New use of dextran sulfate

Abstract

To provide a drug having positive effects in subjects suffering from neurological diseases, including neurodegenerative diseases, demyelinating diseases, neuro ischemic diseases and neuromuscular diseases.SOLUTION: Dextran sulfate, or a pharmaceutically acceptable derivative thereof, is used to induce differentiation of glial cells and neurons.SELECTED DRAWING: Figure 12

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), and 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 compromised, as seen in reduced metabolic function and mitochondrial energy metabolism and elevated oxygen stress. Damaged neurons further release glutamate, which has an excitotoxic effect on nearby neurons, which in turn causes further cell damage and cell 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 drugs that are useful 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 pharma- ceutically 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 specifically induce the differentiation of glial cells and neurons, reduce the oxidative stress of neurons and glial cells, reduce glutamate excitotoxicity, improve the metabolic function and energy metabolism of the mitochondria of neurons and glial cells, and activate the body's endogenous repair mechanisms.Dextran sulfate, or its pharmaceutically acceptable derivatives, can also prevent fibrosis by suppressing fibrogenic factors such as TGF-β, activating fibrolysis, and thereby inducing the decomposition of existing scar tissue and the realization of 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 dissipating 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, in which: [Brief description 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. Since DNA content changes during the cell cycle, PI staining can indicate cell cycle progression. The data showed that most cells remain in the G1 phase of the cell cycle (dotted arrows), but low molecular weight dextran sulfate (LMW-DS) appears to increase the number of cells in the G2 / M phase (solid arrows). [Diagram 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). [Diagram 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 is a representative photograph of βIII-tubulin expression in mouse cortical neurons. [Figure 5A] 5A and 5B show the effect of LMW-DS on βIII-tubulin expression in mouse cortical neurons. The graphs show the overall intensity (FIG. 5A) and average size (FIG. 5B) of positive cells. [Figure 5B] 5A and 5B show the effect of LMW-DS on βIII-tubulin expression in mouse cortical neurons. The graphs show the overall intensity (FIG. 5A) and average size (FIG. 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 following EAE induction in mice for the negative control (vehicle), the positive control cyclosporine 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-13D show the change in levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a function of mitochondrial phosphorylation capacity. [Figure 13B] 13A-13D show the change in levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a function of mitochondrial phosphorylation capacity. [Figure 13C] 13A-13D show the change in levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a function of mitochondrial phosphorylation capacity. [Figure 13D]13A-13D show the change in levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a function of mitochondrial phosphorylation capacity. [Figure 14A] 14A-14D show changes in levels of oxidized and reduced nicotine coenzyme. [Figure 14B] 14A-14D show changes in levels of oxidized and reduced nicotine coenzyme. [Figure 14C] 14A-14D show changes in levels of oxidized and reduced nicotine coenzyme. [Figure 14D] 14A-14D show changes in levels of oxidized and reduced nicotine coenzyme. [Figure 15A] 15A-15C show changes in levels of biomarkers indicative of oxidative stress. [Figure 15B] 15A-15C show changes in levels of biomarkers indicative of oxidative stress. [Figure 15C] 15A-15C show changes in 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 changing levels of N-acetylaspartate (NAA) and its substrates. [Figure 17B] 17A-17C show changing levels of N-acetylaspartate (NAA) and its substrates. [Figure 17C] 17A-17C show 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 in subjects with primary open angle glaucoma (POAG) and treated with saline control or LMW-DS. [Figure 21] FIG. 21 shows changes in fibronectin immunoreactivity in the angle in subjects with POAG and treated with saline control or LMW-DS. [Figure 22] Figure 22 shows amyloid beta monomer and oligomer preparations. Oligomeric (lanes 1, 2, 5-7) or monomeric (lanes 3 and 4) preparations of amyloid beta (1-42)A or amyloid beta biotin (B). 50 pmole (lane 5), 100 pmole (lanes 1, 3 and 6) or 200 pmole (lanes 2, 4 and 7) of each peptide preparation were loaded onto the gel. Proteins in the ongoing Western blot were immunolabeled with anti-amyloid beta. Expected oligomer 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] FIG. 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 min after trauma induction. Controls are sham-operated animals. Values ​​are the mean of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control at p<0.01. **Significantly different from 2 days after sTBI at p<0.01. [Diagram 25] FIG. 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 at p<0.01. **Significantly different from 2 days after sTBI at p<0.01. [Figure 26] FIG. 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 are sham-operated animals. Values ​​are the mean of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control at p<0.01. **Significantly different from 2 days after sTBI at p<0.01. [Figure 27] FIG. 27 shows the concentration of glutathione (GSH) 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 at p<0.01. **Significantly different from 2 days after sTBI at p<0.01. [Figure 28] FIG. 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 are sham-operated animals. Values ​​are the mean of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control at p<0.01. **Significantly different from 2 days after sTBI at p<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 produce a range of symptoms. Although the brain and spinal cord are surrounded by tough membranes and confined in the bones of the skull and spine, and are chemically isolated by the blood-brain barrier, they are highly susceptible to compromise. Although nerves are often thought to be located deep under the skin, they can still be exposed to damage. Individual neurons, as well as the neural networks and nerves they form, are susceptible to electrochemical and structural destruction. Nerve regeneration can occur in the peripheral nervous system, thus overcoming or functioning around damage to some extent, but 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 anomalies or disorders, infections, lifestyle habits, environmental health issues including nutritional disorders, and brain, spinal cord or nerve injuries. Problems can also begin in another body system that interacts with the nervous system. For example, cerebrovascular disorders involve brain damage due to problems related to the blood vessels, i.e., the cardiovascular system that supplies the brain; autoimmune disorders involve damage caused by the body's own immune system; lysosomal storage diseases such as Niemann-Pick disease can lead to neurological symptoms.

[0015] A neurodegenerative disease, disorder or condition is one that causes progressive loss of neuronal structure and / or function, 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 neuronal and synaptic loss 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 seen in AD on neuropathological examination. It is defined as near total tissue loss with loss of myelin, axons, and oligodendrocytes, and 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 the axons begins to erode. Neuroinflammation is the pathological hallmark of PD and is characterized by activated microglia and infiltrating T cells at the sites 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 repeat 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 the nerves. Increased activated T cells in the peripheral blood have been identified in HD patients.

