrosis, and radiation-induced lung injury following cancer treatment; liver fibrosis, such as cirrhosis and biliary atresia; cardiac fibrosis, such as atrial fibrosis, endomyocardial fibrosis, and previous myocardial infarction; brain fibrosis, such as glial scarring; pancreatitis; arthrofibrosis; Crohn's disease; Dupuytren's contracture; keloids; mediastinal fibrosis; myelofibrosis; Peyronie's disease; nephrogenic systemic fibrosis; retroperitoneal fibrosis; scleroderma or systemic sclerosis.

[0102] Fibrosis can also occur in association with organ transplants, such as kidney, lung, liver, and heart transplants, and in association with cell therapy and cell transplants, such as islets of Langerhans, hepatocytes, insulin-producing cells, stem cells, and progenitor cells.

[0103] Interestingly, gene expression data also show that dextran sulfate of embodiments activates the production of a natural scar-reducing molecule called decorin, which further inhibits scar formation by "cleaning up" growth factors that stimulate scar formation by fibroblasts.

[0104] Decorin is a glycoprotein with an average molecular weight of 90-140 kD. It belongs to the small leucine-rich proteoglycan (SLRP) family and consists of a protein core containing leucine repeats with glycosaminoglycan (GAG) chains composed of chondroitin sulfate or dermatan sulfate. It binds to type I collagen fibrils via the decorin type I collagen binding region.

[0105] Decorin acts as a transforming growth factor beta 1 or 2 (TGFβ1 / 2) antagonist and reduces scar formation. Reports indicate that in acute scar formation, the primary effect of decorin is antifibrogenic via suppression of inflammatory fibrosis by neutralizing TGFβ1 / 2. Decorin also binds directly to collagen, and one of its functions is to affect collagen organization during wound healing.

[0106] Decorin has been described for the inhibition of scar formation in models of brain lesions, hydrocephalus, and chronic spinal cord injury. Decorin also induces fibrolysis of pre-existing trabecular meshwork scars in models of glaucoma.

[0107] In summary, the anti-scarring effects of dextran sulfate of the embodiments indicate its potential use in treating all clinical conditions where scar formation is a problem. Dextran sulfate should work effectively on both new and old scars. This is confirmed by experimental data showing that dextran sulfate of the embodiments was able to induce the degradation of established scar elements in the trabecular meshwork in glaucomatous eyes. This is an important advantage of dextran sulfate of the embodiments, as it can not only be used to inhibit or at least suppress fibrosis and harmful scar formation, but can also degrade established scars. This means that dextran sulfate of the embodiments enables scar degradation and tissue remodeling for better repair.

[0108] Dextran sulfate was evaluated in a panel of complex tissue and human primary cell-based assays that model disease and general tissue biology. Results from the assays indicate that dextran sulfate is involved in modulating immune activation and / or immune resolution responses in inflammation and wound healing biology.

[0109] Modulation of inflammatory markers indicates the utility of dextran sulfate in the treatment of multiple chronic and acute inflammatory conditions and diseases that have an inflammatory component, such as ALS.

[0110] Initially following injury, select components of the innate / pro-inflammatory and adaptive immune responses are upregulated to maintain defense against foreign pathogens, remove tissue debris present at the injury site, and orchestrate the tissue remodeling, cell proliferation, and angiogenesis processes associated with the wound response. However, for proper wound healing to proceed, this initial inflammatory response must be modulated or halted to allow matrix reconstruction, recellularization, and tissue remodeling. Such immune resolution activity, induced by dextran sulfate, includes activation of MMP-1, PAR-1, and uPAR, demonstrating induced immune resolution useful for treating tissues damaged by trauma, including neurotrauma, that would otherwise result in deleterious fibrosis.

[0111] The experimental data showing the effect of dextran sulfate on inflammation resolution indicates that dextran sulfate may be useful in preventing, treating, or at least suppressing autoimmune diseases, particularly those affecting the central and / or peripheral nervous system. The resolution of inflammation by dextran sulfate is also important from the perspective of preventing fibrosis. Furthermore, the experimental data showing the resolution of inflammation and the suppression of microglial responses are also important in neurodegenerative diseases, disorders, and conditions.

[0112] Thus, dextran sulfate, or a pharmaceutically acceptable derivative thereof, may be useful in preventing, treating, or at least suppressing neuroinflammation and neuroinflammatory conditions, such as PD, ALS, MS, ADEM, myelitis, and GDS.

[0113] In conclusion, dextran sulfate appears to normalize and resolve inflammation present in tissues following trauma or disease, thereby these results are consistent with the effects of dextran sulfate observed in gene array and animal studies.

[0114] Generally, the function of nervous system depends on the number of nerve cells, healthy energy metabolism of nerve cells and healthy connection between nerve cells.Neurodegenerative diseases and disorders and the damage that causes neurodegeneration usually have different causes and causes, but all lead to the same final result, that is, neurodegeneration.The functional effect of such diseases, disorders or damage is often only observed after a relatively large number of nerve cells die, while the cause of disease or disorder may exist several years before symptoms occur.

[0115] Therefore, there is a need for a new method for treating or suppressing neurodegeneration.This method should include enhancing the viable function of the nervous system, including healthy energy metabolism of nerve cells and healthy connections between nerve cells.In addition, by reducing the triggers that lead to neuronal death, and even if triggers exist, by preventing further pathology, further neurodegeneration should be prevented or at least slowed down.In addition, the regenerative capacity of the nervous system should be enhanced.

[0116] Therefore, during neurodegeneration and injury, there are multiple triggers of neuronal apoptosis, all of which contribute to neuronal loss. These triggers include neurotransmitter dysregulation, which leads to glutamate excitotoxicity, and oxidative stress, which leads to mitochondrial dysfunction, thereby limiting the energy supply to neurons. In addition, dysregulated neurofilaments lead to reduced motility and limited supply of factors necessary for neuronal survival. Additional triggers include the release of inflammatory mediators, which cause secondary cell damage and scar formation. In addition, vascular disorders are commonly seen in neurodegenerative conditions.

[0117] Glutamate is produced in neurons and is important for the signaling mechanisms that support neuronal learning and memory. Excess glutamate released in healthy brain tissue is removed by glial cells, preventing it from reaching toxic levels. Dextran sulfate induced increased glutamate uptake by glial cells, whereas glutamate production in neurons was unchanged by dextran sulfate. Thus, glutamate necessary for learning and memory is unaffected by dextran sulfate administration, while harmful toxic amounts of glutamate are removed by glial cells. Therefore, dextran sulfate attenuates neurotransmitter dysregulation that leads to glutamate excitotoxicity.

[0118] Oxidative stress in neurodegeneration leads to mitochondrial dysfunction, thereby limiting the energy supply to neurons.Dextran sulfate reduces the production of molecules that induce oxidative stress, including amyloid beta and Lewy bodies, and reduces oxidative stress.Therefore, dextran sulfate prevents neuronal death induced by oxidative stress and prevents mitochondrial dysfunction in neurons.This means that in the presence of oxidative stress, dextran sulfate promotes the normalization of mitochondrial function and prevents neuronal energy crisis in the presence of such oxidative stress.Therefore, dextran sulfate attenuates the oxidative stress in neurodegeneration that would otherwise lead to mitochondrial dysfunction.

[0119] Another contributing factor in neurodegeneration is dysregulated neurofilaments, which leads to reduced mobility and limited supply of survival factors.Dextran sulfate enhances the effects of growth factors present in neurons, increases migration and migration of nerve cells, reduces the production of degeneration-related protein products, and induces cell differentiation.Therefore, dextran sulfate reduces dysregulated neurofilaments.

[0120] Neurodegeneration also induces the release of inflammatory mediators, which cause secondary cell damage and scar formation. Scar formation is promoted by inflammatory cytokines, particularly TGF-β. Dextran sulfate induces metallopeptidase expression, induces expression of the natural anti-scarring molecule decorin, and suppresses TGF-β activity. Furthermore, dextran sulfate inhibits immune cell adhesion, cell aggregation, cell activation, and fibrosis, even in the presence of excess TGF-β. Thus, dextran sulfate reduces the negative effects, including scar formation, resulting from the release of inflammatory mediators. Dextran sulfate also acts to inhibit fibrosis and activate fibrolysis, which together contribute to the beneficial effects of dextran sulfate in reducing or degrading scars.

[0121] Dextran sulfate protects HUVECs against endothelial cell apoptosis, induced angiogenesis, and increased migration and migration. Thus, dextran sulfate enhances the physiological repair response in hypoxic tissues caused by neurodegenerative diseases, disorders, or injuries, but does not affect healthy vasculature.

[0122] Accordingly, aspects of the embodiments relate to a method of inducing differentiation of a cell selected from the group consisting of a glial cell and a neuron, the method comprising contacting the cell with dextran sulfate, or a pharmaceutically acceptable derivative thereof, to induce differentiation of the cell.

[0123] In some embodiments, the method is an in vitro method. In such cases, contacting the cells comprises contacting the cells with dextran sulfate or a pharmaceutically acceptable derivative thereof in vitro. Thus, the cells are treated with and interact with dextran sulfate or a pharmaceutically acceptable derivative thereof in vitro.

[0124] In certain embodiments, neurons are obtained from stem cells, i.e., by differentiation of stem cells into neurons, which can be treated with dextran sulfate or a pharmaceutically acceptable derivative thereof and further differentiated.

[0125] Such in vitro methods may have important applications in research and diagnostics, where neurons and / or glial cells are cultured in vitro. Dextran sulfate or a pharmaceutically acceptable derivative thereof may be added to the medium of such neuronal or glial cell cultures to induce differentiation of the cells, e.g., as described herein.

[0126] The method may also be an ex vivo method, in which neurons and / or glial cells are extracted from a subject and contacted with dextran sulfate or a pharmaceutically acceptable derivative thereof outside the subject's body.

[0127] In the above-mentioned in vitro or ex vivo method, the neuron and / or glial cell treated with dextran sulfate or its pharmaceutically acceptable derivative can be transplanted into the subject to induce differentiation.The differentiated neuron and / or glial cell will then perform their intended function in the subject's body.In this method, the subject can be suffering from neurological disease, as further described herein.

[0128] In an alternative embodiment, dextran sulfate or a pharmaceutically acceptable derivative thereof is administered to a subject, such as a subject suffering from a neurological disease, disorder, or condition. The dextran sulfate or a pharmaceutically acceptable derivative thereof then contacts neurons and / or glial cells within the subject's body to induce cell differentiation. In this embodiment, the method is an in vivo method.

[0129] Another aspect of the embodiment relates to dextran sulfate or a pharmaceutically acceptable derivative thereof for use in inducing differentiation of cells selected from the group consisting of glial cells and neurons.

[0130] In certain embodiments, dextran sulfate or a pharmaceutically acceptable derivative thereof is for use in inducing differentiation of cells in a subject suffering from a neurological disease, disorder or condition.

[0131] In certain embodiments, the dextran sulfate or a pharmaceutically acceptable derivative thereof is for use in inducing differentiation of cells in a subject suffering from a neurological disease, disorder or condition selected from the group consisting of a neurodegenerative disease, disorder or condition; a demyelinating disease, disorder or condition; a neuroischemic disease, disorder or condition; a neuromuscular disease, disorder or condition; a traumatic nerve injury and a post-operative neurological condition.

[0132] In one embodiment, the subject is a human subject suffering from a neurodegenerative disease, disorder or condition selected from the group consisting of AD, PD, HD and ALS.

[0133] In one embodiment, the subject is a human subject suffering from a demyelinating disease, disorder or condition selected from the group consisting of MS, ADEM, CNS neuropathy, CPM, myelopathy, leukoencephalopathy, leukodystrophy, GBS, peripheral neuropathy and Charcot-Marie-Tooth disease, preferably selected from the group consisting of MS, ADEM, CPM and GBS.

[0134] Dextran sulfate or a pharmaceutically acceptable derivative thereof can also, or instead, be used to induce cell differentiation in other types of neurological diseases, disorders, or conditions, including, but not limited to, neuroischemic diseases such as stroke, cerebral ischemic conditions, and critical limb ischemia (CLI); neuromuscular disorders such as ALS, botulism, congenital myasthenic syndromes, congenital myopathies, twitching-fasciculation syndrome, cerebral palsy, elevated creatine kinase, fasciculation, inclusion body myositis, Lambert-Eaton syndrome, mitochondrial myopathy, motor neuron disease, myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, neuromuscular junction disorders, neuromyotonia, peripheral neuropathy, and polymyositis; traumatic nerve injury, and postoperative neurological conditions.

[0135] A further aspect of the embodiment relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, for use in treating, inhibiting or preventing glutamate excitotoxicity in a subject.

[0136] In certain embodiments, dextran sulfate or a pharmaceutically acceptable derivative thereof is effective in treating, inhibiting, or preventing glutamate excitotoxicity in neurons of a subject.

[0137] In certain embodiments, the subject is suffering from a neurological disease, disorder or condition that causes cell damage and / or cell death to neurons, as previously described.

[0138] This aspect also relates to a method for treating, inhibiting, or preventing glutamate excitotoxicity, the method comprising administering dextran sulfate, or a pharmaceutically acceptable derivative thereof, to a subject to treat, inhibit, or prevent glutamate excitotoxicity.

[0139] Other aspects of the embodiments relate to dextran sulfate or a pharmaceutically acceptable derivative thereof for use in protecting neurons from oxidative stress induced by a neurological disease, disorder or condition, for use in restoring deleterious changes in neuronal metabolic homeostasis induced by a neurological disease, disorder or condition, for use in protecting mitochondrial function and mitochondrial energy metabolism in neurons in subjects afflicted with a neurological disease, disorder or condition.

[0140] Dextran sulfate or a pharmaceutically acceptable derivative thereof can thereby be used to treat, inhibit or prevent the neurological diseases, disorders or conditions described herein.

[0141] Dextran sulfate or a pharmaceutically acceptable derivative thereof can also be used to treat, inhibit, or prevent ischemic, oxidative, or traumatic damage to neurons and the CNS, or PNS, such as stroke, ALS, MND, MS, dementia, TBI, SCI, retinal injury, etc.

[0142] A further aspect relates to dextran sulfate or a pharmaceutically acceptable derivative thereof for use in the treatment, inhibition or prevention of fibrosis in a subject, and in particular for use in the treatment or inhibition, such as by breaking down established scars, in a subject suffering from fibrosis or a fibrotic disease, disorder or condition.

[0143] Thus, dextran sulfate embodiments with anti-scarring effects may be effective in wound treatment and tissue remodeling, where degradation of established scars is necessary to allow proper wound healing. This anti-scarring effect of dextran sulfate embodiments is believed to be the result of previously described mechanisms of action of dextran sulfate, including, for example, inhibition of cell adhesion, induction of cell recruitment, induction of metalloproteinases and scar-degrading enzymes, and inhibition of TGFβ, particularly TGFβ1, via induction of decorin. This latter effect obtained with dextran sulfate embodiments is further associated with the prevention or at least inhibition of fibrosis and scar formation via induction of decorin.

[0144] Another aspect relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, for use in the treatment, inhibition, or prevention of neuroinflammation in a subject, particularly a subject suffering from a neurological disease, disorder, or condition that causes neuroinflammation.

[0145] Related aspects of the embodiments provide for the use of dextran sulfate or a pharmaceutically acceptable derivative thereof for various medical applications disclosed herein, e.g., for the manufacture of a medicament for the treatment, suppression, or prevention of any of the diseases, disorders, or conditions disclosed herein.

[0146] Further aspects relate to methods for treating, inhibiting, or preventing the various diseases, disorders, or conditions described above for various uses of dextran sulfate or a pharmaceutically acceptable derivative thereof, in which dextran sulfate or a pharmaceutically acceptable derivative thereof is administered to a subject to treat, inhibit, or prevent the disease, disorder, or condition disclosed herein.

[0147] In the following, the (average) molecular weight and sulfur content of dextran sulfate mentioned also apply to any pharmaceutically acceptable derivative of dextran sulfate. Thus, a pharmaceutically acceptable derivative of dextran sulfate preferably has the average molecular weight and sulfur content discussed in the following embodiments.

[0148] Dextran sulfate outside the preferred range of embodiments is believed to have less effect and / or negative side effects on cells or subjects.

[0149] For example, dextran sulfates with molecular weights greater than 10,000 Da (10 kDa) typically have a lower efficacy-to-side effect profile than dextran sulfates with lower molecular weights. This means that the maximum dose of dextran sulfate that can be safely administered to a subject is lower for larger dextran sulfate molecules (>10,000 Da) than for dextran sulfate molecules with average molecular weights within the preferred range. As a result, when dextran sulfate is to be administered to a subject in vivo, such larger dextran sulfate molecules are less suitable for clinical use.

[0150] Dextran sulfate is a sulfated polysaccharide, particularly a sulfated glucan, i.e., a polysaccharide made from many glucose molecules. The average molecular weight defined herein indicates that individual sulfated polysaccharides may have molecular weights different from this average molecular weight, but the average molecular weight represents the mean molecular weight of the sulfated polysaccharide. This further means that there is likely to be a natural distribution of molecular weights of dextran sulfate samples around this average molecular weight.

[0151] The average molecular weight of dextran sulfate, or more precisely, the weight average molecular weight (M w) is usually determined using indirect methods such as gel exclusion / permeation chromatography, light scattering, or viscosity. Determination of average molecular weight using such indirect methods depends on many factors, including the choice of column and eluent, flow rate, calibration procedure, etc.

[0152] Weight average molecular weight (M w ):

number

[0153] In one embodiment, the dextran sulfate or a pharmaceutically acceptable derivative thereof has an M of 10,000 Da or less. w In certain embodiments, the dextran sulfate or a pharmaceutically acceptable derivative thereof has an M in the range of 2,000 Da to 10,000 Da. w It has.

[0154] In another embodiment, the dextran sulfate or a pharmaceutically acceptable derivative thereof has an M in the range of 2,500 Da to 10,000 Da, preferably in the range of 3,000 Da to 10,000 Da. w In certain embodiments, the dextran sulfate or a pharmaceutically acceptable derivative thereof has an M in the range of 3,500 Da to 9,500 Da, for example, in the range of 3,500 Da to 8,000 Da. w It has.

[0155] In another particular embodiment, the dextran sulfate or a pharmaceutically acceptable derivative thereof has an M in the range of 4,500 Da to 7,500 Da, for example, in the range of 4,500 Da to 5,500 Da. w It has.

[0156] Thus, in some embodiments, dextran sulfate or a pharmaceutically acceptable derivative thereof has an M of 10,000 Da or less, 9,500 Da or less, 9,000 Da or less, 8,500 Da or less, 8,000 Da or less, 7,500 Da or less, 7,000 Da or less, 6,500 Da or less, 6,000 Da or less, or 5,500 Da or less. w It has.

[0157] In some embodiments, the dextran sulfate or pharmaceutically acceptable derivative thereof has an M of 1,000 Da or more, 1,500 Da or more, 2,000 Da or more, 2,500 Da or more, 3,000 Da or more, 3,500 Da or more, 4,000 Da or more, or 4,500 Da or more. w Any of these embodiments may be used in combination with any of the above-presented M w and these may be combined with an upper limit of 10,000 Da or less.

[0158] In certain embodiments, the M of dextran sulfate or a pharmaceutically acceptable derivative thereof as set forth above is w is the average M w and is preferably determined by gel exclusion / permeation chromatography, size exclusion chromatography, light scattering or viscosity-based methods.

[0159] Number average molecular weight (M n ):

number

[0160] In one embodiment, the dextran sulfate or a pharmaceutically acceptable derivative thereof has an M as measured by NMR spectroscopy in the range of 1,850 to 3,500 Da. n It has.

[0161] In certain embodiments, the dextran sulfate or a pharmaceutically acceptable derivative thereof has an M as measured by NMR spectroscopy in the range of 1,850 Da to 2,500 Da, preferably in the range of 1,850 Da to 2,300 Da, for example in the range of 1,850 Da to 2,000 Da. n It has.

[0162] Thus, in some embodiments, the dextran sulfate or pharmaceutically acceptable derivative thereof has an M of 3,500 Da or less, 3,250 Da or less, 3,000 Da or less, 2,750 Da or less, 2,500 Da or less, 2,250 Da or less, or 2,000 Da or less. n Furthermore, dextran sulfate or a pharmaceutically acceptable derivative thereof has an M of 1,850 Da or more. n It has.

[0163] In one embodiment, the dextran sulfate or pharmaceutically acceptable derivative thereof has an average number of sulfates per glucose unit in the range of 2.5 to 3.0.

[0164] In certain embodiments, dextran sulfate or a pharmaceutically acceptable derivative thereof has an average number of sulfates per glucose unit in the range of 2.5 to 2.8, preferably in the range of 2.6 to 2.7.

[0165] In one embodiment, the dextran sulfate or pharmaceutically acceptable derivative thereof has an average number of glucose units in the range of 4.0 to 6.0.

[0166] In a particular embodiment, the dextran sulfate or pharmaceutically acceptable derivative thereof has an average number of glucose units in the range of 4.5 to 5.5, preferably in the range of 5.0 to 5.2.

[0167] In one embodiment, the dextran sulfate or a pharmaceutically acceptable derivative thereof has an M as measured by NMR spectroscopy in the range of 1,850 to 3,500 Da. n , has an average number of sulfates per glucose unit in the range of 2.5 to 3.0, and the average sulfation at the C2 position in the glucose unit of dextran sulfate is at least 90%.

[0168] In one embodiment, the dextran sulfate has an average glucose unit number of about 5.1, an average sulfate number per glucose unit in the range of 2.6 to 2.7, and an M in the range of 1,850 Da to 2,000 Da. n It has.

[0169] In one embodiment, the pharmaceutically acceptable derivative of dextran sulfate is the sodium salt of dextran sulfate. In a specific embodiment, the sodium salt of dextran sulfate has an average glucose unit number of about 5.1, an average sulfate number per glucose unit in the range of 2.6 to 2.7, and a NaOH concentration in the range of 2,100 Da to 2,300 Da. + M containing counter ions n It has.

[0170] In one embodiment, the dextran sulfate has an average glucose unit count of 5.1, an average sulfate count per glucose unit of 2.7, and a Na concentration of about 1,900-1,950 Da as measured by NMR spectroscopy. + Excluded average M n and Na by NMR spectroscopy measurement of approximately 2,200-2,250 Da. + Content average M n It has.

[0171] Dextran sulfate according to embodiments can be provided as a pharmaceutically acceptable derivative of dextran sulfate, such as a pharmaceutically active derivative of dextran sulfate, including pharmaceutically acceptable salts and pharmaceutically acceptable solvates of dextran sulfate, e.g., sodium or potassium salts.

[0172] The subject is preferably a mammalian subject, more preferably a primate, and especially a human subject. However, dextran sulfate or its pharmaceutically acceptable derivatives can also be used for veterinary purposes. Non-limiting examples of animal subjects include primates, cats, dogs, pigs, horses, mice, and rats.

[0173] Dextran sulfate or a pharmaceutically acceptable derivative thereof is preferably administered to a subject by injection, particularly by intravenous (iv), subcutaneous (sc) or intraperitoneal (ip) injection, preferably by iv or sc injection. Other possible parenteral administration routes include intramuscular and intraarticular injection. Alternatively or additionally, dextran sulfate or a pharmaceutically acceptable derivative thereof may be injected, for example, directly into a tissue or organ or other site in the subject's body where the desired effect is to occur.

[0174] Alternatively or additionally, dextran sulfate or its pharmaceutically acceptable derivatives can be administered intrathecally.For example, dextran sulfate or its pharmaceutically acceptable derivatives can be injected into the spinal canal or intrathecally with suitable aqueous carrier or solution so as to reach cerebrospinal fluid (CSF).Another administration route is intraocular administration.