[0020] ALS, also called Lou Gehrig's disease, is a debilitating disease with various etiologies, characterized by rapidly progressive weakness, muscle atrophy and fasciculation, muscle spasticity, dysarthria, dysphagia and dyspnea. 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 characteristic of the ALS pathology is the loss of motor neurons in the anterior horn of the spinal cord and in the motor nuclei of the brainstem. This results in a secondary atrophy of the corresponding muscles (muscle atrophy). Neuroinflammation is the pathological hallmark of ALS, characterized by activated microglia and infiltrating T cells at the site of neuronal damage. "Lateral sclerosis" refers to corticospinal tract degeneration (lateral to the site of the spinal cord). In fact, myelin loss 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 the signal conduction of the affected nerve, thereby causing 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) diseases (all affecting the peripheral nervous system (PNS)).

[0023] MS is an inflammatory disease in which the fatty myelin sheath around the axons in the brain and spinal cord is damaged, leading to demyelination and scarring and a wide range of signs and symptoms. MS involves T cells directing 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 do occur in multiple locations. However, the disease only affects central nervous system myelin, not peripheral nerve myelin. Thus, the condition is a CNS-specific disorder.

[0024] ADEM is an immune-mediated disease of the brain. It occurs after viral, bacterial, or parasitic infections or appears spontaneously. ADEM attacks nerves in the CNS and damages their myelin insulation, which results in the destruction of white matter. It is similar to MS as it involves autoimmune demyelination and is considered part of the MS field disease. ADEM produces multiple inflammatory lesions in the brain and spinal cord, especially 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, which may reduce sensation, movement, glandular or organ function, or other health conditions, depending on the type of nerve affected. Common causes include systemic diseases such as diabetes or leprosy; vitamin deficiencies; drugs, such as chemotherapy agents, commonly prescribed antibiotics; trauma; ischemia; radiation therapy; excessive alcohol consumption; immune system disorders; celiac disease; or viral infections. Neuropathies may be or are acute. Acute neuropathy requires urgent diagnosis. Motor nerves that control muscles, sensory nerves, or autonomic nerves that control autonomic functions such as heart rate, body temperature, and breathing may be affected. Two or more nerves may be affected simultaneously.

[0026] CPM is a neurological disorder caused by severe damage to the myelin sheath of nerve cells in the brainstem, or more precisely, in an area called the pons, mainly of iatrogenic etiology. It is characterized by acute paralysis, dysphagia, and dysarthria, as well as 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 diseases. It applies to all cerebral white matter diseases, whether the molecular cause is known or not. Leukoencephalopathy specifically refers to 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 one of a group of disorders characterized by the degeneration of white matter in the brain. Leukodystrophies are due to the 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 result in inflammation in the CNS along with loss of myelin. Leukodystrophies are characterized by specific symptoms including decreased motor function, muscle stiffness, and eventually decreased vision and hearing. Specific types of leukodystrophies include adrenomyeloneuropathy, Alexander disease, cerebrotendinous xanthomatosis, inherited CNS demyelinating diseases, Krabbe disease, metachromatic leukodystrophy, Pelizaeus-Merzbach disease, Canavan disease, vanishing white matter disease, adrenoleukodystrophy, and Refsum disease.

[0030] GBS, also called Landry's palsy or Guillan-Barre-Strohl syndrome, is an acute polyneuropathy affecting the PNS. In GBS, immune cells attack the myelin sheath (a fatty substance that covers nerve fibers). Ascending anesthesia is a common symptom. GBS is thought to be an immune-mediated disease with an abnormal T-cell response induced by infection. Cellular and humoral immune mechanisms are probably involved in its development. Most patients report an infectious illness several weeks prior to the onset of GBS. It is believed that many identified infectious agents induce the production of antibodies that cross-react with specific gangliosides and glycolipids, such as GM1 and GD1b, which are 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 differentiation of neurons and glial cells, such as oligodendrocytes and Schwann cells, is one of the first steps in disease progression, which is generally followed by 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 may be useful, for example, in the treatment of neurological diseases, disorders, or conditions.

[0034] The experimental data presented herein show that the dextran sulfate of the embodiment can induce the differentiation of neurons and glial cells. This effect of dextran sulfate 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 cells.

[0035] Dextran sulfate of the 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 quite surprising in view of US 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 inhibits them. Thus, it appears that the cell differentiation ability of the embodiment dextran sulfate is cell type specific, and thus potentially may be limited to neurons and glial cells. Prior art data shows that dextran sulfate actually has the opposite effect on other cell types, as shown by the progenitor cells in the above mentioned US 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 the core components of the brain and spinal cord of the CNS and the ganglia of the PNS. Special types of neurons include sensory neurons, which respond to touch, sound, light and all other stimuli that affect cells in the sense organs that later send signals to the spinal cord and brain; motor neurons, which receive signals from the brain and spinal cord, 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 dendrites or the axon, especially in their undifferentiated stages. Dendrites are thin structures arising from the cell body, often extending for 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 a site called the axon hillock and extend for a certain distance. Nerve fibers are often bundled into fascicles, and in the PNS, bundles of fibers constitute the nerve. 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 a specialized type of stem cell. Astrocytes are astroglial cells that have also been observed to turn into neurons due to the multipotency characteristic of stem cells. In humans, neurogenesis is largely halted during adulthood, but in two brain regions, the hippocampus and the olfactory bulb, there is strong evidence of the generation of substantial numbers of new neurons.

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

[0041] βIII-tubulin, also called class III β-tubulin, is a microtubule element 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, with their "minus ends" usually pointing toward the centrosome in the cell body, while their "plus ends" project toward the tips of the axons, in neurons. Microtubule polarity serves important functions in both differentiated and adult neurons. During differentiation, tubulin increases in cells and constructs microtubules that allow differentiating neurons to extend or retract growing axons in response to guidance cues to maintain directional growth toward postsynaptic targets. Their activity is essential for cell migration, axon development and guidance, and is also required for the function and viability of adult neurons (Bioscience Reports (2010), 30:319-330).

[0042] 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, mainly muscles and glands. The axons of motor neurons are efferent nerve fibers that carry effector signals from the spinal cord to produce an effect.

[0045] Motor neuron diseases (MNDs) are neurological disorders that selectively affect motor neurons. These MNDs are 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 called hereditary spastic paraplegia, familial spastic paraplegia, French settlement disease, or Strampel-Lorraine disease, is a group of inherited disorders whose main feature is a progressive disturbance in walking. The disease presents with progressive stiffening (spasticity) and contraction of the legs. The symptoms are the result of dysfunction of long axons in the spinal cord. The affected cells are primary motor neurons, and therefore the disease is an upper motor neuron disease. HSP is caused by impaired transport through the cells of proteins, structural proteins, protein-maintaining cells, lipids, and other substances.

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

[0048] Also known as Duchenne-Allan muscular dystrophy, PMA 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 tracts, including 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 have increased reflexes and spasticity of the tongue and bulbar area, slurred speech, and sometimes uncontrolled emotional outbursts. The condition is usually caused by damage to and bilateral degeneration of neurons in the brainstem, particularly the corticobulbar tract (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 function of neurons in the anterior horn of the spinal cord and subsequent tissue-wide skeletal muscle atrophy.

[0052] Also called post-polio syndrome or post-polio syndrome, PPS is a condition that affects approximately 25-40% of people who survive an initial acute attack from a previous polio (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 may cause a decline in muscle strength.

[0053] Cortical neurons are cells of the cerebral cortex of the brain. Most of the complex activities of the brain 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, providing nutrients and oxygen to neurons, 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. The glial cell types present in the CNS include astrocytes, oligodendrocytes, ependymal cells, radial glia and microglia. The glial cell types present in the PNS include Schwann cells, satellite cells and enteric glial cells.

[0057] Astrocytes, also called astroglia, are the most abundant type of macroglial cell in the CNS. Astrocytes have 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 may regulate vasoconstriction and vasodilation by producing substances such as arachidonic acid, whose metabolites are vasoactive.

[0058] Oligodendrocytes, the cells that coat axons in the CNS with their cell membrane, form 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 called 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 compared to neuroepithelial cells. In the developing nervous system, radial glia function both as neural precursor cells and as a scaffold over 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 main 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, as they constantly clean the CNS of plaques, damaged or unnecessary neuronal synapses, as well as infectious agents.

[0062] Schwann cells are functionally similar to oligodendrocytes, but are present in the PNS rather than in the CNS. Thus, Schwann cells enable myelination of axons in the PNS. They also have phagocytic activity and remove cellular debris, 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 enteric 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 nerves in the nervous system 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 plasma membrane of Schwann cells. 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 increase in 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 embodiment 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] The dextran sulfate of the embodiment not only induces differentiation of cells of the CNS and PNS, but also benefits neurological diseases, disorders and conditions. The experimental data presented herein shows that the dextran sulfate of the embodiment has a favorable effect in combating metabolic changes observed in neurological diseases, disorders and conditions, such as traumatic brain injury (TBI). Thus, many neurological diseases, disorders and conditions are characterized by the regulation of various metabolites related to cellular energy status and mitochondrial function. Furthermore, regulation in amino acid metabolism is observed in many neurological diseases, disorders and conditions. These metabolic changes are early cellular signals that affect enzyme activity and gene and protein expression changes that indicate pathological tissue response. The dextran sulfate of the embodiment functions to favorably regulate cellular metabolism in damaged tissue, thereby inhibiting or at least suppressing any subsequent regulation of enzyme activity and gene and protein expression that contributes to adverse outcomes.

[0071] More specifically, the dextran sulfate of the embodiment can reduce the level of glutamate excitotoxicity, restore the adverse changes in metabolic homeostasis, thereby effectively protecting mitochondrial function and providing neuroprotective effects. The dextran sulfate of the embodiment has 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 measurements of mitochondrial phosphorylation capacity.

[0072] Dextran sulfate of the embodiment also leads to a significant reduction in oxidative stress.In particular, the levels of ascorbic acid as the main water-soluble brain antioxidant and glutathione (GSH) as the main intracellular sulfhydryl group (SH) donor are significantly improved.In addition, the levels of malondialdehyde (MDA), which is used as the end product of polyunsaturated fatty acids in membrane phospholipids and therefore as a marker of reactive oxygen species (ROS)-mediated lipid peroxidation, show a significant reduction after administration of dextran sulfate.After dextran sulfate treatment, all of the above oxidative stress markers show 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 embodiment has a favorable effect on NO-mediated nitrosative stress.

[0074] N-acetylaspartic acid (NAA) is a brain-specific metabolite and a useful biochemical marker for monitoring the deterioration or recovery after neurological diseases, disorders and conditions such as TBI. NAA is synthesized in neurons from aspartic acid and acetyl-CoA by aspartic acid N-acetyltransferase. Dextran sulfate of the embodiment has shown significant improvement in NAA level.

[0075] The experimental data presented herein thereby demonstrate that dextran sulfate of the embodiments can protect against cell loss caused by oxidative stress and / or glutamate excitotoxicity in diseased and damaged nervous systems. By protecting cell metabolism, dextran sulfate of the embodiments can be a useful protective therapeutic agent for 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, retinal injury, etc. These neurological diseases, disorders and conditions have a common link in terms of the death and damage of neuronal function of neurons that occurs in all conditions. There is a commonality in the causes of this neuronal death. Of particular relevance is the toxicity caused by high levels of the neurotransmitter glutamate released from dying neurons. Dextran sulfate of the embodiments induces the scavenging of released glutamate in glial cells, thereby preventing the accumulation of toxic amounts of glutamate in the interneuronal space. This will 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 nerve cells are damaged or killed by excessive stimulation by neurotransmitters, especially 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 extremely rapid benzodiazepine withdrawal, as well as HS. Another common condition that results in excess glutamate concentrations around neurons is hypoglycemia.

[0078] During normal conditions, glutamate concentration can increase to 1 mM in the synaptic cleft, which rapidly declines over the course of a few milliseconds. If glutamate concentration around the synaptic cleft cannot be reduced or reaches high levels, neurons will kill themselves by a process called apoptosis. This pathological phenomenon can also occur after brain injury, such as TBI and SCI. Within minutes of injury, damaged nerve cells in the lesion site spill glutamate into the extracellular space, where it stimulates presynaptic glutamate receptors and enhances the release of further glutamate. Brain trauma or stroke can cause ischemia, where blood flow is reduced to inadequate levels. Following ischemia, accumulation of glutamate occurs in the extracellular fluid, causing cell death, which is exacerbated by the lack of oxygen and glucose. 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 to actively remove glutamate.

[0079] Furthermore, increased extracellular glutamate levels are associated with increased Ca2+ levels in the myelin sheath and on oligodendrocytes. 2+ This leads to activation of permeable N-methyl-D-aspartate (NMDA) receptors, which in turn increases Ca 2+ This leaves the oligodendrocytes susceptible to influx and subsequent excitotoxicity. One of the harmful consequences of excess calcium in the cytosol is the initiation of apoptosis by cleaved caspase processing. Another harmful 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 and release reactive oxygen species and various proteins, which can lead to apoptosis. The pore can also cause mitochondria to release even more calcium. In addition, the production of adenosine triphosphate (ATP) is stopped and ATP synthase can actually start hydrolyzing ATP instead of producing it.