[0175] The dextran sulfate or its pharmaceutically acceptable derivative of the embodiment is preferably formulated as an aqueous injection solution using a selected solvent or excipient. The solvent is conveniently an aqueous solvent, particularly a buffer solution. Non-limiting examples of such buffer solutions include citrate buffers, such as citrate monohydrate (CAM) buffer, or phosphate buffers. For example, the dextran sulfate of the embodiment can be dissolved in saline, such as 0.9% NaCl saline, and then optionally buffered with 75 mM CAM and adjusted to a pH of about 5.9 using sodium hydroxide. Non-buffered solutions are also possible, including aqueous injection solutions such as saline, i.e., NaCl (aqueous). Furthermore, if a buffer solution is desired, buffer systems other than CAM can be used.

[0176] Embodiments are not limited to injection, and other routes of administration can alternatively be used, including oral, nasal, buccal, rectal, transdermal, transbronchial, or topical. The active compound dextran sulfate is then formulated with suitable excipients or carriers, which are selected based on the particular route of administration.

[0177] Suitable dosage ranges for dextran sulfate or its pharmaceutically acceptable derivatives may vary depending on the application, e.g., in vitro versus in vivo, the size and weight of the subject, the condition being treated, and other considerations. For human subjects in particular, possible dosage ranges would be 1 μg / kg to 100 mg / kg body weight, preferably 10 μg / kg to 50 mg / kg body weight.

[0178] In a preferred embodiment, dextran sulfate or a pharmaceutically acceptable derivative thereof is formulated to be administered at a dosage in the range of 0.05 to 50 mg / kg body weight of a subject, preferably 0.05 or 0.1 to 40 mg / kg body weight of a subject, and more preferably 0.05 or 0.1 to 30 mg / kg body weight of a subject, or 0.1 to 25 mg / kg body weight of a subject, or 0.1 to 15 mg / kg body weight of a subject, or 0.1 to 10 mg / kg body weight of a subject.

[0179] Administration of dextran sulfate or a pharmaceutically acceptable derivative thereof is not limited to the treatment or suppression of an existing disease, disorder, or condition, but can alternatively or additionally be used for prophylaxis. In other words, dextran sulfate or a pharmaceutically acceptable derivative thereof can be administered to a subject about to undergo a medical procedure, such as surgery, that may result in nerve injury or damage and / or fibrosis. Dextran sulfate or a pharmaceutically acceptable derivative thereof can also be used to prevent, suppress, or alleviate postoperative neurological complications and conditions in a subject about to undergo a medical procedure, such as surgery and / or fibrosis.

[0180] Dextran sulfate or a pharmaceutically acceptable derivative thereof can be administered in a single dose, such as in the form of a single bolus injection, which can be very quickly infused into a subject, but is conveniently infused over a period of time, such that the dextran sulfate solution is infused into the patient over several minutes, such as 5-10 minutes.

[0181] Alternatively, dextran sulfate or a pharmaceutically acceptable derivative thereof can be administered multiple times, ie, at least twice, during the course of treatment.

[0182] Dextran sulfate or its pharmaceutically acceptable derivatives can be administered sequentially, simultaneously, or in the form of a composition containing dextran sulfate or its pharmaceutically acceptable derivatives and at least one other active agent. The at least one active agent can be selected from any agent useful for any of the above diseases, disorders, or conditions. The at least one active agent can also be in the form of cells for cell therapy, such as stem cells, including, but not limited to, embryonic stem cells (ESCs) and mesenchymal stromal cells (MSCs).

[0183] For example, studies are being conducted on the effects of stem cells in animal models of brain degeneration, such as in Parkinson's disease, MS, ALS, and Alzheimer's disease. Additionally, clinical and animal trials are underway on the use of stem cells in cases of TBI.

[0184] Dextran sulfate or its pharmaceutically acceptable derivatives have beneficial effects on cells in vitro, as demonstrated by experimental data. For example, dextran sulfate or its pharmaceutically acceptable derivatives protect cells from oxidative stress, restore cellular metabolic homeostasis, which is beneficial to cellular energy metabolism, and can act as a differentiation factor for cells. These beneficial effects of dextran sulfate or its pharmaceutically acceptable derivatives can also be used in other types of cell therapy, i.e., not necessarily limited to stem cell therapy. Non-limiting examples of such other types of cell therapy include cardiomyocytes, liver cells, connective tissue cells, optic nerve cells, lymphocytes, macrophages, glial cells, Schwann cells, neurons, etc. In such cases, cells can be treated with dextran sulfate or its pharmaceutically acceptable derivative in vitro before administration to a subject. Alternatively, or in addition, cells can be administered together with dextran sulfate or its pharmaceutically acceptable derivative. In addition, in vitro or ex vivo treatment of tissues and organs with dextran sulfate or a pharmaceutically acceptable derivative thereof may be useful to benefit from the positive effects of the dextran sulfate embodiments, such as protection against oxidative stress and restoration of metabolic homeostasis. Furthermore, treatment of cells, tissues, and organs may be possible in addition to or instead of transplantation of dextran sulfate or a pharmaceutically acceptable derivative thereof.

[0185] In some embodiments, dextran sulfate or a pharmaceutically acceptable derivative thereof is advantageously administered to a subject in the early or acute stage after an injury that causes a disease, disorder, or condition, such as TBI, or in the early or acute stage after diagnosis of a disease, disorder, or condition. This is particularly advantageous because some of the beneficial effects observed with dextran sulfate embodiments are its ability to enhance and amplify endogenous repair mechanisms in the CNS and PNS. This is particularly important for the treatment or prevention of neurological disorders. However, the anti-scarring effects observed with dextran sulfate embodiments indicate that dextran sulfate may also be effective in breaking down existing scar tissue and elements. Therefore, dextran sulfate embodiments may be effective in treating fibrosis and fibrotic conditions during the later or chronic stages.

[0186] Example In the following examples, the sodium salt of dextran sulfate, designated herein as low molecular weight dextran sulfate (LMW-DS), was used (Tikomed AB, Sweden, WO 2016 / 076780).

[0187] Example 1 The aim of the study was to evaluate the effect of LMW-DS on cell survival and expression of differentiation proteins in three cell types: cerebral cortical neurons, motor neurons, and Schwann cells, using two concentrations of LMW-DS, 0.01 and 0.1 mg / ml.

[0188] material and method cell culture All cells were cultured in a dedicated medium appropriate for the cell type. Plasticware was treated with specific adhesion factors to enhance cell adhesion. [Table 1]

[0189] Neurons were cultured at 40,000 cells per well and Schwann cells at 3,000 cells per well. Cells were treated 24 hours later. The number of cells per well depended on growth phenotype, proliferation capacity, etc.

[0190] Coating of tissue culture dishes 96-well plates were coated by adding 100 μl per well of a 50 μg / ml solution of poly-d-lysine (Sigma) in Hank's Balanced Salt Solution (HBSS, Sigma) and incubating overnight at 37°C in the dark. Plates were washed with cell culture water (Fisher) and air-dried in the dark for 30 minutes. Plates were coated by adding 75 μl per well of a 15 μg / ml laminin solution (Sigma) in medium and incubated for 1 hour at 37°C in the dark. Media was specific for each cell type as follows: PNGM™ (Primary Neuron Basal Medium, Lonza) for cortical neurons (Lonza), NeuroBlast (Lonza) for motor neurons (Lonza), and high-glucose Dulbecco's Modified Eagle's Medium (DMEM) for Schwann cells (ATCC). Laminin was removed from plates immediately prior to cell seeding.

[0191] cortical neurons PNGM was prepared by adding PNGM Singlequots (Lonza) to PNBM medium and prewarmed to 37°C. Cells were thawed in a 37°C water bath for less than 2 minutes and gently transferred to a 15 ml tube. Five ml of medium was gently added dropwise. The cell suspension was mixed by carefully inverting the tube twice. Cells were counted using a Cellometer AUTO T4 (Nexcelom Bioscience). 40,000 cells / well were seeded onto precoated 96-well plates. Cells were incubated at 37°C under 5% CO2. After 2 hours of incubation, 80 μl of medium was removed and replaced with 80 μl of fresh medium. The cells were allowed to settle for 24 hours before drug treatment.

[0192] motor neurons NeuroBlast was pre-warmed to 37°C. Cells were thawed in a 37°C water bath for no more than 2 minutes, and 1 ml of medium was gently added dropwise. Cells were resuspended and transferred to a 15 ml tube containing 9 ml of medium. Cells were centrifuged at 200 relative centrifugal force (RCF) for 5 minutes. The pellet was resuspended in 5 ml of medium, and cells were counted using a Cellometer. 40,000 cells / well were seeded into pre-coated 96-well plates. Cells were incubated at 37°C under 5% CO2. Cells were allowed to settle for 24 hours before drug treatment. After 24 hours, the NeuroBlast medium was replaced with MotorBlast (Lonzo).

[0193] Schwann cells Schwann cell growth medium was prepared by adding 10% fetal bovine serum (FBS, PAA) to high-glucose DMEM and pre-warmed to 37°C. Cells were thawed in a 37°C water bath for no more than 2 minutes. Cells were gently transferred to a 10 ml medium-containing tube and centrifuged at 200 RCF for 5 minutes. The pellet was resuspended in 5 ml medium, and cells were counted using a Cellometer. 3,000 cells / well were seeded into pre-coated 96-well plates. Cells were incubated at 37°C under 5% CO2. Cells were allowed to settle for 24 hours before drug treatment.

[0194] Drug treatment and plate preparation LMW-DS was prepared in the optimal medium for each cell line and added to each well at doses of 0.01 and 0.1 mg / ml. For cell viability assays, cells were analyzed after 24 and 48 hours in eight replicate wells / dose / time point. For differentiation and protein expression assays, cells were similarly analyzed in eight replicates after 48 hours.

[0195] PI and immunostaining No adjustment for PI histogram shift Cells were fixed in the wells. Propidium iodide was used for viability assays. For immunohistochemical analysis, neurons were stained with βIII-tubulin, a neuron-specific tubulin. Schwann cells were stained for myelin basic protein (MBP). As a negative control, PBST (0.1% Triton X100 in PBS) was applied instead of the primary antibody.

[0196] Acumen Cytometry The Acumen cytometer allows direct cytometric analysis of adherent cells without prior detachment. Cells were then imaged in situ and classified into different cell cycle phases based on DNA content (PI) or considered as apoptotic or polyploid. Cellular protein content can also be measured directly and expressed as "total protein content" or "average protein content."

[0197] statistics Data are expressed as the mean plus standard deviation (SD) of eight replicates. Comparisons between groups were performed using Student's t-test (two-tailed, equal variance; Excel software). A p value of less than 0.05 was considered statistically significant (*p<0.05, **p<0.01, ***p<0.001).

[0198] result Mouse cortical neurons The DNA histogram in Figure 1 shows that LMW-DS treatment has an effect on cortical neurons, as evidenced by the change in cellular PI uptake and a shift to the right of the histogram. The cell population that has initiated division (G2 / M phase) is shown in the figure.

[0199] Cell numbers were significantly reduced after treatment with LMW-DS. The proportion of apoptotic cells increased slightly, but this does not account for all of the cell loss and is more likely due to cell detachment.

[0200] human motor neurons Data for motor neurons were similar to cortical neurons, with a shift in PI uptake (Fig. 2) and increased proliferation within a very small cell population.

[0201] There was a similarly large cell loss in these cultures, the explanation for which is likely the same as in cortical neurons.

[0202] Schwann cells Schwann cells did not appear to be as affected by LMW-DS as neurons, and there was a similar PI shift (Fig. 3).

[0203] The effect on cell number and cell detachment was not evident in Schwann cells compared to neurons. In contrast to neurons, the proportion of apoptotic cells was reduced upon treatment with LMW-DS.

[0204] Differentiation-associated protein expression Tubulin expression in mouse cortical neurons Cell morphology changed in treated cultures, with cells becoming rounder and larger (Figure 4).

[0205] Tubulin is a family of proteins that are important building blocks in the cytoskeleton of cells. βIII-tubulin is expressed exclusively by neurons. The intensity of tubulin was significantly increased in cells treated with LMW-DS (Figure 5A). Analysis of positive cells showed that these cells were larger than those in control cultures (Figure 5B).

[0206] Tubulin expression in human motor neurons βIII-tubulin expression was significantly increased by LMW-DS (Fig. 6A). Cell morphology was dramatically altered by LMW-DS. Most positive cells were smaller than those in control cultures (Fig. 6B). However, some cells became significantly larger with long neurites (Fig. 7).

[0207] MBP expression in human Schwann cells MBP expression was significantly increased in LMW-DS-treated cultures of Schwann cells (Fig. 8A). Analysis of cell size showed that MBP-positive cells were larger after LMW-DS treatment compared with controls (Figs. 8B and 9).

[0208] conclusion Mouse cortical neurons and human motor neurons The increased expression of βIII-tubulin in the cells and the morphological changes indicated that LMW-DS functions as a differentiation factor. The effect on motor neurons was particularly striking.

[0209] The changes induced by LMW-DS were evident in both mouse and human cells, indicating that this effect is species independent.

[0210] LMW-DS treatment led to significant cell loss in the cultures. This effect of LMW-DS treatment was not thought to be due to a toxic effect; it is more likely that LMW-DS affected neuronal cell adhesion. For example, even though the maximum measure of the rate of apoptotic death (after adjusting for the PI shift) did not account for cell loss in the cultures, significant cell loss was much greater in immunostained preparations (with more washes) than in PI preparations.

[0211] human Schwann cells The increased expression of MBP in the cells and the morphological changes indicated that LMW-DS functions as a differentiation factor in glial cells.

[0212] In dividing Schwann cells, signs of cell detachment upon LMW-DS treatment were not as dramatic as in neuronal cultures, but they were still visible.

[0213] Thus, LMW-DS appeared to promote both neural and Schwann cell differentiation within a very short time period (48 h).

[0214] It is now widely accepted that neurodegenerative diseases, including trauma-related neurodegeneration, AD, and post-stroke dementia, are associated with the reactivation of neuronal cell cycle-related events. In this regard, differentiation-inducing drugs have been proposed to be neuroprotective. Drugs that support Schwann cell differentiation would also be good candidates for the treatment of diseases associated with demyelination.

[0215] Example 2 This study was conducted to investigate the in vivo effects of LMW-DS in a mouse experimental autoimmune encephalomyelitis (EAE) model.

[0216] EAE, sometimes referred to as experimental allergic encephalomyelitis, is an inflammatory demyelinating disease of the CNS mediated by CD4+ T cells. The murine EAE model is currently the most widely accepted animal model of human MS and ADEM (Annals of Neurology, 60:12-21, 2006). Generally, EAE is initiated by administering myelin oligodendrocyte glycoprotein (MOG) emulsified in an adjuvant to mice, which induces an immune response against myelin. 35-55 (MOG 35-55 EAE is induced by a single injection of peptides and proteins containing the agonist ...

[0217] Materials and Methods Freund's Incomplete Adjuvant (IFA) (Difco) ·Mycobacterium tuberculosis H37RA (Difco) MOG 35-55 Rodents (MDBioproducts) ·Pertussis toxin (Sigma Aldrich) Hank's Balanced Salt Solution (HBSS) (Gibco / Invitrogen) Dulbecco's Phosphate Buffered Saline (D-PBS) (Life Technologies) Hydroxypropyl methylcellulose (HPMC) (Sigma Aldrich) 0.9% saline solution (9 mg / ml NaCl, autoclaved) (Scharlau) Cyclosporin A (Sigma Aldrich) LMW-DS dissolved in 0.9% saline Hepatocyte growth factor (HGF) recombinant mice (R&D Systems) ·Isoba vet 3.5%(Schering Plough Animal Health) Methylbutane (Sigma Aldrich)

[0218] C57B1.6 mice (female, 8–10 weeks old) were obtained from Harlan Europe. Mice were housed in a conventional animal facility (Lund University, Sweden) in polystyrene cages containing wood shavings (type II cages, maximum of 7 mice per cage) under a 12-h light / dark cycle and provided with standard rodent chow and water ad libitum.

[0219] Disease induction and boosting On day 0, 150 μg of MOG in a volume of 100 μl per mouse 35-55 EAE was induced by subcutaneous injection into the flank of an emulsion containing 300 μg of H37RA and 300 μg of H37RA in Freund's complete adjuvant (CFA) (H37RA in IFA at a concentration of 6 mg / ml) and MOG. 35-55 An emulsion was prepared by mixing the 500mg / ml IgG antibody (dissolved in PBS at a concentration of 3mg / ml) on ice. Mice were anesthetized during immunization to ensure the correct location of the injection. Pertussis toxin (PTX) was resuspended in mqH2O at a concentration of 50µg / ml and diluted to a final concentration of 1µg / ml in PBS. Mice received intraperitoneal booster injections of 200ng PTX on days 0 and 2.

[0220] Dosage Preparation For Group 3, LMW-DS dilutions were prepared on days 0 and 14. LMW-DS was diluted in 0.9% saline according to the doses listed in Table 2 below and sterilized by filtration through a 0.2 μm filter. The administration vehicle was 0.9% saline. Recombinant HGF was reconstituted with 1 ml of 0.1% bovine serum albumin (BSA) in PBS at a concentration of 25 μg / ml and further diluted to 1 μg / ml in PBS. Cyclosporine A was prepared by dissolving 50 mg of cyclosporine A in 1 ml of 70% ethanol and diluting it to a final concentration of 0.98 mg / ml in HPMC. [Table 2]

[0221] Experimental groups and administration of LMW-DS Treatment began on day 0 for groups 2-3 and was administered intraperitoneally in group 2 and subcutaneously in group 3, three times weekly. Treatment began on day 18 for the remaining groups. Animals in group 4 were dosed intravenously every other day for a total of three doses. Treatment groups were mixed in cages to avoid systematic errors due to cage effects and unequal housing.

[0222] Disease evaluation Disease progression was followed throughout the experiment, and plasma was collected at the end of the experiment, 28 days after disease induction.

[0223] Clinical disease is monitored daily and disease is graded on a scale of 0-8. 0=healthy 1=tail weakness 2=tail motor paralysis 3 = Tail paresis and mild waddling 4 = Tail paralysis and severe wobbling gait 5 = Tail paralysis and paralysis of one limb 6 = Tail paralysis and one pair of limbs paralysis 7 = Quadriplegia or paralysis of three limbs 8 = Pre-disease or death

[0224] Graphs and Statistics Graphical and statistical analyses were performed using Prism 5 for Mac OS X (GraphPad Software, San Diego, CA, USA). All statistical data were calculated using a one-sided nonparametric Mann-Whitney test, and p<0.05 was considered significant. *, # indicate p<0.05, **, ## indicate p<0.01.

[0225] Results and Discussion Figure 10 shows the onset of EAE in mice in the control group (vehicle and cyclosporine A) and the group treated with LMW-DS subcutaneous injections three times a week. Cyclosporine A had significantly (*) lower mean scores on days 13, 14, 16, 20, 21, and 25-27 compared to vehicle controls. Animals treated with 10 mg / kg dextran sulfate subcutaneous injections three times a week had significantly (#) lower mean scores on days 13, 14, 16, 17, 19, 21, and 26 compared to vehicle controls.

[0226] Figure 11 shows mice treated with vehicle and 100 ng / dose HGF intravenously every other day for 5 days starting on day 18 (see arrows). HGF did not produce any significant difference compared to vehicle.

[0227] Thus, LMW-DS produced significantly lower mean scores compared with vehicle controls in the EAE model, indicating that LMW-DS has beneficial effects on neurodegenerative and demyelinating diseases such as MS and ADEM of the CNS.

[0228] Example 3 The effects of daily subcutaneous injections of LMW-DS on glutamate excitotoxicity and mitochondrial function in rats after severe traumatic brain injury (sTBI) were assessed by high-performance liquid chromatography (HPLC) analysis of frozen brain samples. The results suggest that LMW-DS interferes with mitochondrial function, improving energy metabolism and reducing glutamate excitotoxicity.

[0229] material and method sTBI induction and drug administration protocol The experimental protocol used in this study was approved by the Ethics Committee of the Catholic University of Rome in accordance with international standards and guidelines for animal care. Male Wistar rats weighing 300–350 g body weight (bw) were kept in a controlled environment and provided with standard laboratory chow and water ad libitum.

[0230] The rats were divided into three groups: 1) n = 6 animals received sTBI, received drug administration 30 min later, and were sacrificed 2 days after TBI (acute phase 1). 2) n = 6 animals received severe TBI, received drug administration 30 min later, and were sacrificed 7 days after TBI (acute phase 2). 3) n = 6 animals received severe TBI, received drug treatment 3 days after TBI, and were sacrificed 7 days after TBI (chronic phase).

[0231] Animals received an anesthetic mixture of 35 mg / kg body weight ketamine and 0.25 mg / kg body weight midazolam via intraperitoneal injection. According to the "weight-drop" impact acceleration model (Marmarou et al., A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics. J Neurosurg. 1994;80:291-300), sTBI was induced by dropping a 450 g weight from a height of 2 m onto the head of rats protected by a metal disk previously fixed on the skull. Rats that suffered skull fractures, seizures, epistaxis, or did not survive the impact were excluded from the study. At the end of each treatment period, rats were re-anesthetized and then immediately sacrificed.

[0232] Drug treatment was administered by subcutaneous injection of 0.5 ml of LMW-DS (15 mg / kg) according to the outlined protocol described above.

[0233] Brain tissue processing In all animals, an in vivo osteotomy craniotomy was performed while under anesthesia, the rat's skull was carefully removed, and the brain was exposed, removed with a surgical spatula, and quickly placed in liquid nitrogen. After measuring wet weight (ww), tissue preparation was performed as previously described (Tavazzi et al., Cerebral oxidative stress and depression of energy metabolism correlate with severity of diffuse brain injury in rats. Neurosurgery. 2005;56:582-589; Vagnozzi et al., Temporal window of metabolic brain vulnerability to concussions: mitochondrial-related impairment—part I. Neurosurgery. 2007;61:379-388; Tavazzi et al., Temporal window of metabolic brain vulnerability to concussions: oxidative and nitrosative stresses—part II. Neurosurgery. 2007;61:390-395; Amorini et al., Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids. J Cell Mol Med. 2017;21:530-542). Briefly, whole brain homogenization was performed with 7 ml of ice-cold, nitrogen-saturated precipitation solution (CHCN + 10 mM KHPO, pH 7.40 (3:1; v:v) using an Ultra-Turrax set (Janke & Kunkel, Staufen, Germany) at 24,000 rpm / min. After centrifugation at 20,690 x g for 10 min at 4°C, the clear supernatant was saved, and the pellet was supplemented with 3 ml of precipitation solution and homogenized again as described above.A second centrifugation was performed (20,690 x g, 10 min at 4°C), the pellet was saved, and the supernatant was combined with the previous one and extracted by vigorous stirring with twice the volume of HPLC-grade CHCl3 and centrifuged as above. The upper aqueous phase containing water-soluble low molecular weight compounds was collected and subjected to two further chloroform washes (this procedure allows the removal of all organic solvents and any lipid-soluble compounds from the buffered tissue extract), and the volume was adjusted with 10 mM KH2PO4, pH 7.40, to obtain a final aqueous 10% tissue homogenate, which was stored at -80°C until assayed.

[0234] HPLC analysis of purine-pyrimidine metabolites An aliquot of each deproteinized tissue sample was filtered through a 0.45 μm HV Millipore filter and loaded (200 μl) onto a Hypersil C-18, 250 x 4.6 mm, 5 μm particle size column (Thermo Fisher Scientific, Rodano, Milan, Italy) with its own guard column. The column was then coupled to a HPLC system consisting of a Surveyor System (Thermo Fisher Scientific, Rodano, Milan, Italy) equipped with a high-sensitivity diode array detector (equipped with a 5 cm optical path flow cell) set at a wavelength of 200-300 nm. Data acquisition and analysis were performed on a PC using the ChromQuest software package provided by the HPLC manufacturer.