[0080] Insufficient ATP production resulting from brain trauma can eliminate the electrochemical gradients of certain ions. Glutamate transporters require the maintenance of these ionic gradients to remove glutamate from the extracellular space. Loss of the ionic gradient not only results in the cessation of glutamate uptake but also in the reversal of 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 accumulation of glutamate 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 of embodiments to prevent or at least inhibit the accumulation of toxic levels of glutamate effectively protects surrounding neurons from glutamate excitotoxicity, and as a result, dextran sulfate of embodiments protects neurons from damage and cell death that would otherwise result from this glutamate excitotoxicity.

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

[0084] Thus, dextran sulfate of the embodiment is effective in restoring the severely imbalanced mitochondrial-related energy metabolism in subjects with brain injury, such as severe TBI (sTBI), with favorable effects on the concentration of purine triphosphate and pyrimidine nucleotides. In particular, ATP levels were 16% lower than those of healthy control subjects, while untreated sTBI subjects showed a 35% decrease. Of note, NAA concentrations in dextran sulfate-treated sTBI subjects were 16% lower than those of healthy control subjects, while sTBI subjects showed a 48% lower value 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, meaning that dextran sulfate-treated subjects, despite suffering from sTBI, had subnormal coenzymes that compensated for correct redox reactions and allowed good functional operation of the TCA cycle.

[0086] Moreover, the above-mentioned improvement of brain metabolism was responsible for another significant dextran sulfate effect, namely the disappearance of glutamate excitotoxicity. Moreover, dextran sulfate affected sulfur-containing amino acids. Presumably, this effect could be related to the dextran sulfate molecule containing an S atom. The increase in the 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.

[0087] Further positive effects recorded were an increase in antioxidants and a decrease in biochemical signatures of oxidative / nitrosative stress in sTBI subjects receiving dextran sulfate. It is pertinent that the effect of dextran sulfate was more evident 7 days after sTBI than 2 days after sTBI. This strongly suggests that the general restoration of cerebral metabolism by dextran sulfate administration was not a transient phenomenon.

[0088] In an embodiment, dextran sulfate further comprises a β-amyloid oligomer and a PrP C and have competing affinity for a protein-protein interaction between these two proteins, 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 (HUVEC) with respect to protecting against apoptosis; inducing angiogenesis (in HUVEC); 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 damage and promote the development of new blood vessels in damaged or diseased tissue, such as after stroke or other ischemic conditions.

[0091] Analysis of key molecular pathways showed that dextran sulfate reduced oxidative stress effects on mitochondria and increased harmful glutamate uptake in Schwann cells. As a result, 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 in turn leads to reduced mitochondrial oxidative stress. In addition, dextran sulfate of embodiments also induced the expression of the protein complex calmodulin (CALM) (a multifunctional intermediate calcium-binding messenger protein); Gbeta-gamma complex (Gβγ) (a tightly bound 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 diagrammatically in FIG. 19, this protein complex in turn inhibits 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 show that dextran sulfate of the embodiment can prevent and suppress apoptosis, prevent its negative effect on amyloid beta and Lewy body pathology and mitochondrial fragmentation and dysfunction and subsequent damage, and suppress inhibitory fatty acid oxidation.Dextran sulfate of the embodiment also improves mitochondrial function and reduces mitochondrial levels of H2O2 and reactive oxygen species.

[0094] Analysis of upstream regulators of genes regulated by dextran sulfate showed that dextran sulfate of the embodiment enhanced the effect of existing growth factors on cells. As shown in Tables 12-14, dextran sulfate of the embodiment could modulate the effect of some growth factors by increasing their activation or decreasing their inhibition. This means that dextran sulfate of the embodiment can be used for diseases, disorders and conditions where increasing the activity or decreasing 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 disease; and metabolic disorders.

[0095] The 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 contains heparin) was relatively less than in Schwann cells (normal control medium does not contain 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 cell metabolism, dextran sulfate of the embodiment can 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 embodiment can support damaged tissue to restore some lost function while enhancing remaining endogenous repair mechanisms.

[0098] Thus, gene expression data confirms the potential therapeutic utility of dextran sulfate of embodiments in 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] A further interesting effect of the dextran sulfate of the embodiment is that it affects cell adhesion. Cell adhesion was mainly affected in neurons and Schwann cells, and the dextran sulfate of the embodiment promoted cell detachment and migration. The effect on cell adhesion is mainly due to the expression of metalloproteinase-type enzymes. This finding also explains the anti-scarring effect of the dextran sulfate of the embodiment. This result suggests that the anti-scarring effect is mediated by the dextran sulfate of the embodiment by activating degradative enzymes that support tissue remodeling and block fibrogenic (scarring) signals in damaged tissue.

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

[0101] Therefore, the anti-scarring effect of dextran sulfate of the embodiment indicates its possible use in the treatment of fibroproliferative (scarring) conditions. These include, for example, glaucoma, proliferative vitreoretinopathy, cerebrospinal traumatic injury, cerebral subarachnoid hemorrhage, invasive surgery, postoperative adhesions, rotator cuff injury, burns, reconstructive surgery, ulcerative conditions (diabetes), etc. 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 after cancer treatment; hepatic fibrosis, such as liver cirrhosis and biliary atresia; cardiac fibrosis, such as atrial fibrosis, endomyocardial fibrosis, and old myocardial infarction; brain fibrosis, such as glial scar; pancreatitis; arthrofibrosis; Crohn's disease; Dupuytren's contracture; keloid; 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, heart, and in association with cell therapy and cell transplants such as islets of Langerhans, hepatocytes, insulin-producing cells, stem cells, progenitor cells, etc.

[0103] Interestingly, gene expression data also indicates that dextran sulfate of embodiments activates the production of a natural scar-reducing molecule called decorin, which further blocks 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 effect of dextran sulfate of the embodiments indicates its potential use in the treatment of all clinical conditions where scar formation is a problem. Dextran sulfate should work effectively on 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, since dextran sulfate can not only be used to inhibit or at least suppress fibrosis and harmful scar formation, but can also degrade already established scars. This means that dextran sulfate of the embodiments allows scar degradation and tissue remodeling to allow 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 regulating 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 contain an inflammatory component, such as ALS.

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

[0111] The effect of dextran sulfate in resolving inflammation shown in the experimental data 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 in terms of preventing fibrosis. Furthermore, the resolution of inflammation and suppression of microglial responses observed in the experimental data are also important in neurodegenerative diseases, disorders and conditions.