[0235] Metabolites belonging to the purine-pyrimidine profile (described below), those related to tissue energy status, mitochondrial function, and those related to oxidative-nitrosative stress were separated in a single chromatographic run according to a slightly modified, existing ion-pair HPLC method (Lazzarino et al., Single-sample preparation for simultaneous cellular redox and energy state determination. Anal Biochem. 2003;322:51-59; Tavazzi et al., Simultaneous high-performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis of inborn errors of metabolism. Clin Biochem. 2005;38:997-1008). Assignment and calculation of compounds of interest in chromatographic runs of tissue extracts were performed by comparing the retention times, absorption spectra, and areas of the peaks at appropriate wavelengths (206, 234, and 260 nm) with those of peaks in chromatographic runs of freshly prepared ultrapure standard mixtures of known concentrations.

[0236] List of compounds: cytosine, creatinine, uracil, beta-pseudouridine, cytidine, hypoxanthine, guanine, xanthine, cytidine diphosphate-choline (CDP-choline), ascorbic acid, uridine, adenine, nitrite (-NO2-), reduced glutathione (GSH), inosine, uric acid, guanosine, cytidine monophosphate (CMP), malondialdehyde (MDA), thymidine, orotic acid, nitrate (-NO3-), uridine monophosphate (UMP), nicotinamide adenine dinucleotide, oxidized (NAD +), adenosine (ADO), inosine monophosphate (IMP), guanosine monophosphate (GMP), uridine diphosphate-glucose (UDP-Glc), UDP-galactose (UDP-Gal), oxidized glutathione (GSSG), UDP-N-acetyl-glucosamine (UDP-GlcNac), UDP-N-acetyl-galactosamine (UDP-GalNac), adenosine monophosphate (AMP), guanosine diphosphate-glucose (GDP-glucose), cytidine diphosphate (CDP), UDP, GDP, nicotinamide adenine dinucleotide phosphate, oxidized (NADP) + ), adenosine diphosphate-ribose (ADP-ribose), cytidine triphosphate (CTP), ADP, uridine triphosphate (UTP), guanosine triphosphate (GTP), nicotinamide adenine dinucleotide, reduced form (NADH), adenosine triphosphate (ATP), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), malonyl-CoA, coenzyme A (CoA-SH), acetyl-CoA, N-acetylaspartate (NAA).

[0237] HPLC analysis of free amino acids and amino group-containing compounds Simultaneous measurement of primary free amino acids (FAAs) and amino-group-containing compounds (AGCCs) (described below) was performed using precolumn derivatization of samples with a mixture of ortho-phthalaldehyde (OPA) and 3-mercaptopropionic acid (MPA). This method has been described in detail elsewhere (Amorini et al., "Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids." J Cell Mol Med. 2017;21:530-542; Amorini et al., "Metabolic profile of amniotic fluid as a biochemical tool to screen for inborn errors of metabolism and fetal anomalies." Mol Cell Biochem. 2012;359:205-216). Briefly, a derivatization mixture consisting of 25 mmol / L OPA, 1% MPA, and 237.5 mmol / L sodium borate, pH 9.8, was prepared daily and loaded into the autosampler. Automated pre-column derivatization of samples (15 μl) with OPA-MPA was performed at 24°C, and 25 μl of the derivatization mixture was loaded onto an HPLC column (Hypersil C-18, 250 × 4.6 mm, 5 μm particle size, thermostated at 21°C) for subsequent chromatographic separation. In the case of glutamic acid, a 20-fold dilution with HPLC-grade HO was performed prior to the derivatization procedure in deproteinized brain extracts and subsequent injection.Separation of OPA-AA and OPA-AGCC was performed using two mobile phases (mobile phase A = 24 mmol / L CH3COONa + 24 mmol / L Na2HPO4 + 1% tetrahydrofuran + 0.1% trifluoroacetic acid, pH 6.5; mobile phase B = 40% CH3OH + 30% CH3CN + 30% HO) at a flow rate of 1.2 ml / min using an appropriate step gradient (Amorini et al., Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids. J Cell Mol Med. 2017;21:530-542; Amorini et al., Metabolic profile of amniotic fluid as a biochemical tool to screen for inborn errors of metabolism and fetal anomalies. Mol Cell Biochem. 2012;359:205-216).

[0238] The assignment and calculation of OPA-AA and OPA-AGCC in the chromatographic run of the whole brain extract was performed by comparing the retention time and area of ​​the peaks at a wavelength of 338 nm with those of the peaks in the chromatographic run of a freshly prepared ultrapure standard mixture with known concentrations.

[0239] List of FAA and ACGC compounds: aspartic acid (ASP), glutamic acid (GLU), asparagine (ASN), serine (SER), glutamine (GLN), histidine (HIS), glycine (GLY), threonine (THR), citrulline (CITR), arginine (ARG), alanine (ALA), taurine (TAU), gamma-aminobutyric acid (GABA), tyrosine (TYR), S-adenosylhomocysteine ​​(SAH), L-cystathionine (L-Cystat), valine (VAL), methionine (MET), tryptophan (TRP), phenylalanine (PHE), isoleucine (ILE), leucine (LEU), ornithine (ORN), lysine (LYS).

[0240] statistical analysis Normal data distribution was tested using the Kolmogorov-Smirnov test. Differences between groups were estimated by two-way analysis of variance for repeated measures. Fisher's constrained least squares method was used as a post-hoc test. Only two-sided p values ​​less than 0.05 were considered statistically significant.

[0241] result The most obvious result of brain levels of 24 standard and nonstandard amino acids and primary amino group-containing compounds was that LMW-DS treatment significantly suppressed the sTBI-induced increase in glutamate (GLU) (Figure 12), thus ensuring a reduction in excitotoxicity resulting from excess of this compound.

[0242] However, this effect was only observed when the drug was administered early after injury (30 min after sTBI), and there was no effect on this excitotoxicity marker when LMW-DS was injected 3 days after sTBI. It is also worth emphasizing that LMW-DS had a significant beneficial effect on compounds involved in the so-called methyl cycle (Met, L-Cystat, SAH; see Table 3). [Table 3] TIFF0007745261000006.tif76162a p<0.01 (compared with control); b p<0.05 (compared with control); c p<0.01 (compared to 2 days after TBI), d p<0.05 (compared to 2 days after TBI), e p<0.01 (compared to 5 days after TBI), f p<0.05 (compared to 5 days after TBI), g p<0.01 (compared with acute phase 2), h p<0.05 (compared with acute phase 2), i p<0.01 (compared with chronic phase), j p<0.05 (compared to chronic stage) Table 3 lists the compounds in μmol / g (ww).

[0243] As can be seen in Table 4, LMW-DS positively affected various compounds related to energy metabolism and mitochondrial function. Of particular interest were the adenine nucleotide concentration and the ATP / ADP ratio as measures of mitochondrial phosphorylation capacity (Figure 13). [Table 4] TIFF0007745261000008.tif230162TIFF0007745261000009.tif225162 a p<0.01 (compared with control); b p<0.05 (compared with control); c p<0.01 (compared to 2 days after TBI), d p<0.05 (compared to 2 days after TBI), e p<0.01 (compared to 5 days after TBI), f p<0.05 (compared to 5 days after TBI), g p<0.01 (compared with acute phase 2), h p<0.05 (compared with acute phase 2), i p<0.01 (compared with chronic phase), j p<0.05 (compared to chronic stage) Table 4 lists the compounds in nmol / g (ww).

[0244] Significant changes in oxidized and reduced nicotine coenzymes were also observed (Figure 14).

[0245] Parameters related to oxidative stress were also measured, and a significant reduction in oxidative stress was detected after administration of LMW-DS. In particular, ascorbic acid, the major water-soluble brain antioxidant, and GSH, the major intracellular SH donor, were measured. The results, as shown in Table 4 and Figure 15, showed a significant improvement in their levels after administration of LMW-DS.

[0246] In addition, MDA, a polyunsaturated fatty acid end product of membrane phospholipids and therefore used as a marker of ROS-mediated lipid peroxidation, was also measured. MDA levels showed a significant decrease after administration of LMW-DS. After treatment with LMW-DS, all of the above oxidative stress markers showed an improvement in the restoration of antioxidant status (Figure 15).

[0247] Indices representing NO-mediated nitrosative stress (nitrite and nitrate) were also analyzed. LMW-DS administration significantly reduced nitrate concentrations in both acute and chronic sTBI (Figure 16).

[0248] NAA is a brain-specific metabolite and a useful biochemical marker for monitoring deterioration or recovery after TBI. NAA is synthesized in neurons from aspartate and acetyl-CoA by aspartate N-acetyltransferase. To ensure NAA turnover, this molecule must travel between intracellular compartments to reach oligodendrocytes, where it is degraded to acetate and aspartate by aspartoacylase (ASPA). To ensure the availability of the substrate aspartate, upregulation of the catabolic enzymes ASPA and NAA decreases, and acetyl-CoA is an indicator of a metabolically impaired state. In this study, NAA and its substrates were measured after sTBI and showed significant improvement in levels after LMW-DS administration (Figure 17).

[0249] These effects on energy metabolites were particularly evident when animals received LMW-DS treatment early (30 min) after injury. It is important to note that the overall beneficial effects of LMW-DS were observed when animals were sacrificed 2 days after sTBI or 7 days after sTBI. In this group of animals, the overall recovery of metabolism related to AGCC and energy metabolites was more evident, suggesting a sustained positive effect of LMW-DS treatment on cerebral metabolism.

[0250] Consideration TBI is the leading cause of death and disability in the first 40 years of life. The cost to the UK economy alone is estimated at £8 billion per year, making it comparatively more costly to the economy than stroke. In the United States, the combined health care and socioeconomic costs of TBI are estimated to exceed $60 billion per year, excluding military expenditures. Additionally, recent years have seen a sharp increase in interest in sports concussions on both sides of the Atlantic.

[0251] Despite the clear clinical need, there are currently no approved pharmacological treatments for TBI. While the primary insult (contusion) associated with TBI may be amenable to surgical treatment, reduction of the subsequent secondary non-mechanical damage to the surrounding brain tissue (penumbra) may offer greater therapeutic opportunities.

[0252] Using well-established rodent models of severe traumatic brain injury (sTBI), characterized by the diffuse axonal injury of TBI, we have demonstrated that severely injured animals exhibit persistent cellular energy status and mitochondrial function (Vagnozzi et al., Changes of cerebral energy metabolism and lipid peroxidation in rats leading to mitochondrial dysfunction after diffuse brain injury. J Neurotrauma. 1999;16:903-913; Signoretti et al., N-Acetylaspartate reduction as a measure of injury severity and mitochondrial dysfunction following diffuse traumatic brain injury. J Neurotrauma. 2001;18:977-993; Tavazzi et al., Cerebral oxidative stress and depression of energy metabolism correlate with severity of diffuse brain injury in rats. Neurosurgery. 2005;56:582-589; Vagnozzi et al., Temporal window of metabolic brain vulnerability to It has previously been shown that mitochondrial-related impairment (Tavazzi et al., Temporal window of metabolic brain vulnerability to concussions: oxidative and nitrosative stresses—part II. Neurosurgery. 2007;61:379-388; Tavazzi et al., Temporal window of metabolic brain vulnerability to concussions: oxidative and nitrosative stresses—part II. Neurosurgery. 2007;61:390-395) as well as alterations in various metabolites related to amino acid metabolism (Amorini et al.,Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids.J Cell Mol Med.2017;21:530-542). In the complex molecular mechanisms underlying TBI-induced brain injury, metabolic changes are the initial cellular signals that influence enzyme activity and gene and protein expression changes that indicate pathological tissue responses (Di Pietro et al., Potentially neuroprotective gene modulation in an in vitro model of mild traumatic brain injury. Mol Cell Biochem. 2013;375:185-198; Di Pietro et al., The molecular mechanisms affecting N-acetylaspartate homeostasis following experimental graded traumatic brain injury. Mol Med. 2014;20:147-157; Di Pietro et al., Neuroglobin expression and oxidant / antioxidant balance after graded traumatic brain injury in the rat. Free Radic Biol Med. 2014;69:258-264; Amorini et al., Metabolic, enzymatic, and gene involvement in cerebral glucose dysmetabolism after traumatic brain injury. Biochim Biophys Acta Mol Basis of Dis. 2016;1862:679-687). This means that agents that function to favorably regulate cellular metabolism in injured tissue may reduce subsequent TBI-associated changes in enzyme activity and gene and protein expression that contribute to adverse outcomes.

[0253] The data presented herein suggest that early administration of LMW-DS reverses adverse changes in metabolic homeostasis by reducing levels of glutamate excitotoxicity and protecting mitochondrial function, demonstrating the neuroprotective effects of the compound after severe TBI. Thus, LMW-DS has the potential to be used to treat or prevent TBI, including sTBI.

[0254] Example 4 Analysis of gene expression changes induced by LMW-DS was investigated in several cell lines.

[0255] material and method Experimental design For each cell line, n=8x25cm 2 Culture flasks were prepared. On the day of treatment (24 hours after seeding), two flasks were harvested with each cell type. This represents the day 0 time point. From the remaining flasks, three flasks were treated with control medium and three with LMW-DS-containing medium (CM) to achieve a final concentration of 0.01 mg / ml. After 48 hours, cells were harvested from the treated flasks. Thus, the collected data represent (a) untreated cells (day 0 control and day 2 control) and (b) cells treated with LMW-DS for 48 hours (2-day LMW-DS treatment).

[0256] Coating of tissue culture dishes for all cells 25cm 2 Flasks were coated by adding 2 ml of a 50 μg / ml solution of poly-d-lysine in Hank's balanced salt solution (HBSS) per flask and incubating overnight at 37°C in the dark. Flasks were washed with cell culture water and air-dried in the dark for 30 minutes. Flasks were coated by adding 1 ml of a 25 μg / ml solution of laminin in phosphate-buffered saline (PBS) per flask and incubating for 2 hours at 37°C in the dark. Before seeding the cells, the laminin flasks were washed three times with PBS.

[0257] Human umbilical vein endothelial cells (HUVECs) Medium 200 + Large Vessel Endothelial Supplement (M200 + LVES) supplement (1:50) was prepared and pre-warmed to 37°C. Cells were thawed in a 37°C water bath for no more than 2 minutes and gently transferred to a 50 ml tube containing 20 ml of Dulbecco's Modified Eagle's Medium Nutrient Mixture F12 (DMEM-F12). The cell suspension was mixed by carefully inverting the tube twice. The cells were centrifuged at 400 x g for 10 minutes. The supernatant was removed, and the cells were resuspended in 10 ml of medium (M200 + LVES supplement).

[0258] Cells were counted using a Cellometer. 1,000,000 cells / flask was used for 25 cm 2 1000 cells were seeded into flasks (n=8) and medium was added to a total volume of 5 ml per flask. Cells were incubated at 37°C under 5% CO2. Cells were allowed to settle for 24 hours before LMW-DS treatment.

[0259] human Schwann cells Schwann cell growth medium was prepared by adding 10% fetal bovine serum (FBS) to high glucose DMEM and pre-warmed to 37° C. Cells were thawed in a 37° C. water bath for no more than 2 minutes.

[0260] The cells from the 12 vials were gently transferred to a tube containing 10 ml of high-glucose DMEM medium and centrifuged at 400 relative centrifugal force (RCF) for 10 minutes. The pellet was resuspended in medium. The cells from the 12 vials were mixed and spread onto a pre-coated 25 cm 2 The cells were evenly distributed among flasks (n=8). The cells were incubated at 37°C under 5% CO2. The cells were allowed to settle for 24 hours before LMW-DS treatment.

[0261] In mouse cortical neurons (Lonza) The medium was prepared by adding 10 ml of B-27 serum-free supplement and 2.5 ml of GlutaMAX™-I supplement to 500 ml of Neurobasal medium. The medium was preheated to 37°C. Cells from 12 vials were thawed sequentially in a 37°C water bath for no more than 2 minutes and gently transferred to 15 ml tubes. 9 ml of medium was gently added dropwise to each. The cell suspension was mixed by carefully inverting the tube twice.

[0262] The cells were centrifuged at 200xg for 5 minutes. The supernatant was removed (down to the final 0.5 ml) and the cells were gently resuspended by trituration. The cells from the 12 vials were mixed and applied to a pre-coated 25cm 2 The cells were evenly distributed among flasks (n=8). The cells were incubated at 37°C under 5% CO2 for 24 hours.

[0263] Mouse motor neuron (Aruna) The medium was prepared according to Table 5. [Table 5]

[0264] Culture medium (see Table 5) was pre-warmed to 37°C. Cells were thawed in a 37°C water bath for no more than 2 minutes. 9 ml of culture medium was gently added dropwise. The cell suspension was mixed by carefully inverting the tube twice. Cells were counted using a Cellometer. Cells were centrifuged at 200 x g for 5 minutes. The supernatant was removed (down to the last 0.5 ml) and the cells were gently resuspended by trituration. Cells from 8 vials were mixed and plated onto pre-coated 25 cm plates. 2 The cells were evenly distributed among flasks (n=8). Cells were incubated at 37°C under 5% CO2 for 24 hours before treatment.

[0265] Drug Treatment LMW-DS was prepared at a stock concentration of 20 mg / ml and kept in a temperature-monitored refrigerator at 4°C. Fresh 100X LMW-DS stock (1.0 mg / ml) was prepared in sterile DMEM-F12. The concentrated drug stock was filter-sterilized and added to the respective media (19.6 ml of CM and 0.4 ml of LMW-DS stock solution). A control was made using 19.6 ml of CM and 0.4 ml of DMEM-F12. LMW-DS and CM were added to their respective flasks (5 ml each), resulting in a concentration of 0.01 mg / ml LMW-DS in each dish containing a total of 10 ml of CM.

[0266] Culture harvest and cell lysis The CM was aspirated into a clean, labeled 15 ml Falcon tube. The flask (without medium) was placed in a -80°C freezer for 30 minutes. The CM in the Falcon tube was centrifuged at 3000 x g for 5 minutes. The supernatant was removed, and the small pellet was resuspended in 2.5 ml of Trizol:water (4:1) solution at room temperature (RT, approximately 22°C).

[0267] The frozen flasks were removed from the freezer one by one, and Trizol-water was transferred from the appropriate tube into the flask. The flasks were left at room temperature for 5 minutes, after which the contents were aspirated back into 15 ml Falcon tubes (after thoroughly rinsing the bottom of the flask with the solution). The flasks were examined under a microscope to ensure complete removal of cells. The collected lysates in the 15 ml Falcon tubes were placed in a -80°C freezer.

[0268] RNA extraction The homogenate-containing Falcon tube was removed from the freezer and stored at room temperature for 5 minutes to allow complete disintegration of nucleoprotein complexes.

[0269] Two 1 ml aliquots of lysate were removed from each sample, and 200 μl of chloroform was added to each (0.2 ml of chloroform per ml of Trizol reagent was used during the cell lysis step), and the tubes were vigorously shaken. The samples were stored at room temperature for 2–3 min and then centrifuged at 12,000 × g for 15 min at 4°C.

[0270] The mixture separated into three layers: a bottom red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remained in the upper aqueous phase, DNA in the white interphase (interphase), and protein in the pink bottom (organic) phase. Three-quarters of the upper aqueous phase was transferred to a new, clean Eppendorf tube.

[0271] RNA was precipitated from the aqueous phase by adding an equal volume of 100% ethanol. The precipitated RNA was immobilized on a Spin Cartridge, washed twice, and dried. RNA was eluted with 50 μl of warm RNase-free water. The quantity and quality of purified RNA were determined by NanoDrop. RNA was stored at -80°C before being shipped to Source Bioscience for array analysis.

[0272] Expression data analysis plan Expression data were downloaded into separate files for each cell line. "Background-corrected" expression is data from the array's "gProcessedSignal," which is the result of subtracting background signal from the actual signal of the associated probe. This is the most commonly used variable in array analysis. Background-corrected signals were log2-transformed for statistical analysis across all samples. To reduce the false positive rate in a sample, signals below the "expression level" were removed. The "below expression" level was set to 5 of the log2-transformed expression value.

[0273] statistical analysis To reduce variability in the results, it was decided to perform median centering on all arrays prior to analysis based on the expression pattern of the control probes for each array. Data were grouped by cell type, and each cell type was analyzed using the following algorithm: Comparison of D0 control samples with D2 control samples - Expression changes observed in cells in normal cultures Comparison of D0 control samples with D2 LMW-DS-treated samples - Expression changes observed in cells in LMW-DS-treated cultures Comparison of D2 LMW-DS-treated samples with D2 control samples - Differential expression induced by LMW-DS in culture

[0274] A preliminary analysis was performed to screen out genes that were not differentially expressed between any combination of the three datasets. A simple, non-stringent analysis of variance (p<0.05) was performed to look for expression patterns. Probes that showed no change across the three datasets were removed. A volcano plot was used to analyze the remaining probe sets for fold change and significance. To allow for the detection of expression patterns, in the first case, a change in probe expression of more than 20% (FC≧1.2 or FC≦0.84) was considered significant.

[0275] Quality Parameters The seeding density was calculated from the number of cells taken from the Schwann cell stock. HUVECS were seeded at their optimal density.

[0276] Additional quality control from the array service provider indicated that the RNA was of high quality (no degradation) and the quantity was within the parameters of the low input RNA microarray from Agilent.

[0277] Analysis of the raw data indicated that significant differences existed between arrays, as expected. However, these differences (reflecting differences among the same control samples included on all arrays) were easily removed with normalization techniques. Median-centering the data to remove selected inter-array variations did not affect the overall differences expected to be observed between controls representing different concentrations of RNA.

[0278] Expression analysis of Schwann cells As previously described, genes not expressed in Schwann cells were removed prior to data analysis. The "under-expressed" level was set at 5 log2-transformed expression values. This left 15,842 unique analytical probes in Schwann cell cultures. In the next step of the analysis, three sets of data (comparison of D0 control samples to D2 control samples; comparison of D0 control samples to D2 LMW-DS-treated samples; comparison of D2 control samples to D2 LMW-DS-treated samples) were analyzed to determine the effects of CM on cells and the relative changes induced by LMW-DS.

[0279] Comparing D0 control samples to D2 control samples, 585 genes were differentially expressed in Schwann cell cultures. The molecular functions affected by these genes were cell motility (1.14E-07 to 2.49E-03); cell morphology (5.56E-07 to 2.36E-03); cell development (7.3E-06 to 2.48E-03); cell growth and proliferation (7.3E-06 to 2.48E-03); cell assembly and organization (1.23E-05 to 2.36E-03); cell function and maintenance (1.23E-05 to 2.47E-03); cell death and survival (1.53E-05 to 2.51E-03); lipid metabolism (8.14E-05 to 1.6E-03); and small molecule biochemistry (8.14E-05 to 1.6E-03). molecular transport (1.18E-04 to 2.29E-03); protein transport (1.62E-04 to 1.6E-03); glucose metabolism (3.22E-04 to 1.78E-03); gene expression (3.98E-04 to 2.2E-03); intracellular signaling (4.39E-04 to 2.25E-03); cell-cell signaling and interaction (5.05E-04 to 2.48E-03); cell damage (7.69E-04 to 1.58E-03); cell cycle (1.12E-03 to 1.8E-03); amino acid metabolism (1.6E-03 to 1.6E-03); and nucleic acid metabolism (1.6E-03 to 1.6E-03).