[0112] Thus, dextran sulfate, or a pharma- ceutical acceptable derivative thereof, would 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] In general, the function of the nervous system depends on the number of nerve cells, healthy energy metabolism of nerve cells and healthy connections between nerve cells.Neurodegenerative diseases and disorders and injuries that cause neurodegeneration usually have different triggers and causes, but all lead to the same final result, namely, neurodegeneration.The functional effects of such diseases, disorders or injuries are often only observed after a relatively large number of nerve cells die, while the trigger of disease or disorder may exist several years before symptoms occur.

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

[0116] Thus, during neurodegeneration and injury, there are multiple triggers of neuronal apoptosis that all contribute to neuronal loss. These triggers include oxidative stress leading to mitochondrial dysfunction and neurotransmitter dysregulation leading to glutamate excitotoxicity, thereby limiting energy supply to neurons. Dysregulated neurofilaments also lead to reduced motility and limited supply of factors required for neuronal survival. Further triggers include the release of inflammatory mediators that cause secondary cell damage and scar formation. In addition, vascular disorders are common in neurodegenerative conditions.

[0117] Glutamate is produced in neurons and is important for signaling mechanisms that support learning and memory in neurons. Excess glutamate released in healthy brain tissue is removed by glial cells to prevent toxic levels. Dextran sulfate induced an increase in glutamate uptake by glial cells, whereas glutamate production in neurons was not altered by dextran sulfate. Thus, glutamate required for learning and memory is not affected by dextran sulfate administration, whereas harmful toxic amounts of glutamate are removed by glial cells. Thus, dextran sulfate attenuates neurotransmitter dysregulation that leads to glutamate excitotoxicity.

[0118] Oxidative stress in neurodegeneration leads to mitochondrial dysfunction, which limits 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 the energy crisis of neurons in the presence of such oxidative stress.Therefore, dextran sulfate weakens the oxidative stress in neurodegeneration that would otherwise have led to mitochondrial dysfunction.

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

[0120] Neurodegeneration also induces the release of inflammatory mediators that cause secondary cell damage and scar formation. Such 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 inhibits TGF-β activity. In addition, 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, that result from the release of inflammatory mediators. Dextran sulfate also acts to inhibit fibrosis as well as activate fibrolysis, which together result in the beneficial effects observed with dextran sulfate in reducing scarring or decomposing scarring.

[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] Thus, an aspect of the embodiment relates 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 pharma- ceutically 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 pharma- ceutically acceptable derivative thereof in vitro. Thus, the cells are treated with and interact with dextran sulfate or a pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically acceptable derivative thereof may be added to the medium of such neuronal or glial cell cultures, e.g., as described herein, to induce differentiation of the cells.

[0126] The method may also be an ex vivo method, where neurons and / or glial cells are extracted from a subject and contacted with dextran sulfate or a pharma- ceutically 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 pharma- ceutically acceptable derivative can be transplanted into a subject to induce differentiation.The differentiated neuron and / or glial cell should then perform their intended function in the subject's body.In this method, the subject can suffer from a neurological disease, as described herein further.

[0128] In an alternative embodiment, dextran sulfate or a pharma- ceutically 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 pharma- ceutically acceptable derivative thereof is then contacted with neurons and / or glial cells in 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 pharma- ceutically 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 pharma- ceutically 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, dextran sulfate or a pharma- ceutically 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 certain embodiments, 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 pharma- ceutically acceptable derivative thereof may also or alternatively 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, twitch-fasciculations syndrome, cerebral palsy, elevated creatine kinase, fasciculation, inclusion body myositis, Lambert-Eaton syndrome, mitochondrial myopathies, motor neuron diseases, myopathy, muscular dystrophies, 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 pharma- ceutically acceptable derivative thereof, for use in treating, inhibiting or preventing glutamate excitotoxicity in a subject.

[0136] In certain embodiments, dextran sulfate, or a pharma- ceutically 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 of treating, inhibiting or preventing glutamate excitotoxicity, the method comprising administering to a subject dextran sulfate or a pharma- ceutically acceptable derivative thereof to treat, inhibit or prevent glutamate excitotoxicity.

[0139] Other aspects of the embodiments relate to dextran sulfate or a pharma- ceutically 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 of neurons in a subject suffering from a neurological disease, disorder or condition.

[0140] Dextran sulfate, or a pharma- ceutically 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 pharma- ceutically acceptable derivative thereof may 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 pharma- ceutically 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 scarring, in a subject suffering from fibrosis or a fibrotic disease, disorder or condition.

[0143] Thus, dextran sulfate of the embodiment having an anti-scarring effect may be effective in wound treatment and tissue remodeling, where already established scars need to be broken down to allow proper wound healing. This anti-scarring effect of dextran sulfate of the embodiment is believed to be the result of the previously described mechanism of action of dextran sulfate, including, for example, inhibition of cell adhesion, induction of cell recruitment, induction of metalloproteases and scar-degrading enzymes, and inhibition of TGFβ, particularly TGFβ1, via induction of decorin. This latter effect obtained with dextran sulfate of the embodiment 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 pharma- ceutically 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 pharma- ceutically acceptable derivative thereof for the manufacture of a medicament for the treatment, suppression or prevention of any of the diseases, disorders or conditions disclosed herein, for various medical applications as disclosed herein.

[0146] Further aspects relate to methods for treating, inhibiting or preventing the various diseases, disorders or conditions mentioned above for various uses of dextran sulfate or a pharma- ceutically acceptable derivative thereof, in which dextran sulfate or a pharma- ceutically 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 applies to any pharma- ceutically acceptable derivative of dextran sulfate. Thus, a pharma- ceutically 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 efficacy and / or negative side effects on cells or subjects.

[0149] For example, dextran sulfate with a molecular weight of more than 10,000 Da (10 kDa) usually has a lower effect-to-side effect profile than dextran sulfate with a lower molecular weight.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, specifically 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 sulfated polysaccharides. 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 pharma- ceutical acceptable derivative thereof has an M of 10,000 Da or less. w In certain embodiments, dextran sulfate or a pharma- ceutically acceptable derivative thereof has an M in the range of 2,000 Da to 10,000 Da. w has.

[0154] In another embodiment, the dextran sulfate or a pharma- ceutically 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, dextran sulfate or a pharma- ceutically 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 has.

[0155] In another particular embodiment, the dextran sulfate or a pharma- ceutically 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 has.

[0156] Thus, in some embodiments, dextran sulfate or a pharma- ceutically 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 has.

[0157] In some embodiments, the dextran sulfate or a pharma- ceutically 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 These may be combined with an upper limit value of 10,000 Da or less.

[0158] In certain embodiments, the M of dextran sulfate or a pharma- ceutical 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 pharma- ceutical acceptable derivative thereof has an M as measured by NMR spectroscopy in the range of 1,850 to 3,500 Da. n has.