[0280] The values ​​shown above are p-values ​​representing the statistical significance associated with these genes in different pathways. The two p-values ​​represent the lower and upper bounds of the observed statistical significance (p<0.05 is significant).

[0281] LMW-DS induced differential expression of 1244 genes in Schwann cell cultures, as assessed by comparing D0 control samples with D2 LMW-DS-treated samples. The molecular functions affected by these genes included cell morphology (1.43E-08 to 8.39E-04); cell motility (1.4E-07 to 9.6E-04); post-translational modifications (3.93E-07 to 6.71E-05); protein synthesis (3.93E-07 to 1.08E-04); protein transport (3.93E-07 to 1.26E-06); cell death and survival (2.13E-06 to 8.65E-04); cell assembly and organization (7.46E-06 to 8.24E-04); DNA replication, recombination, and repair (7.46E-06 to 7.46E-06); and cell function and maintenance (9.53E-06 to 9.53E-06). -06~6.46E-04); gene expression (1.27E-05~4.92E-04); cell development (1.29E-05~9.06E-04); cell growth and proliferation (1.29E-05~9.06E-04); cell-cell signaling and interaction (1.97E-05~8.81E-04); amino acid metabolism (4.22E-05~8.24E-04); small molecule biochemistry (4.22E-05~8.24E-04); lipid metabolism (4.81E-05~3.64E-04); molecular transport (3.64E-04~3.64E-04); and cell cycle (4.53E-04~4.86E-04).

[0282] LMW-DS induced differential expression of 700 genes in Schwann cell cultures, as assessed by comparing D2 LMW-DS-treated samples with D2 control samples. The molecular functions affected by these genes included cell morphology (1.49E-07 to 5.62E-03); cell assembly and organization (1.49E-07 to 5.95E-03); cell motility (7.24E-07 to 6.06E-03); cell death and survival (9.41E-06 to 5.95E-03); amino acid metabolism (2.56E-05 to 3.7E-03); and cell morphology (1.49E-07 to 5.62E-03). Post-translational modifications (2.56E-05 to 1.05E-03); small molecule biochemistry (2.56E-05 to 3.7E-03); cell signaling and interactions (5.05E-05 to 5.76E-03); gene expression (7.18E-05 to 4.94E-03); cell cycle (1.06E-04 to 5.95E-03); cell development (1.06E-04 to 5.95E-03) ); Cellular function and maintenance (1.96E-04 to 5.95E-03); Cellular growth and proliferation (2.35E-04 to 5.95E-03); DNA replication, recombination, and repair (2.75E-04 to 5.95E-03); Intracellular signal transduction (5.92E-04 to 2.54E-03); Cellular damage (6.26E-04 to 6.26E-04); Lipid metabolism (6.26E -04 to 1.85E-03); molecular transport (6.26E-04 to 5.95E-03); protein synthesis (1.05E-03 to 1.93E-03); cellular response to therapeutic drugs (1.85E-03 to 1.85E-03); protein transport (2.66E-03 to 5.95E-03); and RNA post-transcriptional modification (4.32E-03 to 4.32E-03).

[0283] Mechanistic molecular network models simulate the effects of differentially regulated molecules by LMW-DS and allow for the evaluation of the functional consequences of these changes. In silico models show that LMW-DS suppresses neuronal cell death, apoptosis, and protein synthesis, and suppresses angiogenesis, cell migration, cell viability, cell survival, cell motility, cell proliferation, cell differentiation, cell homeostasis, cell cycle progression, cell transformation, and RNA expression.

[0284] Table 6 summarizes the results of changes in gene expression in cultured Schwann cells. [Table 6]

[0285] Twenty-one genes whose expression changed in the control cultures for 2 days showed no change whatsoever in the LMW-DS-treated cultures for the same 2 days. One gene whose expression increased in the control cultures was downregulated in the LMW-DS-treated cultures for the same 2 days. Thirteen genes that were downregulated in the control cultures were upregulated in the LMW-DS-treated cultures for the same 2 days. 122 genes were significantly downregulated by growth factors in the medium, and this downregulation was even stronger than in the LMW-DS-treated cultures. 441 genes were upregulated in the control cultures, and the addition of LMW-DS significantly strengthened this upregulation.

[0286] Expression analysis of HUVECs As previously described, genes not expressed in HUVECs were removed before any analysis was attempted. The "under-expressed" level was set to a log2-transformed expression value of 5. This left 15,239 unique probes for analysis in HUVEC cultures. In the next step of the analysis, the three sets of data were analyzed to determine the effects of CM on gene expression in the cells and the differences induced by LMW-DS. A preliminary analysis was performed to select and remove genes that were not differentially expressed between any combination of the three datasets. A simple, non-stringent analysis of variance (p<0.05) was performed to look for expression patterns. Genes that did not change across the three datasets were removed, leaving a total of 12,313 probes (10,368 genes) for analysis.

[0287] Comparing the D0 control samples to the D2 control samples, 1551 genes were differentially expressed in HUVEC cultures. The molecular functions affected by these genes are cell assembly and organization (2.55E-15 to 1.29E-03); cell function and maintenance (2.55E-15 to 1.29E-03); cell cycle (1.98E-11 to 1.32E-03); cell morphology (3.18E-10 to 1.29E-03); gene expression (1.05E-08 to 2.01E-04); cell development (1.66E-07 to 1.37E-03); cell growth and proliferation (1.66E-07 to 1.37E-03); DNA replication, recombination, and repair (2.04E-07 to 9.84E-04); cell death and survival (2.09E-07 to 1.3E-03); and RNA post-transcriptional modification (4.86E-06 ~6.53E-04); cell motility (9.9E-06~1.18E-03); post-translational modifications (1.92E-05~1.34E-03); cell-cell signaling and interactions (2.19E-05~9.1E-04); protein synthesis (5.49E-05~1.14E-03); cell damage (8.16E-05~8.16E-05); molecular transport (6.27E-04~6.27E-04); protein transport (6.27E-04~6.27E-04); intracellular signaling (8.86E-04~8.86E-04); cellular response to therapeutic drugs (9.84E-04~9.84E-04); and protein degradation (1.14E-03~1.14E-03).

[0288] LMW-DS induced differential expression of 1779 genes in HUVEC cultures, as assessed by comparing D0 control samples with D2 LMW-DS-treated samples. The molecular functions affected by these genes included cell assembly and organization (4.14E-17 to 9.7E-04); cell function and maintenance (4.14E-17 to 8.05E-04); cell cycle (5.83E-14 to 9.85E-04); cell morphology (1.69E-10 to 7.48E-04); gene expression (7.99E-09 to 8.62E-04); cell death and survival (2E-08 to 8.4E-04); cell development (1.28E-07 to 8.88E-04); cell growth and proliferation (1.28E-07 to 8.88E-04); DNA replication, recombination, and repair (3.07E-07 to 9.7E-04); and RNA replication, recombination, and repair (3.07E-07 to 9.7E-04). post-transcriptional modifications (1.13E-06 to 6.31E-04); cell motility (1.42E-06 to 8.34E-04); post-translational modifications (3.4E-05 to 9.17E-04); cell-cell signaling and interactions (6.97E-05 to 9.56E-04); molecular transport (7.43E-05 to 9.7E-04); protein transport (7.43E-05 to 7.43E-05); RNA transport (1.57E-04 to 5.72E-04); protein synthesis (1.92E-04 to 9.02E-04); cell damage (2.47E-04 to 6.28E-04); and intracellular signaling (4.64E-04 to 9.02E-04).

[0289] LMW-DS induced differential expression of 76 genes in HUVEC cultures, as assessed by comparing D2 control samples with D2 LMW-DS-treated samples. The molecular functions affected by these genes were DNA replication, recombination, and repair (9.62E-05 to 2.57E-02); cell cycle (1.22E-04 to 2.4E-02); cell development (1.59E-04 to 2.67E-02); cell morphology (4.64E-04 to 2.42E-02); cell function and maintenance (4.64E-04 to 2.57E-02); lipid metabolism (9.49E-04 to 1.07E-02); and molecular transport (9.62E-05 to 2.57E-02). .49E-04-1.61E-02); Small Molecule Biochemistry (9.49E-04-1.87E-02); Cell Damage (1.6E-03-2.62E-02); Cell Death and Survival (2.06E-03-2.67E-02); Amino Acid Metabolism (2.7E-03-2.7E-03); Glucose Metabolism (2.7E-03-1.07E-02); Cell Signaling and Interaction (2.7E-03-2.4E-02); Cell Assembly and Organization Origenation (2.7E-03 to 2.57E-02); Cell growth and proliferation (2.7E-03 to 2.4E-02); Cell motility (2.7E-03 to 2.4E-02); Energy production (2.7E-03 to 2.7E-03); Nucleic acid metabolism (2.7E-03 to 1.07E-02); Post-translational modification (2.7E-03 to 1.61E-02); Gene expression (5.39E-03 to 2.36E-02); RNA post-transcriptional modification (5.39E-03 to 2.4E-02); drug metabolism (8.07E-03 to 1.61E-02); vitamin and mineral metabolism (8.07E-03 to 8.07E-03); protein synthesis (1.07E-02 to 1.07E-02); RNA transport (1.07E-02 to 1.07E-02); cellular response to therapeutic drugs (1.24E-02 to 1.24E-02); and free radical scavenging activity (1.43E-02 to 1.43E-02).

[0290] Although the overall differences between control and LMW-DS-treated cultures after 2 days of treatment do not immediately appear to be large, the effect of LMW-DS on gene expression changes was significant, especially considering the modulation of growth factor-induced gene expression by LMW-DS.

[0291] Using mechanistic molecular network models, we can simulate the effects of differentially regulated genes by LMW-DS and explore the functional consequences of these changes. In silico models show that LMW-DS suppresses neuronal cell death, apoptosis, and protein synthesis, and activates angiogenesis, cell migration, cell viability, cell survival, cell motility, cell proliferation, cell differentiation, cell homeostasis, cell cycle progression, cell transformation, and RNA expression.

[0292] HUVEC control cultures contained growth factors. In treated cultures, LMW-DS was added to medium already containing growth factors.

[0293] Table 7 summarizes the results of gene expression changes in cultured HUVECs. Sixty-seven genes whose expression changed (due to the effects of growth factors) in control cultures for 2 days showed no change whatsoever in LMW-DS-treated cultures for the same 2 days. Four genes whose expression increased in control cultures containing growth factors were downregulated in LMW-DS-treated cultures for the same 2 days. Eleven genes that were downregulated by growth factors in control cultures were upregulated in LMW-DS-treated cultures for the same 2 days. 120 genes were significantly downregulated by growth factors, and this downregulation was stronger in LMW-DS-treated cultures. 229 genes were upregulated in control cultures, and the addition of LMW-DS significantly strengthened this upregulation. [Table 7]

[0294] We analyzed the effects of LMW-DS on several molecular pathways important for different disease states and therapeutic applications. For this analysis, we compared the effect of LMW-DS addition on gene expression to that observed in cells in CM, and predicted functional effects based on the observed changes in expression patterns.

[0295] Expression analysis of motor neurons As previously described, genes not expressed in motor neurons were removed before any analysis was attempted. The "under-expressed" level was set at a log2-transformed expression value of 5. This left 12,240 unique probes that met the expression threshold in at least three samples in the series. In the next step, the three sets of data were analyzed to determine the effects of CM on cells and the differences induced by LMW-DS.

[0296] The changes in gene expression under normal culture conditions mimic the normal developmental process of motor neurons when they develop a motor neuron phenotype from a set of dissociated cells. The growth factors in the normal medium are necessary for these cells to differentiate. The stressor present in these cultures is oxidative stress (normal in tissue culture conditions).

[0297] Comparing the D0 control samples to the D2 control samples, 485 genes were differentially expressed in the motor neuron cultures. The molecular functions affected by these genes were: cell death and survival (1.99E-17 to 1.98E-04); cell motility (1.14E-16 to 1.91E-04); cell assembly and organization (1.22E-16 to 1.93E-04); cell function and maintenance (1.22E-16 to 1.95E-04); cell morphology (6.46E-16 to 1.74E-04); cell-cell signaling and interaction (3.16E-12 to 1.95E-04); cell development (1.59E-10 to 1.93E-04); cell growth and proliferation (1.59E-10 to 1.9E-04); molecular transport (4.27E-10 to 1.89E-04); protein synthesis (9.85E-09 to 5.03E-05); and lipid metabolism (1.08E-04). -08~1.61E-04; Small Molecule Biochemistry (1.08E-08~1.89E-04); Gene Expression (8.45E-08~3.8E-05); Cell Cycle (4.55E-07~1.09E-04); Free Radical Scavenging (7.12E-07~1.65E-04); Intracellular Signal Transduction (1.23E-05~1.89E-04); Vitamins and Minerals neural metabolism (1.23E-05 to 1.89E-04); protein degradation (3.07E-05 to 1.31E-04); glucose metabolism (3.32E-05 to 1.61E-04); drug metabolism (4.16E-05 to 4.16E-05); post-translational modification (7.1E-05 to 1.31E-04); and protein folding (7.1E-05 to 7.1E-05).

[0298] LMW-DS induced differential expression of 315 genes in motor neurons, as assessed by comparing D0 control samples with D2 LMW-DS-treated samples. The molecular functions affected by these genes were cell death and survival (6.54E-08 to 9.06E-03); cell motility (8.21E-08 to 5.42E-03); cell assembly and organization (8.36E-08 to 9.01E-03); cell function and maintenance (8.36E-08 to 9.01E-03); cell morphology (2.9E-06 to 8.75E-03); and cell development. Biogenesis (1.04E-05 to 9.01E-03); Cell growth and proliferation (1.04E-05 to 7.83E-03); DNA replication, recombination and repair (2.79E-05 to 8.01E-03); Cell-cell signaling and interaction (8.18E-05 to 7.11E-03); Post-translational modification (1.32E-04 to 7.56E-03); Protein degradation (1.32E-04 to 4. 35E-03); protein synthesis (1.32E-04~5.09E-03); gene expression (1.9E-04~9.01E-03); cell damage (3.58E-04~9.01E-03); cell cycle (6.08E-04~9.01E-03); free radical scavenging (7.41E-04~7.31E-03); amino acid metabolism (7.67E-04~6.61E-03); small Molecular biochemistry (7.67E-04 to 9.01E-03); vitamin and mineral metabolism (7.67E-04 to 1.13E-03); lipid metabolism (1.05E-03 to 9.01E-03); molecular transport (1.05E-03 to 9.01E-03); intracellular signal transduction (1.13E-03 to 5.09E-03); and glucose metabolism (4.71E-03 to 4.71E-03).

[0299] LMW-DS induced differential expression of 425 genes in motor neurons as assessed by comparison of D0 control samples to D2 LMW-DS treated samples. The molecular functions affected by these genes are cell death and survival (2.87E-08 to 6.27E-03); cell motility (4.73E-07 to 6.47E-03); cell morphology (4.95E-07 to 7.47E-03); cell development (1.02E-06 to 7.13E-03); cell growth and proliferation (1.02E-06 to 7.48E-03); cell assembly and organization (7.03E-06 to 7.47E-03); cell function and maintenance (7.03E-06 to 7.47E-03); gene expression (1.95E-05 to 6.18E-03); cell cycle (2.88E-05 to 7.48E-03); DNA replication, recombination, and repair (3.39E-05 to 5.16E-03); and amino acid metabolism (7.75E-05 ~4.68E-03);Small Molecule Biochemistry (7.75E-05~4.68E-03);Cellular Damage (8.23E-05~4.61E-03);Cellular Signaling and Interaction (3.27E-04~7.48E-03);Vitamin and Mineral Metabolism (3.27E-04~3.27E-04);Protein Synthesis (8.94E-04~5.29E-03); post-translational modifications (9.67E-04 to 9.67E-04); molecular transport (9.7E-04 to 4.68E-03); protein transport (9.7E-04 to 9.7E-04); glucose metabolism (1.44E-03 to 1.92E-03); cellular response to therapeutic drugs (1.92E-03 to 1.92E-03); and lipid metabolism (4.68E-03 to 4.68E-03). [Table 8]

[0300] Expression analysis of cortical neurons As previously described, genes not expressed in motor neurons were removed before any analysis was attempted. The "under-expressed" level was set at a log2-transformed expression value of 5. This left 10,653 unique probes that met the expression threshold in at least three samples in the series. In the next step, the three sets of data were analyzed to determine the effects of CM on cells and the differences induced by LMW-DS.

[0301] Under normal culture conditions, changes in gene expression mimic the normal development of cortical neurons when they develop a cortical neuron phenotype from a set of dissociated cells. The growth factors in the normal medium are necessary for these cells to differentiate. The stressor present in these cultures is oxidative stress (normal in tissue culture conditions).

[0302] Comparing the D0 control samples to the D2 control samples, 1101 genes were differentially expressed in the motor neuron cultures. The molecular functions affected by these genes included cell assembly and organization (3.57E-25 to 6.65E-04); cell function and maintenance (3.57E-25 to 6.65E-04); cell morphology (4.28E-22 to 6.36E-04); cell development (4.28E-22 to 6.53E-04); cell growth and proliferation (4.28E-22 to 6.6E-04); and intercellular signaling. Nuclear communication and interaction (2.16E-13 to 6.65E-04); molecular transport (5.18E-12 to 4.95E-04); cell motility (1.86E-11 to 6.65E-04); cell death and survival (3.37E-11 to 6.41E-04); gene expression (1.27E-08 to 8.96E-05); protein synthesis (3.84E-07 to 8.69E-05); small molecule biochemistry (6 0.65E-07 to 5.18E-04; cell damage (7.12E-06 to 4.54E-04); protein degradation (1.62E-05 to 1.62E-05); amino acid metabolism (2.11E-05 to 4.25E-04); protein transport (3.4E-05 to 3.4E-05); intracellular signal transduction (8.69E-05 to 3E-04); post-translational modification (8.69E-05 to 2.15E-0 4); protein folding (2.15E-04 to 2.15E-04); cell cycle (2.69E-04 to 3.07E-04); DNA replication, recombination and repair (2.69E-04 to 4.77E-04); nucleic acid metabolism (2.69E-04 to 2.69E-04); lipid metabolism (3.12E-04 to 5.18E-04); and glucose metabolism (5.18E-04 to 5.18E-04).

[0303] LMW-DS induced differential expression of 609 genes in motor neurons as assessed by comparison of D0 control samples to D2 LMW-DS treated samples. The molecular functions affected by these genes are cell assembly and organization (3.91E-15 to 1.83E-03); cell function and maintenance (3.91E-15 to 1.83E-03); cell morphology (2.53E-13 to 1.43E-03); cell development (2.53E-13 to 1.81E-03); cell growth and proliferation (2.53E-13 to 1.83E-03); cell motility (4.95E-09 to 1.2E-03); cell-cell signaling and interaction (5.96E-09 to 1.47E-03); cell death and survival (2.25E-08 to 1.77E-03); molecular transport (7.08E-08 to 1.79E-03); DNA replication, recombination, and repair (3.03E-06 to 1.71E-03); and cell damage (9.23E-06 to 7. 0.65E-04); Amino Acid Metabolism (1.75E-05 to 1.64E-03); Cell Cycle (1.75E-05 to 1.77E-03); Small Molecule Biochemistry (1.75E-05 to 1.79E-03); Protein Synthesis (2.77E-05 to 1.5E-03); Protein Transport (2.77E-05 to 1.9E-04); Intracellular Signal Transduction (7.65E-05 to 1.73E-0 3); post-translational modification (3.01E-04 to 1.4E-03); gene expression (3.65E-04 to 1.15E-03); drug metabolism (6.49E-04 to 6.49E-04); glucose metabolism (6.95E-04 to 7.69E-04); vitamin and mineral metabolism (1.09E-03 to 1.09E-03); and nucleic acid metabolism (1.44E-03 to 1.73E-03).

[0304] LMW-DS induced differential expression of 247 genes in motor neurons as assessed by comparison of D0 control samples to D2 LMW-DS treated samples. The molecular functions affected by these genes were: cell morphology (6.01E-08 to 1.01E-02); cell development (7.46E-08 to 1.01E-02); cell growth and proliferation (7.46E-08 to 1.01E-02); cell death and survival (4.23E-07 to 1.01E-02); cell motility (2.69E-06 to 9.91E-03); cell assembly and organization (1.57E-05 to 1.01E-02); cell function and maintenance (1.57E-05 to 1.01E-02); cell cycle (1.01E-04 to 1.01E-02); cell signaling and interaction (1.01E-04 to 1.01E-02); and lipid metabolism (1.56E-0 4 to 1.01E-02); small molecule biochemistry (1.56E-04 to 1.01E-02); gene expression (2.28E-04 to 3.38E-03); RNA damage and repair (2.28E-04 to 2.28E-04); RNA post-transcriptional modification (2.28E-04 to 2.28E-04); molecular transport (4.18E-04 to 8.32E-03); cellular damage (4.47E-04 to 2.2E-03); protein synthesis (2.66E-03 to 7.29E-03); protein transport (4.11E-03 to 8.32E-03); protein degradation (5.64E-03 to 7.29E-03); and DNA replication, recombination and repair (7.31E-03 to 1.01E-02). [Table 9]

[0305] Effects of LMW-DS on mitochondrial oxidative stress pathway Oxidative stress pathways occurring in mitochondria are important not only in cancer but also in aging and age-related degenerative diseases. Normal growth conditions trigger a certain amount of oxidative stress in cells, which contributes to the aging process both in vivo and in vitro.

[0306] In Schwann cells cultured under normal conditions, complex I (NADH dehydrogenase), labeled A in Figure 18, was inhibited, whereas complex IV (cytochrome c oxidase), labeled B in Figure 18, was activated. When LMW-DS was added to the cultures, complex III (cytochrome bc1), labeled C in Figure 18, was inhibited. Inhibition of complex III suppresses oxidative stress phenomena involved in the pathogenesis of cancer and neurological diseases.

[0307] Complex III, sometimes called coenzyme Q:cytochrome c oxidoreductase or cytochrome bc1 complex, is the third complex in the electron transport chain (EC 1.10.2.2) and plays a key role in the biochemical production of ATP (oxidative phosphorylation). Complex III is a multisubunit transmembrane protein encoded by both the mitochondrial (cytochrome b) and nuclear genomes (all other subunits). Complex III is present in the inner membrane mitochondria of all animals, all aerobic eukaryotes, and most eubacteria. Mutations in complex III result in exercise intolerance and multisystem diseases. The bc1 complex contains 11 subunits: three respiratory subunits (cytochrome B, cytochrome C1, and Rieske protein), two core proteins, and six low-molecular-weight proteins.

[0308] In HUVECs, no significant modulation of the mitochondrial effects of oxidative stress was detected after treatment with LMW-DS.

[0309] Under normal culture conditions, motor neurons appear to undergo significant oxidative stress, which leads to the activation of certain apoptotic mechanisms, as indicated by F in Figure 18, including activation of cytochrome C, AIF, and caspases 3, 8, and 9. Furthermore, motor neurons are characterized by the production of amyloid-β, as indicated by E in Figure 18, and fatty acid oxidation, as indicated by G in Figure 18, which further exacerbate oxidative stress and FIAS1-mediated mitochondrial fragmentation in cells. Furthermore, complex V, as indicated by D in Figure 18, is activated.

[0310] Addition of LMW-DS to the cultures ameliorated these negative effects by blocking the pathway marked F in Figure 18 and preventing its negative effects on amyloid-β production and mitochondrial fragmentation and dysfunction, and subsequent damage, marked E in Figure 18, and by suppressing fatty acid oxidation, marked G in Figure 18, thereby preventing and suppressing apoptosis. LMW-DS also inhibited the pathway involving TRAK1 and PINK1, marked H in Figure 18, thereby contributing to improved mitochondrial function. LMW-DS further reduced the levels of HO, marked I in Figure 18. An additional effect was the inhibition of HtrA2, marked J in Figure 18, which contributed to the suppression of apoptosis.

[0311] Under normal culture conditions, cortical neurons are exposed to significant oxidative stress, leading to amyloid-β production and Lewy body formation accompanied by synuclein-α activation and increased levels of ROS (labeled K in Figure 18 ); apoptosis (labeled F in Figure 18 ); mitochondrial fragmentation (labeled E in Figure 18 ); and impaired mitochondrial function accompanied by C161 (labeled L in Figure 18 ). Addition of LMW-DS to the cultures prevented and reversed most of these adverse effects, including the accumulation of amyloid-β and Lewy body pathologies (labeled E and K in Figure 18 ) and mitochondrial dysfunction (labeled L in Figure 18 ). Some apoptosis-inducing mechanisms (labeled F in Figure 18 ) remained active in the cultures, likely due to their robust activation.

[0312] Effect of LMW-DS on glutamate excitotoxicity Glutamate is an essential excitatory amino acid involved in long-term potentiation (LTP), i.e., learning and memory function. However, excess glutamate is also associated with excitotoxicity, leading to neuronal death. This latter phenomenon is postulated to be involved in neuronal death caused in chronic neurodegenerative conditions (including TBI). Genes involved in glutamate signaling were not expressed in HUVECs but are present in the Schwann and neuronal cell lines used in this study (see Figure 19).

[0313] Glutamate production is suppressed by baseline conditions in motor neuron cultures. The suppression is unaffected by LMW-DS. Glutamate production is elevated in cortical neurons at baseline. Addition of LMW-DS did not alter glutamate production in these cells.

[0314] Addition of LMW-DS to the CM of Schwann cells induced the expression of a protein complex (CALM, Gβγ, GRM7, PICK1) marked as A in Figure 19. More importantly, LMW-DS increased the activity and / or levels of glutamate transporters, particularly SLC1A2 / 3, in Schwann cells, thereby resulting in the removal of glutamate produced and released by presynaptic neurons. Thus, LMW-DS induced Schwann cells to remove toxic glutamate from the synaptic cleft, thereby preventing its excitotoxicity.

[0315] SLC1A3, member 3 of the solute carrier family 1 (glial high-affinity glutamate transporter), is a protein encoded by the SLC1A3 gene in humans. SLC1A3 is also often referred to as glutamate-aspartate transporter (GLAST) or excitatory amino acid transporter 1 (EAAT1). SLC1A3 is primarily expressed in the plasma membrane, where it allows glutamate to be removed from the extracellular space. SLC1A3 is also localized in the inner mitochondrial membrane as part of the malate-aspartate shuttle. SLC1A3 functions as a homotrimer in vivo. SLC1A3 mediates the transport of glutamate and aspartate and transports three Na + and one H + Cotransports cations and one K + It countertransports cations. This symport coupling (or isotropic transport) allows glutamate to be transported into cells against its concentration gradient. SLC1A3 is expressed throughout the CNS and is highly expressed in astrocytes and Bergmann glia in the cerebellum. In the retina, SLC1A3 is expressed in Müller glial cells. SLC1A3 is also expressed in many other tissues, including cardiac myocytes.

[0316] SLC1A2, solute transporter family 1 member 2, also known as excitatory amino acid transporter 2 (EAAT2) and glutamate transporter 1 (GLT-1), is a protein encoded by the SLC1A2 gene in humans. SLC1A2 is a member of the solute transporter family of proteins. This membrane-bound protein is the primary transporter for the removal of the excitatory neurotransmitter glutamate from the extracellular space of synapses in the CNS. Glutamate removal is necessary for proper synaptic activation and to prevent neuronal damage due to excessive activation of glutamate receptors. SLC1A2 is responsible for over 90% of glutamate reuptake in the brain.

[0317] These findings indicate that LMW-DS may be useful in preventing glutamate excitotoxicity under conditions where its high extracellular levels are harmful, such as after TBI.

[0318] Effect of LMW-DS on cell adhesion One of the most striking phenotypic effects of LMW-DS was its effect on cell adhesion, which was cell type specific: cell adhesion was most strongly affected in neurons, followed by Schwann cells, but not in HUVECs.

[0319] Gene expression analysis showed that this was due to the effect of LMW-DS on the expression of enzymes that regulate cell adhesion, including metallopeptidases, also known as matrix metalloproteinases (MMPs) (see Table 10).

[0320] The aggregate effect of these molecules on pathways regulating cell motility and adhesion in Schwann cells (17 molecules, see Table 10) was such that cell adhesion could be inhibited and cell motility could be simultaneously activated, whereas in HUVECs (1 molecule, ADAM11), adhesion was not affected but angiogenesis could be activated. [Table 10]

[0321] The effects of LMW-DS-induced differential gene expression in neurons were analyzed. In motor neurons, the same metallopeptidase-dependent pathway may be involved in the cell detachment observed in Schwann cells (see Table 11). [Table 11]

[0322] However, none of the MMP-related genes were differentially expressed in cortical neurons.

[0323] This finding led to a re-evaluation of all molecular interactions that affect cell adhesion as well as adhesion-related molecules and their effects on cell adhesion in four different cultures. The complete list of 217 adhesion-related molecules (197 genes and 20 drugs) is shown below:

[0324] ACE2, ACP1, ADAM15, ADGRB1, ADGRE2, ADIPOQ, AG490, AMBN, ANGPT1, ANTXR1, ARAP3, ARMS2, batimastat, BCAM, BCAP31, BCAR1, benzyloxycarbonyl-Leu-Leu-Leu-aldehyde, BMP2, BMP4, BTC, C1QBP, Ca 2+, CA9, CADM1, CALR, calyculin A, caspase, CBL, CD209, CD36, CD44, CD46, CDH13, cerivastatin, chloramphenicol, chondroitin sulfate, CLEC4M, colchicine, type I collagen, collagen, COMP, CRK, CRP, CSF1, CSF2RB, CTGF, curcumin, CXCL12, cyclic AMP, DAB2, DAG1, DCN, DDR1, Desferriexochelin 772SM, DOCK2, DSG2, DSG4, Durapatite, Efna, EFNA1, EFNB, EFNB1, EGF, EGFR, EGR1, ELN, ENG, EP300, Eph receptor, EPHA8, EPHB1, eptifibatide, ethylenediaminetetraacetic acid, ETS1, F11R, F3, FBLN5, FBN1, Fc receptor, FCN2, FERMT2, FES, FGF2, FG FR1, fibrin, FN1, focal adhesion kinase, FSH, FUT3, FUT6, FUT7, FYN, HACD1, heparin, histone h3, histone h4, HRAS, HSPG2, HTN1, hyaluronic acid, hydrocortisone, hydrogen peroxide, ICAM1, ICAM2, IGF1R, IgG, Igg3, IL1, IL1B, IL6, ILK, integrin, integrin alpha 4 beta 1, integrin α, I PO9, ITGA1, ITGA2, ITGA3, ITGA5, ITGA6, ITGB1, ITGB2, ITGB3, ITGB5, JAK2, Jnk, KP-SD-1, LAMC1, laminin, laminin 1, levothyroxine, LGALS3, LIF, lipopolysaccharide, LOX, LRP1, LRPAP1, MAD1L1, mannose, MAPK7, MBL2, MERTK, metronidazole, MGAT5, MMP2, Mn 2+, NCK, NEDD9, NRG1, okadaic acid, OLR1, P38 MAPK, PDGF BB, phosphatidylinositol, PKM, platelet-activating factor, PLD1, PLG, PMP22, PODXL, POSTN, PRKCD, PTAFR, PTEN, PTGER2, PTK2, PTK2B, PTN, PTPN11, PTPRZ1, pyrrolidine dithiocarbamate, Rac, RALB, RANBP9, RHOA, RHOB, RPSA, SDC3, SELE, selectin, SELL, SEMA3A, simvastatin, SIRPA , SPARC, sphingosine-1-phosphate, SPI1, SPP1, SPRY2, SRC, STARD13, SWAP70, TEK, TFPI, TFPI2, TGFA, TGFB1, TGFBI, TGM2, THBS2, THY1, thyroid hormone, TIMP2, tirofiban, TLN1, TLN2, TNF, TP63, tretinoin, VAV1, VCAM1, VCAN, Vegf, VHL, VTN, VWF, and WRR-086.

[0325] In HUVECs, none of the 197 cell adhesion-regulating genes were differentially regulated by LMW-DS. In Schwann cell cultures, the 17 differentially expressed molecules collectively led to slightly increased adhesion. However, in neurons, the expression pattern led to a significant inhibition of cell adhesion in these cells.

[0326] This result explains the cell-type-specific effect of LMW-DS on cell adhesion, and this finding also applies to the anti-scarring effect of LMW-DS (see Example 5) by reducing tissue fibrosis and adhesion signals of immune cells.

[0327] Upstream regulatory pathways affected by LMW-DS As shown in Table 12, in Schwann cells, upstream regulator analysis revealed that LMW-DS modulated the effects of several growth factors by enhancing their activation or reducing their inhibition in the system. [Table 12]

[0328] In HUVECs, the number of growth factors whose effects were enhanced by LMW-DS was relatively small, but still highly significant (see Table 13). [Table 13]

[0329] As shown in Table 14, in motor neurons, upstream regulator analysis revealed that LMW-DS affected the effects of several growth factors that either enhanced the activation of growth factors present in the system or reduced their inhibition. [Table 14]

[0330] In normal culture conditions of cortical neurons, most pathway-dependent growth factors were significantly activated by normal medium. In most cases, this activation was unchanged by LMW-DS. However, LMW-DS activated molecules that are downstream effectors of GDF7, and the effects of this growth factor were enhanced by LMW-DS. GDF7 is a potent differentiation factor for neurons, and the additional activation of these growth factors in response to BDNF and NT3 activation provides a good explanation for the enhanced differentiation of these cells in culture.

[0331] Consideration Normal culture conditions for HUVECs mimic the environment following tissue hypoxia and reperfusion and include high nutrient content and growth factors similarly supplemented with heparin. LMW-DS-treated cultures mimicked the effects of LMW-DS after 24 hours of hypoxia and reperfusion. A relevant real-life scenario is that of angiogenesis following an ischemic condition such as stroke.

[0332] In Schwann cells, control cultures with high nutrient content and glucose mimicked Schwann cell activation. LMW-DS-treated cultures mimicked the effects of LMW-DS added 24 hours later on glial activation. This mimics a real-life scenario: glial activation after nervous system injury, such as after TBI.

[0333] Normal culture conditions for neurons, both motor neurons and cortical neurons, containing high nutrient content and growth factors mimic the environment during normal neuronal differentiation. The only negative effect in these cultures is the oxidative stress experienced by the cells. A relevant real-life scenario is a degenerative state driven by oxidative stress in the presence of sufficient growth and differentiation factors. This corresponds to early-stage neurodegenerative diseases or conditions in which oxidative stress plays a central role.

[0334] The molecular effects observed in Schwann cells and HUVECs support a role for LMW-DS in protecting against apoptosis, inducing angiogenesis, increasing cell migration and migration, increasing cell viability and survival, and inducing cell differentiation across cell types. Analysis of key molecular pathways indicated that in neurons, LMW-DS should reduce the effects of oxidative stress on mitochondria and reduce neurodegeneration-related molecules such as amyloid-β and Lewy bodies.

[0335] Thus, the results from the HUVEC cell model indicate that LMW-DS can protect against cell injury and promote the development of new blood vessels in injured or affected tissues following stroke, and the results from Schwann cells indicate that LMW-DS can protect against cell loss in the diseased or injured nervous system, such as that resulting from TBI or neurodegenerative diseases.

[0336] Analysis of key molecular pathways showed that in Schwann cells, LMW-DS reduced the effects of oxidative stress on mitochondria and increased glutamate uptake. The results in Schwann cells indicate that LMW-DS can protect against cell loss caused by oxidative stress and / or glutamate excitotoxicity in the diseased and injured nervous system, which is relevant, for example, to neurodegenerative diseases and TBI.

[0337] Of particular importance, LMW-DS increased glial cell glutamate uptake, as demonstrated by Schwann cells. However, LMW-DS did not alter neuronal glutamate production. This is important because glutamate is necessary for LTP, i.e., learning and memory. Therefore, it is informative that LMW-DS did not alter neuronal glutamate production, since glutamate is necessary for normal neurotransmission in the processes described above. However, increased levels of glutamate released from damaged or dying cells are efficiently taken up by surrounding glial cells due to the effects of LMW-DS. Therefore, activation of glutamate transporters in glial cells by LMW-DS effectively removed glutamate released by damaged or dying neurons from the neuronal gap. This, in turn, prevented glutamate from causing excitotoxicity and further neuronal damage. Thus, LMW-DS induced the uptake of potentially harmful neurotoxic amounts of glutamate by glial cells.

[0338] Thus, the neuronal results confirm the potential therapeutic utility of LMW-DS in neurodegenerative diseases, disorders, and conditions by reducing secondary tissue damage due to oxidative stress, promoting repair, and reducing degeneration-associated protein accumulation.

[0339] Taken together, the results support a role for LMW-DS in protecting against apoptosis in general and neuronal cell death in particular, in inducing angiogenesis, increasing cell migration and migration, increasing cell viability and survival, inducing cell differentiation, reducing the effects of oxidative stress, reducing glutamate excitotoxicity and reducing the production of degeneration-associated protein products such as amyloid-β and Lewy bodies.

[0340] Cell adhesion was primarily affected in neurons and Schwann cells, where LMW-DS promoted cell detachment and migration. Cell adhesion was unaffected in HUVECs. The effect on cell adhesion was primarily due to the expression of metalloproteinase-type enzymes, although modulation of other adhesion molecules also contributed to this effect.

[0341] This finding also explains the anti-scarring effect of LMW-DS, as observed in Example 5. The results suggest that the anti-scarring effect observed in Example 5 is mediated by LMW-DS, which activates degradative enzymes that support tissue remodeling and block fibrogenic (scar-forming) signals in injured tissue.

[0342] Scar formation as a pathological response is promoted by TGFβ. TGFβ induces a large interconnected network of 171 molecules that results in immune cell adhesion, cell activation, cell motility, cell aggregation, fibrosis, and TGFβ induction. Administration of LMW-DS completely abolished the TGFβ-induced effects on immune cell adhesion, cell activation, cell aggregation, fibrosis, and TGFβ autoactivation. These inactivating effects of LMW-DS on the molecular network promoted by TGFβ in Schwann cells were also observed even when TGFβ was activated, i.e., in the presence of excess TGFβ.

[0343] The effects demonstrated by the gene expression data confirm the phenotypic changes observed in Example 1 with respect to cell adhesion, as well as phenotypic changes on differentiation and cell survival.

[0344] Thus, these studies establish the potential therapeutic utility of LMW-DS in post-ischemic conditions by promoting revascularization, reducing secondary tissue damage, and promoting repair for neurodegenerative diseases, disorders, and conditions, where LMW-DS can promote neuronal survival, differentiation, and ultimately repair.

[0345] Analysis of upstream regulators of genes regulated by LMW-DS showed that, similar to the effects of heparin, LMW-DS enhanced the effects of existing growth factors on cells. The hypothesis is that LMW-DS binds to growth factor molecules and promotes their binding to their receptors.

[0346] This hypothesis is also supported by the observation that LMW-DS-induced differential gene expression in HUVECs (normal CM already contained heparin) was relatively less than in Schwann cells (normal CM did not contain heparin).

[0347] The mechanism of action also explains why LMW-DS is effective in the acute phase of TBI seen in Example 3 when growth factors are present, but less effective in the later phase when early repair attempts have already diminished.

[0348] Therefore, at least some of the therapeutic effects of LMW-DS may depend on pre-existing repair mechanisms that are amplified by LMW-DS. In such cases, it is generally recommended that LMW-DS be administered in the early stages of any neurodegenerative condition, when the tissue has sufficient repair potential.

[0349] By protecting cellular metabolism, LMW-DS may be a useful protective therapeutic agent for many degenerative conditions in which cells are progressively lost due to ischemic, oxidative, or traumatic injury. Non-limiting examples of such degenerative conditions include stroke, ALS, MS, dementia, TBI, SCI, retinal injury, AD, etc. LMW-DS may support these damaged tissues and restore some lost function while enhancing remaining endogenous repair mechanisms.

[0350] The anti-scarring activity of LMW-DS indicates its potential use in the treatment of fibroproliferative (scar-forming) conditions, including glaucoma, proliferative vitreoretinopathy, SAH, traumatic brain and spinal cord injury, invasive surgery, postoperative adhesions, rotator cuff injuries, burns, reconstructive surgery, and ulcerative conditions (diabetes). Experimental results support a role for LMW-DS in both preventing the development of fibroproliferative (scar-forming) conditions and in degrading existing fibrotic scars in such conditions.

[0351] Example 5 This study investigated the effect of LMW-DS on trabecular meshwork (TM) scar formation on glaucomatous eyes.

[0352] material and method Test Plan Glaucoma was induced in adult male Sprague-Dawley rats by increasing intraocular pressure (IOP) with repeated twice-weekly intracameral (IC) injections of transforming growth factor-β (TGF-β). A sustained increase in IOP (after 2 weeks) resulted in retinal ganglion cell death (30-40%). From the start of the experiment, 15 mg / kg LMW-DS was administered by daily subcutaneous injection, and RGC protection was assessed compared to control. Group 1: n = 12 rats; 24 eyes IOP + IC TGF-β (twice weekly for 28 days) days 0-28 + daily subcutaneous administration of dextran sulfate on days 14-28. Group 2: n = 8 rats; 16 eyes. IOP + IC TGF-β (twice weekly for 28 days) days 0-28 + daily subcutaneous administration of vehicle (saline) on days 14-28. Group 3: n=8 rats; 8 eyes IOP+untreated (uninjured eyes) and 8 eyes IOP+IC PBS daily for 28 days.

[0353] Measurement evaluation items IOP was measured twice weekly throughout the study period from day 0 to day 28; Immunohistochemistry counts retinal ganglion cells (RGCs) immunoreactive for brain-specific homeobox / POU domain protein 3A (Brn3a) at day 28 (RGC survival); Immunohistochemistry to assess scar formation in the trabecular meshwork by laminin and fibronectin in groups 1 and 2 at day 28; Anterior segment optical coherence tomography (OCT) imaging was performed on day 28 to examine the angle and thickness of the retinal nerve fiber layer containing RGC axons; Weight, day 28.

[0354] Animals and surgery Sixteen 8- to 10-week-old male Sprague-Dawley rats (Charles River, Kent, UK), weighing 175-200 g, were housed under a 12-h light / dark cycle with food and water ad libitum and used for these experiments. Surgeries were performed at the Biomedical Services Unit at the University of Birmingham in accordance with the Home Office guidelines set out in the Animal Act 1986 (UK) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All ocular surgeries and IOP measurements were performed under inhalation anesthesia using 2-5% isoflurane in 95% O2 (National Vet Supplies, Stoke, UK) at a flow rate of 1.5 L / min. The postoperative health of all rats was closely monitored.

[0355] On day 0, a single self-sealing wound was created through the cornea into the anterior chamber of both eyes using a 15° disposable blade through a tunnel created with a homemade, disposable, sterile glass micropipette (Harvard Apparatus, Kent, UK), allowing repeated twice-weekly (twice-weekly) IC injections (every Monday and Thursday) of 3.5 μl of active human recombinant TGFβ1 (5 ng / μl; Peprotech, London, UK) for 28 days.

[0356] Tissue preparation for immunohistochemistry (IHC) Rats were sacrificed by exposure to increasing concentrations of CO2 and transcardially perfused with 100 ml of phosphate-buffered saline (PBS) to flush out blood, followed by further perfusion with 100 ml of 4% paraformaldehyde (PFA) in PBS, pH 7.4. Dissected eyes for IHC were postfixed in 4% PFA in PBS for 2 hours at 4°C, then cryoprotected by immersion in increasing concentrations of sucrose solutions (10%, 20%, and 30% sucrose in PBS; all from Sigma, Poole, UK) for 24 hours at 4°C, and then embedded in optimal cutting temperature embedding medium (Thermo Shandon, Runcorn, UK) in peel-mold containers (Agar Scientific, Essex, UK). Eyes immersed in embedding medium were flash-frozen in crushed dry ice and stored at −80°C before being cut at 15 μm thickness in the parasagittal plane through the optic nerve head at −22°C using a Bright cryostat microtome (Bright, Huntingdon, UK). Sections were mounted on positively charged glass slides (Superfrost plus; Fisher Scientific, Pittsburgh, USA), placed at 37°C for 2 hours, and stored at −20°C.

[0357] Immunohistochemistry Frozen sections were allowed to thaw for 30 minutes, then washed 3x5 minutes in PBS, followed by permeabilization with 0.1% Triton X-100 (Sigma) for 20 minutes. Sections were blocked for 30 minutes in 0.5% bovine serum albumin (BSA) and 0.3% Tween 20 in PBS (all from Sigma) and incubated overnight with primary antibodies (Table 11). Then, sections were washed 3x5 minutes in PBS and incubated with secondary antibodies (Table 11) for 1 hour at room temperature (RT; 20-25°C). Sections were then washed 3x5 minutes in PBS and mounted with Vectorshield mounting medium (Vector Laboratories) containing 4',6-diamidino-2-phenylindole (DAPI). Control tissue sections incubated with secondary antibodies alone were all negatively stained (not shown). [Table 15]

[0358] Quantification of immunohistochemistry After immunofluorescence staining, sections were viewed under a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss Ltd.), and images were acquired using a Zeiss AxioCam HRc using the same exposure time for each antibody. IHC was quantified as previously described (Hill et al., Decorin reduces intraocular pressure and retinal ganglion cell loss in rodents through fibrolysis of the scarred trabecular meshwork. Invest Ophthalmol Vis Sci. 2015, 56(6):3743-3757). Briefly, the region of interest used to quantify TM fibrosis was defined as a quadrant of the same predetermined size within the TM for all eyes / treatments. ECM deposition was quantified within this defined TM quadrant, and the percentage of immunofluorescent pixels above a normalized background threshold was calculated using ImageJ software (National Institutes of Health, USA). For each antibody, a threshold level of brightness in the region of the TM was established using intact, untreated eye sections to define the reference level for test group analysis of pixel intensity. Images were assigned randomized numbers to ensure blinding of treatment group during assessor quantification.

[0359] For quantification of RGCs in retinal sections, RPBMS + / DAPI + RGCs were counted in 15-μm-thick parasagittal sections of the retina from a 250-μm straight section through the neuronal layer on both sides of the optic nerve. Four retinal sections from each eye in the control and treatment groups were quantified. Images were randomized to ensure blinding of the treatment group during quantification by assessors.

[0360] statistics All statistical analyses were performed using SPSS20 (IBM, USA). Normal distribution tests were performed to determine the most appropriate statistical analysis for comparing treatments. Statistical significance was determined at p<0.05. Significant differences in TM fibrosis were tested using Student's t-test or one-way analysis of variance for two-group comparisons ± SEM and are presented textually or graphically as mean ± SEM.

[0361] result LMW-DS treatment significantly attenuated TM scar formation as evidenced by significantly reduced (P<0.001 laminin; P<0.01 fibronectin) levels of immunoreactive laminin (Figure 20) and fibronectin (Figure 21) angles.

[0362] Consideration Since laminin and fibronectin levels in the angles of dextran sulfate-treated rats were significantly lower, LMW-DS treatment induced degradation of existing TM scar elements. Therefore, the anti-scarring effect of LMW-DS indicates that the drug can be used to degrade existing scars, thereby enabling, for example, tissue remodeling and wound healing in fibrotic conditions.