[0161] In certain embodiments, dextran sulfate or a pharma- ceutically acceptable derivative thereof has an M 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 has.

[0162] Thus, in some embodiments, dextran sulfate or a pharma- ceutically 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 pharma- ceutically acceptable derivative thereof has an M of 1,850 Da or more. n has.

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

[0164] In a particular embodiment, dextran sulfate or a pharma- ceutically 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 a pharma- ceutically acceptable derivative thereof has an average number of glucose units in the range of 4.0 to 6.0.

[0166] In a particular embodiment, dextran sulfate or a pharma- ceutically 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 pharma- ceutical 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 units of dextran sulfate is at least 90%.

[0168] In one embodiment, dextran sulfate has an average glucose unit number of about 5.1, an average number of sulfates 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 has.

[0169] In one embodiment, the pharma- ceutically acceptable derivative of dextran sulfate is the sodium salt of dextran sulfate. In a particular 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 ratio in the range of 2,100 Da to 2,300 Da. + M with counter ion n has.

[0170] In one embodiment, dextran sulfate has an average glucose unit count of 5.1, an average number of sulfates 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 at about 2,200-2,250 Da. + Content average M n has.

[0171] Dextran sulfate according to embodiments can be provided as a pharma- ceutically acceptable derivative of dextran sulfate, such as a pharma- ceutically active derivative of dextran sulfate, including pharma- ceutically acceptable salts and pharma- ceutically acceptable solvates of dextran sulfate, such as 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 pharma- ceutically acceptable derivatives can also be used for veterinary applications.Non-limiting examples of animal subjects include primates, cats, dogs, pigs, horses, mice, and rats.

[0173] Dextran sulfate or a pharma- ceutically acceptable derivative thereof is preferably administered to a subject by injection, in particular 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 pharma- ceutically 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 derivative can be administered intrathecally.For example, dextran sulfate or its pharmaceutically acceptable derivative 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 pharma- ceutically acceptable derivative of the embodiment is preferably formulated as an aqueous injection solution using a selected solvent or excipient. The solvent is advantageously an aqueous solvent, particularly a buffer. Non-limiting examples of such buffers are 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 about pH 5.9 with sodium hydroxide. Non-buffered solutions are also possible, including aqueous injection solutions such as saline, i.e., NaCl (aqueous). Furthermore, if a buffer is desired, buffer systems other than CAM can also be used.

[0176] Embodiments are not limited to injection, and other routes of administration may 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 a pharma- ceutically acceptable derivative thereof may vary depending on the application, e.g., in vitro versus in vivo, the size and weight of the subject, the condition the subject is being treated for, 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 pharma- ceutically acceptable derivative thereof is formulated to be administered at a dosage in the range of 0.05-50 mg / kg body weight of subject, preferably 0.05 or 0.1-40 mg / kg body weight of subject, and more preferably 0.05 or 0.1-30 mg / kg body weight of subject, or 0.1-25 mg / kg body weight of subject, or 0.1-15 mg / kg body weight of subject, or 0.1-10 mg / kg body weight of subject.

[0179] Administration of dextran sulfate or its pharmaceutically acceptable derivatives is not limited to treatment or suppression of existing diseases, disorders or conditions, but can be used instead or in addition for prevention.In other words, dextran sulfate or its pharmaceutically acceptable derivatives can be administered to subjects who are about to undergo a medical procedure, such as surgery, that may cause neurological injury or damage and / or fibrosis.Dextran sulfate or its pharmaceutically acceptable derivatives can also be used to prevent, suppress or reduce postoperative neurological complications and conditions in subjects who are about to undergo a medical procedure, such as surgery and / or fibrosis.

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

[0181] Alternatively, dextran sulfate or a pharma- ceutically acceptable derivative thereof may be administered multiple times, ie, at least twice, during the treatment period.

[0182] Dextran sulfate or its pharmaceutically acceptable derivatives can be administered together with other active agents, either sequentially, simultaneously, or in the form of a composition comprising 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 of cell therapy, such as stem cells, including, but not limited to, embryonic stem cells (ESCs) and mesenchymal stromal cells (MSCs).

[0183] For example, the effects of stem cells are being investigated 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 regarding 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 shown by experimental data. For example, dextran sulfate or its pharmaceutically acceptable derivatives protect cells from oxidative stress, restore metabolic homeostasis of cells, which is beneficial for the energy metabolism of cells, 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 derivatives in vitro before administration to a subject. Alternatively, or in addition, cells can be administered with dextran sulfate or its pharmaceutically acceptable derivatives. Also, in vitro or ex vivo treatment of tissues and organs with dextran sulfate or a pharma- ceutically acceptable derivative thereof may be useful to benefit from the positive effects of the dextran sulfate of the embodiments, such as protection against oxidative stress and restoration of metabolic homeostasis.Furthermore, treatment of cells, tissues and organs may additionally or alternatively be possible after transplantation of dextran sulfate or a pharma- ceutically acceptable derivative thereof.

[0185] In some embodiments, dextran sulfate or its pharma- ceutically acceptable derivatives are advantageously administered to subjects in the early or acute state after injury that causes a disease, disorder or condition, such as TBI, or in the early or acute state after diagnosis of a disease, disorder or condition.This is particularly advantageous, since some beneficial effects observed with dextran sulfate of the embodiments are its ability to enhance and amplify endogenous repair mechanisms in the CNS and PNS.This is particularly important for treating or suppressing neurological diseases.However, the anti-scarring effects observed with dextran sulfate of the embodiments indicate that dextran sulfate may also be effective in breaking down existing scar tissue and elements.Therefore, dextran sulfate of the embodiments may also have a therapeutic effect during late or chronic states against fibrosis and fibrotic conditions.

[0186] Working 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 dedicated medium appropriate for the cell type, and plasticware was treated with specific adhesion factors to enhance cell attachment. [Table 1]

[0189] Neurons were plated 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 solution of 50 μg / ml 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 min. Plates were coated by adding 75 μl per well of a solution of laminin (Sigma) at 15 μg / ml in medium and incubated at 37° C. in the dark for 1 h. 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 made by adding PNGM Singlequots (Lonza) to PNBM medium and pre-warmed to 37°C. Cells were thawed in a 37°C water bath for no more than 2 min and gently transferred to a 15 ml tube. 5 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 into pre-coated 96-well plates. Cells were incubated at 37°C under 5% CO2. After 2 h of incubation, 80 μl of medium was removed and replaced with 80 μl of fresh medium, and cells were allowed to settle for 24 h 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. Pellet was resuspended in 5 ml of medium and cells were counted on 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, NeuroBlast medium was replaced with MotorBlast (Lonzo).