[0363] Example 6 Alzheimer's disease (AD) is devastating for patients and their families, requires significant economic resources, and places a heavy burden on the healthcare system. Current strategies that attempt to provide small, often temporary improvements in symptoms can provide some therapeutic benefit to patients, but many patients fail to benefit at all. Disease-modifying drugs have the potential to transform treatment and achieve deep market penetration.

[0364] The pathological hallmark of AD is the presence of senile plaques composed of β-amyloid protein, which oligomerizes to form neurotoxic complexes that adversely affect physiological neurotransmission. The harmful effects of oligomeric β-amyloid protein are due in part to the formation of cellular prion protein (PrP). C), thus pharmacological strategies that inhibit this protein-protein interaction have potential as disease-modifying therapeutic agents.

[0365] This study aims to evaluate the efficacy and safety of oligomeric beta-amyloid and PrP in LMW-DS to determine their disease-modifying potential as therapeutic agents for treating AD. C The ability of the inhibitors to inhibit protein-protein interactions between the two was investigated.

[0366] material and method Chemicals and antibodies Streptavidin-HRP was obtained from BioLegend; β-amyloid-(1-42)-biotin was obtained from Innovagen; normal human cellular prion protein (PrP C ) from Merck; TMB from eBioscience; 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) from Sigma; anti-amyloid beta antibody clone 6E10 from BioLegend; anti-mouse HRP from Cell Signaling; dextran sulfate sodium salt (DSSS) with an average MW >500,000 Da from Sigma; dextran (MW 450,000-650,000 Da) from Sigma; Maxisorp plates from Sigma.

[0367] Preparation of amyloid beta oligomers The oligomerization of β-amyloid was optimized based on previous methods (Stine et al., Methods Mol. Biol. 2011, 670:13-32; Aimi et al., J. Neurochem. 2015, 134:611-617). Briefly, amyloid β was dissolved in HFIP to a final concentration of 1.0 mM, subjected to protective sonication, and the HFIP was carefully evaporated. The resulting peptide film was stored in a sealed container at -20°C. Prior to use, the peptide film was slowly dissolved in DMSO to a final concentration of 5.0 mM and subjected to protective sonication for 10 minutes. To prepare oligomers, the DMSO solution was diluted in ice-cold DMEM medium to a final concentration of 100 μM and incubated at 37°C for 16 hours (β-amyloid-biotin). To prepare monomers, the DMSO solution was diluted in ice-cold 18 M Ohm water to a final concentration of 100 μM and used immediately.

[0368] Identification of amyloid beta monomers and oligomers Preparations optimized for the generation of amyloid-β monomers or oligomers were solubilized in 5% SDS-containing gel sample buffer without reducing agents. Proteins were measured on a 15% Bis-Tris gel using MES running buffer without reducing agents. The gels were transferred to PVDF, blocked in 10% nonfat milk, and then incubated with anti-amyloid-β antibody overnight at 4°C. They were developed with anti-mouse HRP followed by ECL and exposed to film.

[0369] Oligomeric amyloid beta and PrP C ELISA method to quantify protein-protein interactions between PrP C Dilute in carbonate coating buffer to 10x coating volume (100 μl; 500 ng PrP per well) CA final volume of 100 μl of 100 μL of 100 μL of oligomeric amyloid β-biotin peptide preparation (final concentration 200 nM) was applied to a Maxisorp plate. The plate was then sealed and placed at 4°C overnight. The coated plate was then carefully washed in PBS-Tween 20 and blocked with 2% BSA in PBS. The plate was washed, and 100 μl of oligomeric amyloid β-biotin peptide preparation (final concentration 200 nM) was carefully mixed with the test compound and added to each well. The plate was incubated for 60 minutes at room temperature, washed, treated with streptavidin-HRP, and after further washing, developed with TMB (the reaction was stopped with 2N H2SO4). The absorbance was read at 450 nm within 30 minutes.

[0370] All conditions were performed in triplicate. C Amyloid β-biotin binding to β was calculated as described by Aimi et al., J Neurochem. 2015, 134:611-617.

[0371] Curve fitting Quantitative pharmacological analysis was performed by iterative curve fitting to a floating four-parameter logistic equation.

[0372] result Formation of amyloid-β monomers and oligomers Amyloid beta monomers and oligomers were prepared using an optimized protocol, and oligomerization was successful, resulting in higher apparent efficiencies compared to the results described by Aimi et al., J Neurochem. 2015, 134:611-617 (Figure 22).

[0373] Oligomeric amyloid beta and PrP C Optimization of an ELISA method for quantitative assessment of protein-protein interactions between The method reported by Aimi et al., J Neurochem. 2015, 134:611-617 did not specify the amount of protein to be coated per well on the ELISA plate, but 50 ng of PrP per well was used. CHowever, when this amount was coated onto the plate, no specific binding signal of oligomeric amyloid beta was evident. The experiment was repeated using a more effective coating buffer, but the signal was still not clear. The lack of signal, and the known theoretical maximum binding capacity of Maxisorp plates (600-650 ng / cm) are inconsistent with the previous study. 2 ) showed that the coating level was not optimal. C A range of coating levels was evaluated; 250 ng PrP per well C Although a relatively small signal of oligomeric amyloid beta was discernible in the 500 ng of PrP per well, C A larger, more reproducible signal was evident at a coating level of 1 μg / well. This coating amount is consistent with published literature (Beringe et al., Brain. 2003, 126:2065-2073: 500 ng / well; Nakato et al., J Immunol. 2012, 189:1540-1544: 250 ng / well; Souan et al., Eur J Immunol. 2001, 31:2338-2346: 1.0 μg / well of various prion protein constructs).

[0374] Oligomeric amyloid beta and PrP C Ability of DSSS and LMW-DS to compete with protein-protein interactions between DSSS detects oligomeric amyloid beta and PrP C Similar to LMW-DS, dextran competed for the protein-protein interaction between PrP and oligomeric amyloid beta in a concentration-dependent manner (Figure 23; Table 12). Quantitative pharmacological analysis showed that LMW-DS exhibited similar overall affinity to DSSS, despite clear differences in competitive binding and Hill coefficients at the head-to-head level, suggesting a different interaction between the two compounds (Figure 23; Table 12). In contrast to DSSS and LMW-DS, dextran inhibited the binding of oligomeric amyloid beta and PrP to oligomeric amyloid beta. C The protein-protein interaction between the two proteins could not be clearly competed. [Table 16]

[0375] Consideration High molecular weight dextran sulfate (DSSS) binds oligomeric amyloid beta and PrP C It has previously been reported that amyloid-β competes with the protein-protein interaction between amyloid-β and amyloid-β at effective concentrations in the low μg / ml range (Aimi et al., J Neurochem. 2015, 134:611-617). In this study, optimization of the method produced a significantly higher proportion of oligomeric amyloid-β compared to the Aimi et al. study. Optimization of the protein-protein interaction ELISA resulted in a greater degree of specific protein-protein interaction; the larger dynamic range of competition facilitated quantitative pharmacological analysis of the interaction with competing compounds. Thus, this study represents an improvement over the study reported by Aimi et al.

[0376] DSSS and LMW-DS express oligomeric amyloid beta and PrP C and showed comparable affinity for protein-protein interactions with IC values ​​of 0.62 ± 0.07 and 0.42 ± 0.16 μg / mL, respectively. 50 Hill analysis of the nature of competition showed that LMW-DS exhibited a shallower competition curve compared to the relatively high Hill coefficients associated with DSSS, providing evidence for different pharmacological actions between DSSS and LMW-DS.

[0377] Therefore, LMW-DS is a complex of oligomeric amyloid beta and PrP C This effect observed in LMW-DS is due to the fact that oligomeric amyloid beta and PrP compete for protein-protein interactions between these proteins and can be used to prevent or at least inhibit this protein-protein interaction. C This has potential for diseases and disorders such as AD that involve protein-protein interactions between the

[0378] Example 7 The aim of this study was to evaluate the potential neuroprotective effects of LMW-DS against biochemical, molecular, and histoanatomical damage produced by an experimental model of severe closed head diffuse traumatic brain injury (sTBI) in rats. In this investigation, results were obtained by HPLC analysis of low molecular weight metabolites specific to energy metabolism, oxidative / nitrosative stress, antioxidants, and free amino acids in brain tissue extracts of treated animals.

[0379] material and method sTBI induction and drug administration protocol Male Wistar rats (n=160) weighing 300-350 g were used in this study. They were fed standard laboratory chow and water ad libitum in a controlled environment.

[0380] Animals received an intramuscular injection of an anesthetic mixture containing 35 mg / kg of ketamine and 0.25 mg / kg of midazolam. Diffuse sTBI was induced according to the "weight-drop" impact acceleration model established by Marmarou et al. J. Neurosurg. 1994, 80:291-300. This model produces diffuse axonal injury and can reproduce the physical and mechanical characteristics of human diffuse TBI.

[0381] Severe TBI was induced by dropping a 450 g weight from a height of 2 m onto the head of a rat protected by a helmet (a metal disk previously fixed on the skull with dental cement) to distribute the mechanical force evenly to the brain. The rat was placed in a prone position on a special polyurethane foam bed inserted into a special container; this foam dissipates most of the potential energy (from the mechanical force) and prevents any rebound of the animal after the impact, which could result in spinal cord injury.

[0382] Rats that suffered skull fractures, seizures, nosebleeds, or did not survive the impact were excluded from the study. Two or seven days after TBI induction, rats were re-anesthetized and then immediately sacrificed. These time points correspond to the worst biochemical damage (2 days) or, in the case of a minimally damaged brain, full metabolic recovery (7 days).

[0383] Drug treatments were administered by subcutaneous injection of 0.5 ml of LMW-DS (Tikomed) at three different concentrations (1, 5, and 15 mg / kg body weight) according to the outlined protocol described below.

[0384] Sham-operated animals underwent identical procedures of anesthesia except for TBI and were used as a control group.

[0385] Experimental design To test the efficacy of three different concentrations of LMW-DS at two different time points after TBI, the rats used in this study were divided into four groups. As specified below, in each group, animals were subjected to a specific treatment for metabolic analysis, and other animals were used for histomorphometric testing, according to the procedures described below. group 1 Controls (n=12) were reserved for biochemical evaluation. An additional 4 animals were used for histomorphometric studies. Total rats in this group: n=16 group 2 Following the induction of sTBI and without pharmacological treatment, rats were divided into the following subgroups: 1. Twelve animals received sTBI and were sacrificed 2 days after TBI. 2. 12 animals received sTBI and were sacrificed 7 days after TBI. Four additional rats for each subgroup were used for histomorphometric studies. Total rats in this group: n=32. group 3 Rats underwent induction of sTBI, received a single dose of LMW-DS 30 minutes after TBI, and were sacrificed 2 days after TBI. Animals were divided into the following subgroups: 1. Twelve animals received sTBI and were treated with 1 mg / kg body weight of LMW-DS. 2. 12 animals that underwent sTBI induction and were treated with 5 mg / kg body weight of LMW-DS. 3. 12 animals that underwent sTBI induction and were treated with 15 mg / kg body weight of LMW-DS. Four additional rats for each subgroup were used for histomorphometric studies. Total rats in this group: n=48. group 4 Rats underwent induction of sTBI, received a single dose of LMW-DS 30 minutes after TBI, and were sacrificed 7 days after TBI. Animals were divided into the following subgroups: 1. Twelve animals received sTBI and were treated with 1 mg / kg body weight of LMW-DS. 2. 12 animals that underwent sTBI induction and were treated with 5 mg / kg body weight of LMW-DS. 3. 12 animals that underwent sTBI induction and were treated with 15 mg / kg body weight of LMW-DS. Four additional rats for each subgroup were used for histomorphometric studies. Total rats in this group: n=48. group 5 Rats (n = 12) underwent sTBI and received repeated administration of the maximum dose of LMW-DS (15 mg / kg body weight) at 30 min, 3 days, and 5 days after TBI. They were then sacrificed 7 days after TBI. Four additional rats were used for histomorphometric studies. Total rats in this group: n = 16.

[0386] Brain tissue processing for biochemical and gene expression analyses To minimize metabolite loss, an in vivo osteotomy craniotomy was performed on all animals during anesthesia. The rat skull was carefully removed to expose the brain, and a quick incision along the sagittal groove was made to separate the two hemispheres. The hemisphere reserved for biochemical analysis was freeze-clamped with aluminum tongs pre-cooled in liquid nitrogen and immersed in liquid nitrogen. A freeze-clamp procedure was implemented to accelerate tissue freezing, thereby minimizing the potential for metabolite loss.

[0387] The remaining hemisphere, reserved for molecular biology analysis, was placed in 5-10 volumes of RNAlater® solution (Invitrogen Life Technologies), an RNA stabilizing solution that stabilizes and protects RNA from degradation. The brain samples were stored overnight at 4°C to allow the solution to completely penetrate the tissue.

[0388] Tissue homogenization for metabolite analysis was performed as follows: After wet weight (ww) determination, the frozen hemispheres were placed in 7 ml of ice-cold, nitrogen-saturated precipitation solution consisting of CH3CN + 10 mM KH2PO4, pH 7.40 (3:1; v:v) (1:10 w / v), and homogenization was performed using an Ultra-Turrax homogenizer set (Janke & Kunkel, Staufen, Germany) at 24,000 rpm / min. After centrifugation at 20,690 x g for 10 min at 4°C, the clear supernatant was saved, and the pellet was supplemented with an aliquot of 10 ml of KH2PO4, homogenized again as described above, and stored overnight at -20°C to ensure complete recovery of the aqueous phase from the tissue. A second centrifugation was performed (20,690 × g, 10 min at 4 °C), and the supernatant was combined with the previous one and extracted by vigorous stirring with twice the volume of HPLC-grade CHCl3 and centrifuged as above. The upper aqueous phase (containing water-soluble low molecular weight compounds) was collected and subjected to two further chloroform washes (this procedure allows the removal of all organic solvents and any lipid-soluble compounds from the buffered tissue extract), and the volume was adjusted with 10 mM KH2PO4, pH 7.40, to obtain a final aqueous 10% tissue homogenate, which was stored at -80 °C until assayed.

[0389] HPLC analysis of energy metabolites, antioxidants and oxidative / nitrosative stress biomarkers An aliquot of each deproteinized tissue sample was filtered through a 0.45 μm HV Millipore filter and loaded (200 μl) onto a Hypersil C-18, 250 x 4.6 mm, 5 μm particle size column (Thermo Fisher Scientific, Rodano, Milan, Italy) with its own guard column. The column was then coupled to a HPLC system consisting of a Surveyor System (Thermo Fisher Scientific, Rodano, Milan, Italy) equipped with a high-sensitivity diode array detector (equipped with a 5 cm optical path flow cell) set at a wavelength of 200-300 nm. Data acquisition and analysis were performed on a PC using the ChromQuest software package provided by the HPLC manufacturer.

[0390] Metabolites related to tissue energy status, mitochondrial function, antioxidants, and metabolites specific to oxidative / nitrosative stress (described below) were separated in a single chromatographic run according to a slightly modified, existing ion-pair HPLC method (Lazzarino et al., Anal Biochem. 2003;322:51-59; Tavazzi et al., Clin Biochem. 2005;38:997-1008). Assignment and calculation of compounds of interest in chromatographic runs of tissue extracts were performed by comparing the retention times, absorption spectra, and areas of peaks with those of chromatographic runs of freshly prepared ultrapure standard mixtures of known concentrations using appropriate wavelengths (206, 234, and 260 nm).

[0391] List of compounds: cytosine, creatinine, uracil, β-pseudouridine, cytidine, hypoxanthine, guanine, xanthine, CDP-choline, ascorbic acid, uridine, nitrite (NO2), reduced glutathione (GSH), inosine, uric acid, guanosine, CMP, malondialdehyde (MDA), nitrate (NO3), UMP, NAD +, ADO, IMP, GMP, UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetyl-glucosamine (UDP-GlcNac), UDP-N-acetyl-galactosamine (UDP-GalNac), AMP, GDP-glucose, UDP, GDP, NADP + , ADP-ribose, CTP, ADP, UTP, GTP, NADH, ATP, NADPH, malonyl-CoA, coenzyme A (CoA-SH), acetyl-CoA, N-acetylaspartate (NAA).

[0392] HPLC analysis of free amino acids and amino group-containing compounds Simultaneous determination of primary free amino acids (FAAs) and amino-containing compounds (AGCCs) (described below) was performed using precolumn derivatization of the sample with a mixture of OPA and MPA as described by Amorini et al., J Cell Mol Med. 2017;21:530-542; Amorini et al., Mol Cell Biochem. 2012;359:205-216. Briefly, a derivatization mixture consisting of 25 mmol / L OPA, 1% MPA, and 237.5 mmol / L sodium borate, pH 9.8, was prepared daily and introduced into the autosampler. Automated precolumn derivatization of samples (15 μl) with OPA-MPA was performed at 24 °C, and 25 μl of the derivatization mixture was loaded onto an HPLC column (Hypersil C-18, 250 × 4.6 mm, 5 μm particle size, thermostated at 21 °C) for subsequent chromatographic separation. To accurately quantify glutamate, the deproteinized brain extract was diluted 20-fold with HPLC-grade HO before the derivatization procedure and subsequent injection. Separation of OPA-AA and OPA-AGCC was achieved using two mobile phases (mobile phase A = 24 mmol / L CH3COONa + 24 mmol / L Na2HPO4 + 1% tetrahydrofuran + 0.1% trifluoroacetic acid, pH 6.5; mobile phase B = 40% CH3OH + 30% CH3CN + 30% HO) at a flow rate of 1.2 mL / min using an appropriate step gradient.

[0393] The assignment and calculation of OPA-AA and OPA-AGCC in the chromatographic run of the whole brain extract was performed by comparing the retention time and area of ​​the peaks at a wavelength of 338 nm with those of the peaks in the chromatographic run of a freshly prepared ultrapure standard mixture with known concentrations.

[0394] List of FAA and AGCC compounds: aspartic acid (ASP), glutamic acid (GLU), asparagine (ASN), serine (SER), glutamine (GLN), histidine (HIS), glycine (GLY), threonine (THR), citrulline (CITR), arginine (ARG), alanine (ALA), taurine (TAU), gamma-aminobutyric acid (GABA), tyrosine (TYR), S-adenosylhomocysteine ​​(SAH), L-cystathionine (L-Cystat), valine (VAL), methionine (MET), tryptophan (TRP), phenylalanine (PHE), isoleucine (ILE), leucine (LEU), ornithine (ORN), lysine (LYS).

[0395] Brain tissue processing for histomorphometric analysis After adequate anesthesia, rats were transcardially perfused as described by Di Pietro et al., Sci Rep. 2017, 7(1):9189. Briefly, a thoracotomy was performed, and heparin solution was administered into the portal vein to avoid blood clotting throughout the procedure. A right atriotomy was then performed, and a perfusion needle was advanced into the ascending aorta. Perfusion was performed with 100 ml of phosphate buffered saline (PBS) pH 7.4 to flush out the blood, followed by further perfusion with 4% paraformaldehyde (PFA) in PBS pH 7.4. After rapid removal from the skull, each brain was postfixed by immersion in 100 ml of 4% PFA in PBS at 4°C for 2 hours. Cryoprotection was achieved by immersing whole brains in PBS enriched with increasing sucrose solutions (10%, 20%, and 30%) for 24 hours, followed by embedding in optimal cutting temperature embedding medium (OCT) (Thermo Shandon, Runcorn, UK) in peel-mold containers (Agar Scientific, Essex, UK). Brains immersed in OCT were rapidly frozen in crushed dry ice and then stored at -80°C.

[0396] statistical analysis Differences between groups were estimated by Student's t-test. Only two-sided p-values ​​less than 0.05 were considered statistically significant.

[0397] result Summary of biochemical data recorded 2 days after sTBI Effects of increasing doses of LMW-DS on measured cerebral energy metabolism Table 13 summarizes the values ​​for phosphorylated high-energy purine and pyrimidine compounds. It is particularly clear that sTBI caused a depletion of triphosphate nucleotides (ATP, GTP, UTP, and CTP), accompanied by an increase in ADP and N-acetylated derivatives of UDP-glucose (UDP-GlcNac) and UDP-galactose (UDP-GalNac).

[0398] At this time point after injury, treatment with LMW-DS was only partially effective in improving cellular energy metabolism, with significantly elevated levels of high-energy phosphates (ATP, GTP, and CTP) recorded at all three drug doses tested. No effect was observed on UTP and ADP concentrations. It is worth recalling that 48 hours after TBI in rats is a critical time point for cerebral metabolism and coincides with the greatest changes in mitochondrial function, including alterations in mitochondrial quality control. In this experimental TBI model, this time point can be considered a kind of "turning point" at which recovery or non-recovery of cerebral metabolism is determined. [Table 17] TIFF0007745261000023.tif16162

[0399] In Tables 13-31, bold indicates significant difference versus control (p<0.05); bold underline indicates significant difference versus TBI (p<0.05); and bold italic indicates significant difference versus both control and TBI (p<0.05).

[0400] Effects of increasing doses of LMW-DS on nicotine coenzyme Oxidized NAD + and NADP + The values ​​for nicotine coenzymes (NADH and NADPH) and reduced (NADH and NADPH) are summarized in Table 14. Table 14 also shows the calculated NAD + We report the dimensionless value of the / NADH ratio, which is suitable for assessing how dependent metabolism is on glycolysis or mitochondrial oxidative phosphorylation.

[0401] As previously observed herein, sTBI increases NAD + , NADPH + and NAD +At this point, treatment with LMW-DS was effective only at the highest dose tested (15 mg / kg body weight), resulting in a significant protection of the nicotine coenzyme pool and avoiding a metabolic switch towards glycolysis, thereby indirectly suggesting an overall better mitochondrial function. [Table 18]

[0402] Effect of increasing doses of LMW-DS on CoA-SH derivatives Table 15 reports data on free CoA-SH and CoA-SH derivatives. Acetyl-CoA, in particular, is a key compound for mitochondrial metabolism, enabling the correct functioning of the tricarboxylic acid cycle (TCA cycle), thereby ensuring a continuous supply of electrons to the electron transport chain (ETC). TCA is the primary cell cycle pathway for the generation of reduced coenzymes (NADH and FADH2), which fuel the ETC and oxidative metabolism by transferring their electrons to mitochondrial complexes I and II, respectively. All compounds, especially acetyl-CoA, were significantly affected by sTBI. Partial recovery of this compound was observed at 5 or 15 mg / kg body weight. LWM-DS was administered to animals 30 minutes after injury. [Table 19]

[0403] Effects of increasing doses of LMW-DS on antioxidant and oxidative / nitrosative stress biomarkers Table 16 shows the major water-soluble brain antioxidants (ascorbic acid and GSH) and the effects of oxidative (MDA) and nitrosative stress (-NO2 - and -NO3 - ) biomarker concentrations are shown. Malondialdehyde (MDA) originates from the breakdown of unsaturated fatty acids in membrane phospholipids as a result of ROS-mediated lipid peroxidation. Nitrite (-NO2 - ) and nitrate (-NO3- ) is a stable end product of nitric oxide (NO) metabolism, which is produced in excess by inducible nitric oxide synthase (iNOS) under pathological conditions and generates reactive nitrogen species (RNS) through reaction with ROS.