[0193] Schwann cells Schwann cell growth medium was made 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 10ml medium-containing tube and centrifuged at 200 RCF for 5 minutes. The pellet was resuspended in 5ml medium and cells were counted on 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 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 in eight identical wells / dose / time point after 24 and 48 hours. 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 therefore imaged in situ and classified into different phases of the cell cycle based on DNA content (PI) or considered as apoptotic death or polyploid. The protein content of cells 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 replicate determinations. Comparisons between groups were performed using Student's t-test (two-tailed, equal variance; Exel 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 the cells' PI uptake was altered and the histogram shifted to the right, indicating that LMW-DS treatment had an effect on cortical neurons. The cell population that had started to divide (G2 / M phase) is shown in the figure.

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

[0200] Human motor neurons The data for motor neurons were similar to cortical neurons, with a shift in PI uptake (FIG. 2) and increased proliferation within a very small population of cells.

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

[0202] Schwann cells Schwann cells appear to be less affected by LMW-DS than neurons, and there was a similar PI shift (Figure 3).

[0203] No effect on cell number and cell detachment was 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 only by neurons. The intensity of tubulin was significantly increased in cells treated with LMW-DS (FIG. 5A). Analysis of positive cells showed that these cells were larger than those in control cultures (FIG. 5B).

[0206] Tubulin expression in human motor neurons The expression of βIII-tubulin was significantly increased by LMW-DS (Fig. 6A). Cell morphology was dramatically altered by LMW-DS. Most positive cells were smaller than in control cultures (Fig. 6B), but some cells became extremely large 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 to 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 a clear 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, the clear cell loss was much greater in immunostained preparations (more washes) than in PI preparations, even though the maximum measure of the rate of apoptotic death (after adjustment for the PI shift) did not account for the cell loss in the cultures.

[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 visible.

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

[0214] It has become 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 supporting Schwann cell differentiation would also be good candidates for the treatment of diseases associated with demyelination.

[0215] Example 2 The present study was conducted to investigate the in vivo efficacy 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 mouse EAE model is currently the most widely accepted animal model of human MS and ADEM (Annals of Neurology, 60:12-21, 2006). In general, EAE is characterized by the administration of myelin oligodendrocyte glycoprotein emulsified with an adjuvant to mice, which induces an immune response against myelin. 35-55 (MOG 35-55 EAE is induced with a single injection of peptides and proteins, including EGFR-1, EGFR-2, EGFR-3, EGFR-4, EGFR-5, EGFR-6, EGFR-7, EGFR-8, EGFR-9, EGFR-10, EGFR-11, EGFR-12, EGFR-13, EGFR-14, EGFR-15, EGFR-16, EGFR-17, EGFR-18, EGFR-19, EGFR-20, EGFR-21, EGFR-22, EGFR-23, EGFR-24, EGFR-25, EGFR-26, EGFR-27, EGFR-28, EGFR-29, EGFR-30, EGFR-31, EGFR-32, EGFR-33, EGFR-34, EGFR-35, EGFR-40, EGFR-5, EGFR-41, EGFR-42, EGFR-43, EGFR-44, EGFR-45, EGFR-46, EGFR-47, EGFR-48, EGFR-49, EGFR-50, EGFR-49, EGFR-51, EGFR-44, EGFR-45, EGFR-45, EGFR-46, EGFR-47, EGFR-48, EGFR-49, EGFR-52, EGFR-49, EGFR-49, EGFR-53, EGFR-44, EGFR-45, EGFR-46, EGFR-47, EGFR-48, EGFR-49, EGFR-49, EGFR-49, EGFR-51, EGFR-49, EGFR-49, EGFR-49,

[0217] Materials and Methods Incomplete Freund's adjuvant (IFA) (Difco) ·Mycobacterium tuberculosis H37RA (Difco) ·MOG 35-55 Rodents (MDBioproducts) ·Pertussis toxin (Sigma Aldrich) Hanks' 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) Cyclosporine 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 of age) were obtained from Harlan Europe. Mice were housed in a conventional animal facility (Lund University, Sweden) and kept in polystyrene cages containing wood shavings (type II cages, maximum of 7 mice per cage) under a 12-h light / dark cycle and fed 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 complete Freund's adjuvant (CFA) (H37RA in IFA at a concentration of 6 mg / ml) and MOG. 35-55 (dissolved in PBS at a concentration of 3 mg / ml) on ice to prepare an emulsion. Mice were anesthetized during immunization to ensure the exact location of the injection. Pertussis toxin (PTX) was resuspended in mqH2O at a concentration of 50 μg / ml and diluted in PBS to a final concentration of 1 μg / ml. Mice received intraperitoneal booster injections of 200 ng 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 and filter sterilized through a 0.2 μm filter according to the doses listed in Table 2 below. The dosing vehicle was 0.9% saline. Recombinant HGF was reconstituted in 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 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. In the remaining groups, treatment began on day 18. 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 to 8. 0=healthy 1=tail weakness 2=tail motor paralysis 3 = Tail paresis and mild staggering 4 = Tail paresis and severe stagger 5 = caudal paresis and one limb paresis 6 = Tail paresis and paresis of one pair of limbs 7 = Quadriplegia or paralysis of three limbs 8 = Pre-morbidity or death

[0224] Graphs and Statistics Graphical and statistical analyses were performed using Prism5 for Mac OS X (GraphPad Software, San Diego, CA, USA). All statistical data were calculated using 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 EAE onset 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 control. 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 control.

[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 a significantly lower mean score in the EAE model compared to vehicle controls. Thus, the results indicate that LMW-DS has a favorable effect 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 evaluated by high performance liquid chromatography (HPLC) analysis of frozen brain samples. The results suggest that LMW-DS interferes with the function of improving mitochondrial energy metabolism and also reduces glutamate excitotoxicity.

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

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

[0231] The animals received an anesthetic mixture of 35 mg / kg body weight ketamine and 0.25 mg / kg body weight midazolam by 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 the rat, which was 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, the rats were re-anesthetized and then immediately sacrificed.