[0404] Two days after impact, a 25-45% decrease in both water-soluble antioxidants occurred in sTBI-induced rats. A consequent increase in oxidative / nitrosative stress signatures was also noted. Administration of LWM-DS significantly restored both ascorbic acid and reduced glutathione (GSH) concentrations, and reductions in brain tissue nitrite and nitrate were evident. These effects were more pronounced when 15 mg / kg body weight was used. [Table 20]

[0405] Effects of increasing doses of LMW-DS on dephosphorylated purines and pyrimidines Most of the compounds reported in Table 16 originate from the degradation pathways of purine and pyrimidine nucleotides and are indirectly linked to cellular energy metabolism. In rats with sTBI, all of these compounds, except CDP-choline, had higher brain concentrations, and most of these were favorably affected by drug administration. [Table 21]

[0406] Effect of increasing doses of LMW-DS on N-acetylaspartic acid (NAA) NAA is the most abundant N-acetylated amino acid in the mammalian brain, and its concentration is roughly equivalent to that of the neurotransmitter glutamate in humans. Although the biological role of NAA has not yet been fully elucidated, we have clearly demonstrated in both preclinical and clinical studies that TBI reduces NAA concentrations, and that its time course after head injury mirrors that of ATP. In particular, we found that sTBI induces irreversible changes in NAA homeostasis, that NAA is a good surrogate marker of cerebral energy metabolism, and that the decline and recovery of NAA levels in athletes after concussion is much slower than symptom resolution. Therefore, NAA has particular relevance to the study of TBI.

[0407] A 40% decrease in whole-brain NAA was observed in sTBI rats 2 days after impact (Figure 24). LMW-DS had a beneficial effect on NAA concentrations when administered at 5 or 15 mg / kg body weight. Although significantly lower than controls, NAA in rats receiving either of the two drug doses was significantly higher than that found in sTBI rats, with the highest NAA levels found in rats receiving the highest dose of LMW-DS.

[0408] Effects of increasing doses of LMW-DS on free amino acids involved in neurotransmission The compounds listed in Table 17 are amino acids involved in neurotransmission directly (GLU, GABA) or indirectly (GLN, ASP, ASN, GLY, SER, THR, ALA). GLU, in particular, is the major excitatory amino acid, and its effects are counteracted by GABA. The excitotoxicity of GLU is regulated by SER, GLY, THR, and ALA, which is related to the function of the GLU-GLN cycle, including neurons and astrocytes. As shown in a previous study (16), we found that most of these amino acids were increased in sTBI rats 2 days after injury. Treatment of animals with a single dose of LMW-DS was partially effective when the drug was injected subcutaneously at 5 or 15 mg / kg body weight. In most cases, the values ​​of these various compounds were significantly better than those observed in untreated sTBI animals, but not better than control values. [Table 22]

[0409] Effects of increasing doses of LMW-DS on free amino acids involved in the methyl cycle The free amino acids reported in Table 18 are involved in the so-called methyl cycle, which regulates the homeostasis of compounds acting as methyl donors in cellular metabolism, or in the formation of cysteine, the only amino acid with a free -SH group. Severe head trauma caused significant changes in the key players in this important metabolic pathway. Restoration of methionine was achieved by LWM-DS at any dose tested. Drug treatment was partially effective in normalizing the other amino acids. Supplementary information for changes in L-cystathionine (L-Cystat) is provided in the corresponding table 7 days after impact. [Table 23]

[0410] Effects of increasing doses of LMW-DS on free amino acids involved in the production of nitric oxide (NO) Table 19 shows the concentrations of free amino acids directly involved in the production of NO in the reaction catalyzed by nitric oxide synthase (NOS), a family of enzymes that exists in three isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The last isoform (iNOS) is involved in nitrosative stress. Nitric oxide is produced through a complex reaction in which arginine (ARG) undergoes partial oxidation to donate a nitrogen atom, generating citrulline (CITR) and NO. Two days after sTBI, animals showed a concomitant decrease in Arg and an increase in CITR, consistent with data showing an increase in the stable NO end products nitrite and nitrate (Table 15). Administration of LMW-DS was particularly effective when a dose of 15 mg / kg body weight was used. [Table 24]

[0411] Effect of increasing doses of LMW-DS on long-chain free amino acids The free amino acids reported in Table 20 are useful sources of carbon skeletons for the generation of α-keto acids that cells use to replenish the TCA cycle. Of these compounds, only isoleucine (ILE) was significantly affected by sTBI and restored in drug-treated rats. [Table 25]

[0412] Effect of increasing doses of LMW-DS on free amino acids and aromatic free amino acids acting as osmolytes The results summarized in Table 21 clearly demonstrate that sTBI results in increased concentrations of all these free amino acids. In particular, the increase in taurine (TAU) may suggest an attempt to counteract the effects of cellular edema by increasing the levels of one of the most important brain osmolytes. Separately, the increase in aromatic amino acids may suggest a reduction in the biosynthesis of the neurotransmitters serotonin (formed from tryptophan) and dopamine (produced by biotransformation first from phenylalanine and then from tyrosine). No significant effect of LMW-DS administration was observed at this time point after impact. [Table 26]

[0413] Summary of biochemical data recorded 7 days after sTBI Effects of increasing doses of LMW-DS on measured cerebral energy metabolism Table 22 summarizes the values ​​for phosphorylated high-energy purine and pyrimidine compounds. It is particularly clear that no recovery of depletion of triphosphate nucleotides (ATP, GTP, UTP, and CTP) was observed 7 days after sTBI. A concomitant increase in AMP and ADP was accompanied by significant changes in the concentrations of UDP derivatives (UDP-Glc, UDP-Gal, UDP-GlcNac, and UDP-GalNac). It should be emphasized that, in general, longer times after injury are often characterized by exacerbated biochemical, metabolic, and molecular changes induced by sTBI.

[0414] At this time point after injury, treatment with LWM-DS resulted in a more pronounced overall improvement in cerebral energy metabolism when the drug dose was higher than 1 mg / kg body weight. Differences from controls were recorded even in rats receiving repeated doses of 15 mg / kg body weight of LWM-DS, with significantly higher values ​​of nucleotide triphosphates observed in the drug-treated animals. Particularly relevant is the progressive recovery of the calculated dimensionless value of the ATP / ADP ratio (considered a good indicator of mitochondrial phosphorylation capacity), which continuously increased with increasing doses of drug administered to sTBI animals. [Table 27] TIFF0007745261000034.tif34162

[0415] To better demonstrate that drug effects are related to drug dose, we report the results for ATP graphically in Figure 25. It can be observed that the increase in ATP is somehow related to dose, and that at every dose tested, drug administration resulted in a significant increase in the most important high-energy phosphates.

[0416] Effects of increasing doses of LMW-DS on nicotine coenzyme Oxidized (NAD + and NADP + The values ​​for nicotine coenzymes (NADH and NADPH) and reduced (NADH and NADPH) are summarized in Table 23. Table 23 also shows the calculated NAD + We report the dimensionless value of the NADH / NADH ratio, which is suitable for assessing how dependent metabolism is on glycolysis or mitochondrial oxidative phosphorylation.

[0417] As previously observed, nicotine coenzyme and NAD +A significant decrease in the NAD / NADH ratio was recorded in sTBI rats 7 days after injury. Treatment with LMW-DS resulted in significant improvements in nicotine coenzyme levels, except for the lowest dose. In particular, single and repeated doses of 15 mg / kg body weight of LMW-DS significantly improved NAD as measured in control animals. + Normalize levels and ensure proper NAD + / NADH ratio could be restored. [Table 28]

[0418] Effect of increasing doses of LMW-DS on CoA-SH derivatives Table 24 reports data on free CoA-SH and CoA-SH derivatives. A significant positive effect of 5 or 15 mg / kg body weight (both doses as single and repeated administration) was detected for both CoA-SH and acetyl-CoA, suggesting much more favorable metabolic conditions for the functional operation of the TCA cycle. [Table 29]

[0419] Effects of increasing doses of LMW-DS on antioxidant and oxidative / nitrosative stress biomarkers Table 25 shows the major water-soluble brain antioxidants (ascorbic acid and GSH) and the effects of oxidative (MDA) and nitrosative stress (-NO2 - and -NO3 - ) biomarker concentrations. 7 days after impact, no recovery of the concentrations of both water-soluble antioxidants occurred in sTBI-induced rats. Signs of extremely high levels of oxidative / nitrosative stress were also recorded. The effects of single and repeated administration of LWM-DS were particularly beneficial in restoring the concentrations of both ascorbic acid and reduced glutathione (GSH), with a clear reduction in brain tissue nitrite and nitrate. These effects were also significant when 5 mg / kg body weight was used. [Table 30]

[0420] To better understand how drug effects relate to drug dose, we report the results for ascorbic acid and GSH graphically in Figures 26 and 27.

[0421] Effects of increasing doses of LMW-DS on dephosphorylated purines and pyrimidines Further deterioration in most of the compounds reported in Table 26, originating from the purine and pyrimidine nucleotide degradation pathway and indirectly linked to cellular energy metabolism, was observed 7 days after injury in rats subjected to sTBI. Most of these compounds were favorably affected by drug administration. [Table 31]

[0422] Effect of increasing doses of LMW-DS on N-acetylaspartic acid (NAA) As previously mentioned, sTBI produces irreversible changes in NAA homeostasis. In this study, we again found that 7 days after sTBI, whole-brain NAA was approximately 50% lower than that measured in control rats (see Figure 28). Interestingly, a dose-dependent increase in NAA was detected in rats receiving a single, increasing dose of LMW-DS or repeated administration of the highest dose tested.

[0423] Effects of increasing doses of LMW-DS on free amino acids involved in neurotransmission The compounds listed in Table 27 are amino acids directly (GLU, GABA) or indirectly (GLN, ASP, AASN, GLY, SER, THR, ALA) involved in neurotransmission. Most of these amino acids were still higher in sTBI rats 7 days after injury compared to controls. From this table, it is clear that administration of LMW-DS was effective, especially when the drug was injected subcutaneously at 15 mg / kg body weight in single or repeated doses. Of particular relevance is the normalization of Glu, thus allowing LMW-DS to eliminate the cause of excitotoxicity by excessive Glu release after sTBI. [Table 32]

[0424] Effects of increasing doses of LMW-DS on free amino acids involved in the methyl cycle As shown in Table 28, the levels of free amino acids involved in the so-called methyl cycle or the formation of cysteine ​​were still different in sTBI rats 7 days after impact when compared with the corresponding values ​​in controls. An increase in MET was observed in animals receiving the maximum dose of LWM-DS (both single and repeated administration). As already observed 2 days after injury, these drug levels resulted in a significant increase in L-cystathionine (L-Cystat). Since this compound is an intermediate in the production of cysteine ​​(CYS), it is reasonable to assume that an increase in L-Cystat could result in an increase in CYS. It is worth recalling that the measurement of CYS requires a specific additional HPLC assay with an additional derivatization using F-MOC, a fluorescent compound that reacts with secondary amines and CYS. [Table 33]

[0425] Effects of increasing doses of LMW-DS on free amino acids involved in the production of nitric oxide (NO) Table 29 shows the concentrations of free amino acids directly involved in the production of NO. Seven days after sTBI, animals showed a concomitant decrease in ARG and an increase in CITR, consistent with data showing the stable NO end products nitrite and nitrate (Table 15). Administration of LMW-DS was particularly effective when doses of 5 or 15 mg / kg body weight (single and repeated) were used. [Table 34]

[0426] Effect of increasing doses of LMW-DS on long-chain free amino acids Animals in any other groups treated with the free amino acids reported in Table 30, which are useful sources of carbon skeletons for the production of α-keto acids that cells use to replenish the TCA cycle, and the drug of interest, were virtually normal 7 days after sTBI. [Table 35]

[0427] Effect of increasing doses of LMW-DS on free amino acids and aromatic free amino acids acting as osmolytes The results summarized in Table 31 clearly demonstrate that sTBI resulted in increased levels of taurine (TAU) 7 days after injury. LMW-DS administration normalized tau levels and resulted in an increase in aromatic amino acids. [Table 36]

[0428] Consideration TBI is one of the most common neurodegenerative diseases and the leading cause of death in people under 45 in Western countries. Its incidence is increasing, and by 2020, the World Health Organization estimates that TBI will be the leading cause of disability worldwide. Depending on the severity of symptoms associated with TBI (as assessed by the Glasgow Coma Scale), three distinct types of TBI can be identified: mild TBI (mTBI), moderate TBI, and severe TBI (sTBI). The ratio of mTBI to sTBI occurrence has been calculated to be approximately 22:1. Unfortunately, the consequences of TBI are often disabling and may lead to permanent or temporary impairments in cognitive, physical, and psychosocial functioning, with associated reductions or alterations in consciousness. Therefore, patients are affected in several important aspects of functioning properly and maintaining social relationships, primarily their ability to support themselves.

[0429] TBI is considered a complex pathological process consisting of a primary insult (an impact force acting on brain tissue) directly inducing a largely unpredictable secondary insult, characterized by a cascade of biochemical, metabolic, and molecular changes that cause severe mitochondrial dysfunction in brain cells. The severity of the injury depends on the impact force acting on the brain tissue, and this event, in fact, induces the elongation of axons and nerve fibers and triggers biochemical and molecular events that are not simultaneous with the onset of clinical symptoms.

[0430] To date, there are no satisfactory pharmacological treatments that reduce mortality and improve recovery in TBI patients. Putative pharmacological treatments are typically tested for their ability to interfere with the biochemical and molecular changes that occur to brain tissue metabolism and the neurometabolic cascades induced by the primary insult, such as vascular and blood flow changes that are closely correlated with tissue damage.

[0431] Previous studies have demonstrated a significant correlation between the severity of TBI and energy deficits associated with anaerobic metabolism, mitochondrial dysfunction, increased production of reactive oxygen species (ROS) and nitrogen species (RNS), and enhanced excitatory amino acid release. Furthermore, the N-acetylated amino acid N-acetylaspartate (NAA) is a reliable surrogate biomarker useful for monitoring the state of energy metabolism in vivo. Indeed, because mitochondrial NAA biosynthesis has a high indirect energy consumption, changes in NAA brain concentrations are closely related to alterations in the homeostasis of several parameters related to energy metabolism (ATP, GTP, ADP, AMP, acetyl-CoA, CoA-SH, and NAD+) and mitochondrial phosphorylation capacity (ATP / ADP).

[0432] Studies conducted to evaluate the effects of increasing doses of LMW-DS on brain metabolites in rats subjected to the induction of sTBI at different times after injury in a large panel demonstrated that administration of this compound resulted in a general recovery of brain metabolism.

[0433] LMW-DS was effective in restoring the severely imbalanced mitochondrial-associated energy metabolism in untreated sTBI animals, with favorable effects on the concentrations of purine triphosphate and pyrimidine nucleotides. Notably, ATP levels 7 days after impact were only 16% lower than control values, while a 35% decrease was observed in sTBI rats (Table 21 and Figure 25). Notably, NAA concentrations in LMW-DS-treated animals at the same time point were only 16% lower than control values, while sTBI animals showed 48% lower levels of this compound. This finding again strongly confirms the strict link between NAA homeostasis and proper mitochondrial energy metabolism and emphasizes the importance of pharmacological interventions that can favorably affect mitochondrial function.

[0434] The overall recovery of brain metabolism obtained by LMW-DS administration was also accompanied by the metabolism of nicotine coenzymes and free CoA-SH and CoA-SH derivatives, indicating that drug-treated animals, despite suffering from sTBI, had subnormal coenzymes that ensured proper redox reactions and allowed good functioning of the TCA cycle.

[0435] The above-mentioned improvement in brain metabolism was certainly responsible for another significant drug effect, namely the disappearance of GLU excitotoxicity. Furthermore, the drug affected sulfur-containing amino acids. This effect was probably related to the drug molecule containing the S atom. The increased bioavailability of this atom resulted in a net increase in the biosynthesis of these amino acids, one of which (MET) is crucial for methylation reactions and the so-called methyl cycle.

[0436] Further favorable effects recorded in this study were an increase in antioxidants and a decrease in biochemical signatures of oxidative / nitrosative stress in sTBI rats administered LMW-DS. Because dysfunctional mitochondria are a major intracellular source of both ROS and RNS, this phenomenon may even be closely related to the normalization of mitochondrial function. It is plausible that the effects of LMW-DS were more evident 7 days after sTBI than 2 days after. This strongly suggests that the overall recovery of cerebral metabolism by drug administration is not a transient phenomenon. It is also worth emphasizing that under the current experimental conditions, drug effects are often related to the administered dose, with repeated administration of 15 mg / kg body weight often being similar to a single administration of the same dose. Thus, repeated drug administration was not necessarily advantageous.

[0437] These contradictory results may be explained by the following: 1) it is well known that sTBI induces disruption of the blood-brain barrier (BBB); 2) the uptake of LMW-DS by brain tissue during the period of BBB alteration / disruption may be highly favorable; 3) if the hypothesis in point 2) is correct, administration performed 30 min after injury may have occurred while the BBB was still open / altered; 4) if the hypotheses in points 2) and 3) are correct, administration early after injury may have resulted in the compound's penetration into the brain compartment when the BBB was still open / altered. 5) If what we reported in point 4) is correct, it means that the administration of 15 mg / kg body weight of LMW-DS 30 min after sTBI, in addition to initiating normalization of cerebral metabolism, also led to closure of the BBB, thereby limiting the possibility of obtaining additional benefits from repeated drug administration protocols, as the second (on the third day) and third (on the fifth day) drug administrations were performed under conditions unfavorable for further significant passage into brain compartments.

[0438] Example 8 In this study, LMW-DS will be profiled with the BioMAP® Diversity PLUS Panel. The BioMAP® Panel consists of human primary cell-based systems designed to model various aspects of the human body in an in vitro format. The 12 systems in the BioMAP® Diversity PLUS Panel (Table 32) enable unbiased characterization of test agents across a broad set of systems modeling various human disease states. BioMAP® systems are constructed with one or more primary cell types derived from healthy human donors, with stimuli such as cytokines or growth factors added to capture relevant signaling networks that naturally occur in human tissues or pathological conditions. Vascular biology is modeled in both Th1 (3C system) and Th2 (4H system) inflammatory environments, as well as a Th1 inflammatory state specific to arterial smooth muscle cells (CASM3C system). Additional systems recapitulate aspects of the systemic immune response, including monocyte-promoted Th1 inflammation (LPS system) or T cell stimulation (SAg system), chronic Th1 inflammation driven by macrophage activation (IMphg system), and T cell-dependent activation of B cells occurring in germinal centers (BT system). The BE3C system (Th1) and BF4T system (Th2) represent pulmonary airway inflammation, while the MyoF system models myofibroblast-mediated lung tissue remodeling. Finally, skin biology is addressed with the KF3CT system, which models Th1 skin inflammation, and the HDF3CGF system, which models wound healing.

[0439] Each test drug generates a signature BioMAP® profile created from changes in protein biomarker readouts within the individual system environment. Biomarker readouts (7–17 per system) are selected for therapeutic and biological relevance, predict disease outcomes or specific drug effects, and are validated using reagents with known mechanisms of action (MoA). Each readout is quantitatively measured using immunologically based methods to detect proteins, such as ELISA, or functional assays that measure proliferation and viability. BioMAP® readouts are diverse and include cell surface receptors, cytokines, chemokines, matrix molecules, and enzymes. Altogether, the BioMAP® Diversity PLUS panel contains 148 biomarker readouts that capture biological changes occurring within the physiological context of a specific BioMAP® system.

[0440] material and method Four concentrations of LMW-DS (150 nM, 440 nM, 1.3 μM, 4 μM) were tested on a BioMAP® Diversity PLUS panel by Eurofins.

[0441] How to get Diversity PLUS Human primary cells in the BioMAP system were used at an early passage (passage 4 or earlier) to minimize adaptation to cell culture conditions and maintain physiological signaling responses. All cells were derived from a pool of multiple donors (n = 2–6), purchased commercially, and handled according to the manufacturer's recommended conditions. CD14 cells were transfected with GFP and GFP-GFP prior to addition to the Mphg system. + Human blood derived monocytes are differentiated into macrophages in vitro. Abbreviations are used as follows: human umbilical vein endothelial cells (HUVEC), peripheral blood mononuclear cells (PBMC), human neonatal dermal fibroblasts (HDFn), B cell receptor (BCR), T cell receptor (TCR), and toll-like receptor (TLR).

[0442] The cell types and stimuli used in each system were as follows: 3C system [HUVEC + (IL-1β, TNFα, and IFNγ)], 4H system [HUVEC + (IL-4 and histamine)], LPS system [PBMC and HUVEC + LPS (TLR4 ligand)], SAg system [PBMC and HUVEC + TCR ligand], BT system [CD19 + B cells and PBMCs + (α-IgM and TCR ligands)], BF4T system [bronchial epithelial cells and HDFn + (TNFα and IL-4)], BE3C system [bronchial epithelial cells + (IL-1β, TNFα, and IFNγ)], CASM3C system [coronary artery smooth muscle cells + (IL-1β, TNFα, and IFNγ)], HDF3CGF system [HDFn + (IL-1β, TNFα, IFNγ, EGF, bFGF, and PDGF-BB)], KF3CT system [keratinocytes and HDFn + (IL-1β, TNFα, IFNγ, and TGFβ)], MyoF system [differentiated lung myofibroblasts + (TNFα and TGFβ)], and / Mphg system [HUVECs and M1 macrophages + zymosan (TLR2 ligand)].

[0443] The systems are derived from single-cell or coculture systems. Adherent cell types are cultured to confluence in 96- or 384-well plates, followed by the addition of PBMCs (SAg and LPS systems). BT systems consist of CD19+ B cells cocultured with PBMCs and stimulated with BCR activators and low levels of TCR stimulation. Test agents prepared in DMSO (small molecules; final concentration ≤0.1%) or PBS (biologicals) are added at the indicated concentrations 1 hour before stimulation and allowed to remain in culture for 24 hours or the following time periods: 48 hours, MyoF system; 72 hours, BT system (soluble reading); 168 hours, BT system (secreted IgG)). Each plate contains a drug control appropriate for each system (e.g., 1.1 μM legacy control test agent colchicine), a negative control (e.g., unstimulated conditions), and a vehicle control (e.g., 0.1% DMSO). Biomarker levels of cell-associated and plasma membrane targets are measured using direct ELISA. Soluble factors from the supernatant are quantified using HTRF® detection, bead-based multiplex immunoassays, or capture ELISA. Overt adverse effects of test agents on cell proliferation and viability (cytotoxicity) are detected by sulforhodamine B (SRB) staining for adherent cells and alamarBlue® reduction for suspension cells. For proliferation assays, individual cell types are cultured at subconfluence and measured at optimized time points for each system (48 hours: 3C and CASM3C systems; 72 hours: BT and HDF3CGF systems; 96 hours: SAg system). Cytotoxicity against adherent cells is measured at the indicated time points by SRB (24 h: 3C, 4H, LPS, SAg, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, and / Mphg system; 48 h: MyoF system) and by alamarBlue staining of suspended cells (24 h: SAg system; 42 h: BT system).

[0444] Data analysis Biomarker measurements in test drug-treated samples were divided by the mean value of the control samples (at least six vehicle controls from the same plate) to generate a ratio, followed by log10 The significance prediction envelope is calculated using the previously collected vehicle control data with 95% confidence intervals.

[0445] Profile Analysis Two or more consecutive concentration changes in the same direction relative to the vehicle control were outside the significance envelope, with an effect size (|log 10 Biomarker activity is annotated if there is at least one concentration with a ratio |>0.1. If these activities are increased in some systems but decreased in others, the primary activity of the biomarker is called modulated. If total protein levels are reduced by more than 50% (log of SRB), 10 If the ratio or alamarBlue level <-0.3), the cytotoxicity status is recorded and indicated by a thin black arrow above the x-axis. If cytotoxicity is detected in three or more systems, the compound is considered to have broad cytotoxicity. Concentrations of test agents with detectable broad cytotoxicity are excluded from biomarker activity annotation and downstream benchmarking, similarity searching, and cluster analysis. Antiproliferative effects are observed when the SRB or alamarBlue log from cells seeded at lower densities is higher. 10 Defined by a ratio value <-0.1 and indicated by a gray arrow above the X-axis, cytotoxic and antiproliferative arrows only require one concentration to meet the indicated threshold for profile annotation.

[0446] Benchmark Analysis Common biomarker readouts are annotated if readouts for both profiles are outside the significance envelope with an effect size of >20% in the same direction. Differentiation biomarkers are annotated if one profile has readouts outside the significance envelope with an effect size >20% and the other profile has readouts inside the envelope or in the opposite direction. Unless otherwise specified, the highest non-cytotoxic concentrations of both the test agent and the benchmark agent are included in the benchmark overlay analysis.

[0447] Similarity analysis If the readouts for both profiles are outside the significance envelope and have an effect size greater than 20% in the same direction, common biomarker readouts are annotated. Test agent concentrations with three or more systems with detectable cytotoxicity are excluded from the similarity analysis. Test agent concentrations with one to two systems with detectable cytotoxicity are included in the similarity search analysis, along with a database overlay of the highest test agent concentration match. This is followed by an additional overlay of the next highest test agent concentration that does not contain a system with detectable cytotoxicity and its respective database match. To determine the degree of similarity between BioMAP® profiles of compounds performed on the Diversity PLUS panel, we developed a custom similarity measure (BioMAP Z-Standard), a combinatorial method with improved performance in mechanistic classification of reference agents compared to other measures tested (including Pearson and Spearman correlation coefficients). This method more efficiently accounts for variations in the number of data points, systems, active biomarker readouts, and amplitude of biomarker readout changes characteristic of BioMAP® profiles. A Pearson correlation coefficient (r) is first generated to measure the linear association between the two profiles based on the similarity in the direction and magnitude of the relationship. Because Pearson correlation can be affected by the magnitude of either biomarker activity, a per-system weighted average Tanimoto measure is used as a filter to account for the under-representation of less robust systems. The Tanimoto measure does not consider the amplitude of biomarker activity, but rather addresses whether the identity and number of readings are common to the weights on a per-system basis. The real-valued Tanimoto measure first maps each profile to a unit vector (e.g.,

number

number

number

number

number

number

[0448] Cluster analysis Cluster analysis (functional similarity map) uses the results of pairwise correlation analysis to project the "similarity" of drug profiles from multidimensional space to two dimensions. The functional clustering of drug profiles generated during this analysis uses Pearson correlation values ​​for pairwise comparisons of profiles for each concentration of each drug, and then subjects the pairwise correlation data to multidimensional scaling. Profiles that are similar with a Pearson correlation coefficient (r) ≥ 0.7 are connected by lines. Drugs that do not cluster with each other are interpreted as mechanistically distinct. This analysis is performed for projects containing three or more test drugs. Cytotoxic concentrations are excluded from the cluster analysis.

[0449] Mechanism heat map analysis Mechanism heat map analysis provides visualization of 19 consensus mechanisms, allowing comparison of biomarker activity across test compounds and all compound concentrations and consensus mechanisms. The composite consensus profile used in mechanism heat map analysis is an average representative BioMAP® profile of multiple compounds from structurally distinct chemical classes. Profiles were calculated by averaging each biomarker endpoint value for all selected profiles (multiple drugs at various concentrations) to construct a consensus mechanism profile. Biomarker activity is colored in the heat map for consensus mechanisms and compounds with expression outside the significance envelope compared to the vehicle control. Red represents increased protein expression, blue represents decreased expression, and white indicates unchanged or levels within the filtering criteria. Darker shades of color represent larger changes in biomarker activity compared to the vehicle control. Mechanism heat maps were generated using R and the gplot package for R.

[0450] Assay Acceptance Criteria BioMAP® assays contain multiparameter datasets generated by the BioMAP® platform for drugs tested in the systems that make up the Diversity PLUS panel. The assays include drug controls appropriate for each system (e.g., the legacy control test drug colchicine), negative controls (e.g., unstimulated conditions), and vehicle controls (e.g., DMSO). BioMAP® assays are plate-based, and data acceptance criteria depend on both plate performance (%CV of vehicle control wells) and system performance across the system's historical controls. A QA / QC Pearson test is performed by first setting a 1% false-negative Pearson cutoff from the historical positive control reference dataset. The process is repeated through all profiles of system biomarker readouts in the positive control reference dataset, and the Pearson value between each profile and the mean of the remaining profiles in the dataset is calculated. The total number of Pearson values ​​used to determine the 1% false-negative cutoff is the total number of profiles present in the reference dataset. The Pearson value at one percentile of all calculated values ​​is the 1% false-negative Pearson cutoff. If the Pearson value between the negative control or drug control profile of the experimental plate and the average of the historical control profiles in the reference dataset exceeds this 1% false negative Pearson cutoff, the system passes the test. If each individual system passes the Pearson test and 95% of all project plates have a %CV<20%, the overall assay is accepted.

[0451] result The BioMAP® Diversity PLUS panel included 12 individual BioMAP human primary cell-based co-culture systems, as shown in Table 32. [Table 37] TIFF0007745261000051.tif81162

[0452] Two or more consecutive concentrations that changed in the same direction relative to the vehicle control were outside the 95% significance envelope, with an effect size (|log 10 Biomarker activity was annotated if it had at least one concentration with a ratio |>0.1). The primary activity of a biomarker was called modulated if its activity was increased in some systems but decreased in others.

[0453] LMW-DS was active in 25 annotated readouts. LMW-DS was not cytotoxic to any of the human primary cells at the concentrations tested in this study. LMW-DS-mediated changes in key biomarker activity included decreased vascular cell adhesion molecule 1 (VCAM-1), monocyte chemotactic protein-1 (MCP1), soluble tumor necrosis factor alpha (sTNFα), interferon-induced T-cell alpha chemoattractant (I-TAC), monokine-induced by gamma interferon (MIG), and inflammation-related activity in the form of interferon gamma-inducible protein 10 (IP-10) and increased eotaxin 3 (Eot3), and interleukin 8 (IL-8). LMW-DS also had immunomodulatory activity in the form of reduced secretion of immunoglobulin G (sIgG) and macrophage colony-stimulating factor (M-CSF) and increased soluble IL-17A (sIL-17A) and cluster of differentiation 69 (CD69). LMW-DS also exhibited tissue remodeling activity in the form of increased matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator receptor (uPAR), and epidermal growth factor receptor (EGFR), and hemostasis-related activity in the form of increased thrombomodulin (TM). Table 33 summarizes the effects of LMW-DS on 12 different human primary cell types in the BioMAP® Diversity PLUS panel. [Table 38]

[0454] The BioMAP® Reference Database contains BioMAP® profiles for over 4,500 bioactive agents (biologics, approved drugs, chemicals, and experimental agents) and can be used to classify and identify the most similar profiles.

[0455] In an unsupervised search of mathematically similar compound profiles in the BioMAP® Reference Database, LMW-DS (4M) was most similar to Clexane (30 μg / ml) (Pearson's correlation coefficient, r=0.701). Clexane (enoxaparin sodium) is an anticoagulant low-molecular-weight heparin used to treat deep vein thrombosis (DVT). There are five common activities annotated in the following systems: BT (sIgG, sIL-17A), CASM3C (MIG), and HDF3CGF (VCAM-1, IP-10).

[0456] Consideration In the study, LMW-DS was characterized by profiling with the BioMAP® Diversity PLUS panel of human primary cell-based assays that model the complex tissue and disease biology of organs (vasculature, immune system, skin, lung) as well as general tissue biology. The BioMAP® Diversity PLUS panel assessed the biological impact of LMW-DS under conditions that maintain the complex crosstalk and feedback mechanisms associated with in vivo outcomes.

[0457] LMW-DS was active but not cytotoxic at the concentrations tested in this study. LMW-DS was moderately and selectively antiproliferative against human primary endothelial cells only at the highest concentration (4 μM). The LMW-DS profile had 25 annotated readouts and demonstrated modulation of immune and inflammation-related readouts and matrix-related biomarkers. Specific activities included reduced inflammation-related VCAM-1, MCP-1, sTNFα, I-TAC, MIG, and IP-10 and increased IL8. Moderately increased eotaxin-3 was observed only at lower concentrations in the BF4T system. Immunomodulatory activities included reduced sIgG and IL17A and IL17F in the BT system, but no antiproliferative effects on B cells. Reduced M-CSF and increased CD69 were also identified. LMW-DS also modulated tissue remodeling biomarkers, including increased MMP-1, PAI-1, uPAR, EGFR, and hemostasis-related TM. Key inflammatory biomarkers, including MIG, VCAM, IP-10, and ITAC, were reduced across all concentrations tested in the CASM3C and HDF3CGF systems, while increased expression of the chemotactic factor IL-8 was observed in multiple systems. Collectively, these data suggest that LMW-DS is involved in regulating immune activation and / or immune resolution responses in inflammation and wound healing biology.

[0458] Modulation of inflammatory markers indicates the utility of LMW-DS in the treatment of multiple chronic and acute inflammatory conditions and diseases with an inflammatory component, such as ALS.

[0459] Following injury, select components of the innate / pro-inflammatory and adaptive immune responses are initially upregulated to maintain defense against foreign pathogens, remove tissue debris present at the injury site, and orchestrate the tissue remodeling, cell proliferation, and angiogenesis processes associated with the wound response. However, for proper wound healing to proceed, this initial inflammatory response must be modulated or halted to allow matrix reconstruction, recellularization, and tissue remodeling. Such immune-resolving activity, induced by LMW-DS, includes activation of MMP-1, PAR-1, and uPAR, demonstrating induced immune resolution useful for treating tissues damaged by trauma, including neurotrauma, that would otherwise result in deleterious fibrosis.

[0460] LMW-DS modulated many biomarker activities in the HDF3CGF system, but only IL8 in the MyoF system. While both systems contain fibroblasts, HDF3CGF models wound healing and the matrix remodeling associated with such wound healing, whereas MyoF models fibrosis through collagen deposition. Therefore, the results indicate that LMW-DS had immunomodulatory and tissue remodeling activities but did not induce undesirable collagen fibrosis, which can result in harmful fibrotic deposition.

[0461] In conclusion, LMW-DS appears to normalize and resolve inflammation present in tissues following trauma or disease, and these results are therefore consistent with the effects of LMW-DS observed in the previous examples.

[0462] Example 9 The aim of this example was to determine the neuroprotective effects of different doses of LMW-DS (1, 5, and 15 mg / kg) in sTBI using gene expression surveys of differentially regulated genes followed by functional analysis.

[0463] material and method sTBI induction and drug administration protocol The experimental protocol used in this study was approved by the Ethics Committee of the Catholic University of Rome in accordance with international standards and guidelines for animal care. Male Wistar rats weighing 300–350 g were kept in a controlled environment and provided with standard laboratory chow and water ad libitum. Animals received an anesthetic mixture of 35 mg / kg body weight ketamine and 0.25 mg / kg body weight midazolam via intraperitoneal injection. Severe traumatic brain injury (sTBI) was induced by dropping a 450 g weight from a height of 2 m onto the rat's head, which was secured by a metal disk previously fixed on the skull, according to the "weight drop" impact acceleration model (Marmarou et al., A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics. J Neurosurg. 1994;80:291–300). Rats that developed skull fractures, seizures, nosebleeds, or did not survive the impact were excluded from the study. At the end of each treatment period, the rats were re-anesthetized and then immediately sacrificed.

[0464] Test Compound LMW-DS (Tikomed AB) was prepared at a stock concentration of 20 mg / ml and kept in a temperature-monitored refrigerator at 4° C. Aliquots of LMW-DS were diluted in sterile saline to the appropriate dosing concentration and then delivered as a single subcutaneous injection.

[0465] Acute phase-1 Three doses of LMW-DS were administered subcutaneously 30 minutes after TBI. Animals were sacrificed two days after TBI. Animals were divided into the following subgroups: 1. n = 4 animals underwent sTBI induction and received a 0.5 ml subcutaneous injection of LMW-DS at a concentration of 15 mg / kg. 2. n = 4 animals underwent sTBI induction and received a 0.5 ml subcutaneous injection of LMW-DS at a concentration of 5 mg / kg. 3. n = 4 animals underwent sTBI induction and received a 0.5 ml subcutaneous injection of LMW-DS at a concentration of 1 mg / kg.

[0466] Acute phase-2 Three doses of LMW-DS were administered subcutaneously 30 minutes after TBI. Animals were sacrificed 7 days after TBI. Animals were divided into the following subgroups: 4. n = 4 animals underwent sTBI induction and received a 0.5 ml subcutaneous injection of LMW-DS at a concentration of 15 mg / kg. 5. n = 4 animals underwent sTBI induction and received a 0.5 ml subcutaneous injection of LMW-DS at a concentration of 5 mg / kg. 6. n = 4 animals underwent sTBI induction and received a 0.5 ml subcutaneous injection of LMW-DS at a concentration of 1 mg / kg. 7. n = 4 animals underwent sTBI induction and received three repeated 0.5 ml subcutaneous injections of LMW-DS at a concentration of 15 mg / kg.

[0467] sTBI-No Treatment 8. n = 4 animals received sTBI induction only and were sacrificed 2 days after TBI 9. n = 4 animals received sTBI induction only and were sacrificed 7 days after TBI

[0468] Sham surgery (healthy control) 10. n = 4 animals received anesthesia only.

[0469] Brain tissue processing In all animals, an in vivo osteotomy craniotomy was performed while under anesthesia. The rat skull was carefully removed, exposing the brain, which was then removed with a surgical spatula, quickly placed in RNALater, and stored at 4°C for further processing.

[0470] RNA extraction and array analysis RNA extraction and array processing were performed by SourceBioscience. The arrays used were Agilent Rat Expression Arrays.

[0471] statistical analysis Statistical analysis was performed to quantify the effects of sTBI on the brain in this model. Subsequent analyses examined the effects of LMW-DS in this model using different iterations and algorithms. Statistical analysis was performed using the Metaboanalyst software package. A 10% change in gene expression was considered significant at p<0.05.

[0472] result Differential gene expression observed 2 days after sTBI Within 2 days of sTBI, brain gene expression was significantly altered, with a relatively small number of genes (221) being up- and down-regulated.

[0473] Within 2 days of sTBI, administration of 1 mg / kg LMW-DS within 30 min after injury altered TBI-specific gene expression of 372 genes, administration of 5 mg / kg LMW-DS within 30 min after TBI altered TBI-specific gene expression of 702 genes, and administration of 15 mg / kg LMW-DS within 30 min after TBI altered TBI-specific gene expression of 247 genes.

[0474] LMW-DS treated animals differed from healthy controls in 209 genes (1 mg / kg LMW-DS), 258 genes (5 mg / kg LMW-DS), and 47 genes (15 mg / kg LMW-DS).

[0475] Differential gene expression observed 7 days after sTBI Within 7 days of sTBI, brain gene expression was significantly altered, with a large number of genes (2739) being up- and down-regulated.

[0476] Within 7 days of sTBI, administration of 1 mg / kg LMW-DS within 30 min after injury altered TBI-specific gene expression of 3602 genes, administration of 5 mg / kg LMW-DS within 30 min after TBI altered TBI-specific gene expression of 3852 genes, and administration of 15 mg / kg LMW-DS within 30 min after TBI altered TBI-specific gene expression of 3901 genes.

[0477] LMW-DS-treated animals differed from healthy controls in the expression of 282 genes (1 mg / kg LMW-DS), 398 genes (5 mg / kg LMW-DS), and 158 genes (15 mg / kg LMW-DS). LMW-DS-treated animals (three repeated doses of 15 mg / kg LMW-DS) differed from healthy controls in the expression of 234 genes.

[0478] Comparative analysis of expression changes observed in LMW-DS Comparison of significantly affected genes by different statistical replicate runs provided information on how LMW-DS altered TBI-induced gene expression.

[0479] Comparison 2 days after TBI showed that out of 221 genes deregulated by TBI (2 days), only 22 (10%), 51 (23%), and 19 (8.5%) remained deregulated compared to healthy control animals when 1 mg / kg, 5 mg / kg, and 15 mg / kg LMW-DS were administered, respectively.

[0480] Comparisons 7 days after TBI showed that of the 2741 genes deregulated by TBI (7 days), only 124 (4.5%), 169 (6.1%), and 85 (3.1%) remained deregulated when LMW-DS was administered at 1 mg / kg, 5 mg / kg, and 15 mg / kg, respectively, compared to healthy controls. After three repeated doses of 15 mg / kg LMW-DS, the number of genes remaining deregulated was 116 (4.25%) more genes than in healthy controls.

[0481] Pathway analysis and mechanistic investigations Pathway analysis of differentially regulated genes was performed using the Ingenuity Pathway Analysis package, specifically focusing on pathways and molecular processes and diseases related to scar formation and fibrosis, including neurodegenerative diseases, including dementia, Alzheimer's disease, ALS, TBI, and stroke, as well as diseases related to glaucoma and normal pressure hydrocephalus (NPH) after subarachnoid hemorrhage.

[0482] Although the effects induced by TBI within 2 days were relatively small, changes in many neurodegeneration- and scar formation-related canonical pathways were significant. Most of these pathway changes were attenuated by LMW-DS administered within 30 minutes of TBI (Tables 34 and 35). Similar to pathways, the number of molecular processes and diseases significantly affected within 2 days of TBI was moderate. However, the effects of TBI were nearly abolished by LMW-DS administered 30 minutes after injury (Tables 36 and 37). [Table 39] TIFF0007745261000054.tif232162TIFF0007745261000055.tif232162TIFF0007745261000056.tif32162 * Unclear effect [Table 40] TIFF0007745261000058.tif231162TIFF0007745261000059.tif231162TIFF0007745261000060.tif60162 * Unclear effect [Table 41] TIFF0007745261000062.tif230162TIFF0007745261000063.tif229162TIFF0007745261 000064.tif230162TIFF0007745261000065.tif231162TIFF0007745261000066.tif53162 * Unclear effect [Table 42] TIFF0007745261000068.tif228162TIFF0007745261000069.tif229162TIFF0007745261000070.tif229162 TIFF0007745261000071.tif228162TIFF0007745261000072.tif228162TIFF0007745261000073.tif130162 * Unclear effect

[0483] The effects induced by TBI within 7 days were significant, with many genes being deregulated. Accordingly, changes in many neurodegeneration- and scar formation-related canonical pathways were significant. Most of these pathway changes were attenuated by ILB administered within 30 minutes of TBI (Tables 38 and 39). Similar to pathways, the number of molecular processes and diseases significantly affected within 7 days of TBI was large, and the effects were significant. However, the effects of TBI were nearly abolished by LMW-DS administered 30 minutes after injury (Tables 40 and 41). [Table 43] TIFF0007745261000075.tif228162TIFF0007745261000076.tif229162TIFF000 7745261000077.tif228162TIFF0007745261000078.tif231162TIFF00077452610 00079.tif229162TIFF0007745261000080.tif228162TIFF0007745261000081.t if228162TIFF0007745261000082.tif229162TIFF0007745261000083.tif206162 * Unclear effect [Table 44] TIFF0007745261000085.tif231162TIFF0007745261000086.tif232162TIFF0007745261000087.tif220162TIFF0007745261000088.t if214162TIFF0007745261000089.tif230162TIFF0007745261000090.tif231162TIFF0007745261000091.tif233162TIFF00077452610 00092.tif229162TIFF0007745261000093.tif231162TIFF0007745261000094.tif225162TIFF0007745261000095.tif230162TIFF000 7745261000096.tif229162TIFF0007745261000097.tif228162TIFF0007745261000098.tif231161TIFF0007745261000099.tif145162 [Table 45] TIFF0007745261000101.tif230162TIFF0007745261000102.tif227162TIFF0007745261000103.tif231162TIFF000 7745261000104.tif232162TIFF0007745261000105.tif230162TIFF0007745261000106.tif233162TIFF0007745261 000107.tif232162TIFF0007745261000108.tif225162TIFF0007745261000109.tif234162TIFF0007745261000110. tif233162TIFF0007745261000111.tif230162TIFF0007745261000112.tif230162TIFF0007745261000113.tif46162 [Table 46] TIFF0007745261000115.tif232162TIFF0007745261000116.tif227162TIFF0007745261000117.tif232162TIFF000 7745261000118.tif229162TIFF0007745261000119.tif233162TIFF0007745261000120.tif232162TIFF00077452610 00121.tif232162TIFF0007745261000122.tif226162TIFF0007745261000123.tif233162TIFF0007745261000124.t if229162TIFF0007745261000125.tif230162TIFF0007745261000126.tif227162TIFF0007745261000127.tif227162 * Unclear effect

[0484] Consideration LMW-DS was able to attenuate and reverse the effects of TBI on most pathways and molecular processes. Data showed that LMW-DS could normalize tissue gene expression and function after TBI. The functions and pathways investigated were highly relevant to neurodegenerative diseases and fibrosis and scar formation. Results demonstrated that LMW-DS can beneficially influence these pathways, even when they are severely perturbed.

[0485] It will be understood that the above-described embodiments are some illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations, and changes can be made to the embodiments without departing from the scope or spirit of the present invention. In particular, where technically feasible, different measures in different embodiments can be combined into other configurations.

Claims

1. A pharmaceutical composition for the treatment of glutamate excitotoxicity, comprising dextran sulfate or a pharmaceutically acceptable salt thereof, wherein the dextran sulfate or the pharmaceutically acceptable salt thereof has a number average molecular weight (M) in the range of 1,850 to 3,500 Da as measured by nuclear magnetic resonance (NMR) spectroscopy. ｎ ), wherein the average number of sulfates per glucose unit is in the range of 2.5 to 3.0, and the average sulfation at C2 position in the glucose unit of the dextran sulfate is at least 90%.

2. The pharmaceutical composition of claim 1 for the treatment of glutamate excitotoxicity in neurons.

3. 3. The pharmaceutical composition of claim 1 or 2 for the treatment of glutamate excitotoxicity in a subject suffering from a neurological disease, disorder or condition.

4. 4. The pharmaceutical composition of claim 3, wherein the neurological disease, disorder or condition is traumatic brain injury (TBI).

5. 4. The pharmaceutical composition of claim 3, wherein the neurological disease, disorder or condition is amyotrophic lateral sclerosis (ALS).

6. 4. The pharmaceutical composition of claim 3, wherein the neurological disease, disorder or condition is Alzheimer's disease (AD).

7. 4. The pharmaceutical composition of claim 3, wherein the neurological disease, disorder or condition is subarachnoid hemorrhage (SAH).

8. 8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average sulfur content in the range of 15 to 20%.

9. The dextran sulfate or a pharmaceutically acceptable salt thereof has a number average molecular weight (M) in the range of 1,850 to 2,500 Da, preferably in the range of 1,850 to 2,300 Da, as measured by nuclear magnetic resonance (NMR) spectroscopy. ｎ 9. The pharmaceutical composition according to claim 1, wherein

10. The dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 1,850 to 2,000 Da as measured by NMR spectroscopy. ｎ 10. The pharmaceutical composition of claim 9, wherein

11. 11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of sulfates per glucose unit in the range of 2.5 to 2.8, and preferably in the range of 2.6 to 2.

7.

12. 12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average of 5.1 glucose units and an average number of sulfate groups per glucose unit of 2.6 to 2.

7.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutically acceptable salt thereof is the sodium salt of dextran sulfate.

14. 1. Use of dextran sulfate or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of glutamate excitotoxicity, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has a number average molecular weight (M) in the range of 1,850 to 3,500 Da as measured by nuclear magnetic resonance (NMR) spectroscopy. ｎ ), the average number of sulfates per glucose unit is in the range of 2.5 to 3.0, and the average sulfation at C2 position in the glucose unit of the dextran sulfate is at least 90%.

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