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

[0233] Brain tissue processing An in vivo osteotomy craniotomy was performed in all animals while under anesthesia, the rat skull was carefully removed, and the brain was exposed, removed with a surgical spatula, and quickly placed in liquid nitrogen. After wet weight (ww) measurements, 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 (CH3CN + 10 mM KH2PO4, 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 (20,690xg, 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 the 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), the volume was adjusted with 10 mM KH2PO4, pH 7.40 to obtain a final aqueous 10% tissue homogenate and 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, 250x4.6 mm, 5 μm particle size column (Thermo Fisher Scientific, Rodano, Milan, Italy) with its own guard column and coupled to an HPLC instrument consisting of a Surveyor System (Thermo Fisher Scientific, Rodano, Milan, Italy) 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 for oxidative-nitrosative stress were separated in a single chromatographic run according to an existing ion-pair HPLC method with slight modifications (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 the chromatographic runs of tissue extracts was performed by comparing the retention times, absorption spectra, and areas of the peaks at the appropriate wavelengths (206, 234, and 260 nm) with those of the peaks in the chromatographic runs of freshly prepared ultrapure standard mixtures with 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 determination of primary free amino acids (FAA) and amino group-containing compounds (AGCC) (described below) was performed using pre-column 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, 237.5 mmol / l sodium borate, pH 9.8 was prepared daily and introduced 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 of 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% H2O) 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-Smirnoff test. Differences across 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 clear finding in the brain levels of 24 canonical and non-canonical amino acids and primary amino group-containing compounds was that LMW-DS treatment demonstrated a significant suppression of 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 efficacy against this excitotoxicity marker when LMW-DS was injected 3 days after sTBI. It is also worth emphasizing that LMW-DS had a marked beneficial effect on compounds involved in the so-called methyl cycle (Met, L-Cystat, SAH, see Table 3). [Table 3] TIFF2025077046000007.tif76162a p<0.01 (compared to control), b p<0.05 (compared to 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 phase) Table 3 lists the compounds in μmol / g (ww).

[0243] As can be seen in Table 4, LMW-DS positively influenced various compounds related to energy metabolism and mitochondrial function. Of particular interest are the adenine nucleotide concentrations and the ATP / ADP ratio as measures of mitochondrial phosphorylation capacity (Figure 13). [Table 4] TIFF2025077046000009.tif230162TIFF2025077046000010.tif225162 a p<0.01 (compared to control), b p<0.05 (compared to 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 phase) 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, as the major water-soluble brain antioxidant, and GSH, as the major intracellular SH donor, were measured. The results showed a significant improvement in their levels after administration of LMW-DS, as shown in Table 4 and Figure 15.

[0246] In addition, MDA, used as a polyunsaturated fatty acid end product of membrane phospholipids and thus as a marker of ROS-mediated lipid peroxidation, was also measured. MDA levels showed a significant reduction after administration of LMW-DS. After treatment with LMW-DS, all of the above oxidative stress markers showed an improved 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 phases of sTBI (Figure 16).

[0248] NAA is a brain-specific metabolite and a useful biochemical marker to monitor 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 move between intracellular compartments to reach oligodendrocytes, where it is degraded to acetate and aspartate by aspartoacylase (ASPA). Upregulation of the catabolic enzymes ASPA and NAA is reduced to provide availability of the substrates aspartate and acetyl-CoA, indicators of a state of metabolic impairment. 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 especially 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 brain 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 and socio-economic costs of TBI are estimated to be in excess of $60 billion per year, excluding military expenditure. In addition, 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. Although the primary insult associated with TBI (contusion) may be amenable to surgical treatment, reduction of the subsequent secondary non-mechanical damage to the surrounding brain tissue (penumbra) may offer a greater opportunity for treatment.

[0252] Using a well-established rodent model of severe traumatic brain injury (sTBI), characterized by the diffuse axonal injury of TBI, we have demonstrated that severely injured animals show 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 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) as well as changes 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 that cause TBI-induced brain injury, metabolic changes are early cellular signals that affect enzyme activities 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 tissues 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 the deleterious changes in metabolic homeostasis by reducing the 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 give a final concentration of 0.01 mg / ml. After 48 hours, cells were harvested from the treated flasks. Thus, the collected data represents (a) untreated cells (day 0 control and day 2 control) and (b) cells treated with LMW-DS for 48 hours (LMW-DS treatment for 2 days).

[0256] Coating of tissue culture dishes for all cells 25cm 2 Flasks were coated by adding 2 ml per flask of a solution of 50 μg / ml poly-d-lysine in Hank's Balanced Salt Solution (HBSS) and incubating overnight at 37° C. in the dark. Flasks were washed with cell culture water and air-dried in the dark for 30 min. Flasks were coated by adding 1 ml per flask of a solution of 25 μg / ml laminin in phosphate buffered saline (PBS) and incubating for 2 h at 37° C. in the dark. Prior to seeding of cells, laminin flasks were washed 3 times with PBS.

[0257] Human umbilical vein endothelial cells (HUVEC) 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. Cells were centrifuged at 400xg for 10 minutes. The supernatant was removed and cells were resuspended in 10 ml of medium (M200 + LVES supplement).

[0258] Cells were counted using a Cellometer. 1,000,000 cells / flask was placed in a 25 cm 2 flasks (n=8) and medium was added to a total of 5 ml per flask. Cells were incubated at 37°C under 5% CO2. Cells were allowed to settle for 24 hours prior to 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 into 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 GlutaMAX™-I supplement to 500 ml of Neurobasal medium. The medium was pre-warmed 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 min. The supernatant was removed (to the last 0.5 ml) and the cells were gently resuspended by trituration. The cells from 12 vials were mixed and applied to 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 media was prepared according to Table 5. [Table 5]

[0264] Media (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 media 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 200xg 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 25cm 2 The cells were evenly distributed among flasks (n=8). The 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. Concentrated drug stocks were filter sterilized and added to the respective media (19.6 ml CM and 0.4 ml LMW-DS stock solution). Controls were made using 19.6 ml CM and 0.4 ml DMEM-F12. LMW-DS and CM were added to the 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 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 3000xg 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 one by one from the freezer and Trizol-water was transferred from the appropriate tube into the flask. The flasks were left at room temperature for 5 minutes before the contents were aspirated back into the 15 ml Falcon tubes (after thoroughly washing the bottom of the flask with the solution). The flasks were inspected 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 tubes were removed from the freezer and stored at room temperature for 5 min to allow complete disintegration of nucleoprotein complexes.

[0269] Two 1 ml aliquots of lysate were removed from each sample, 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. Samples were stored at room temperature for 2-3 min and then centrifuged at 12,000 x 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 proteins in the pink bottom (organic) phase. 3 / 4 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 in 50 μl of warm RNase-free water. The quantity and quality of purified RNA was determined by NanoDrop. RNA was stored at -80°C before being shipped to Source Bioscience for array analysis.

[0272] Analysis plan for expression data Expression data was downloaded into separate files for each cell line. "Background corrected" expression is data from the "gProcessedSignal" of the array that is the result of background signal subtracted from the actual signal of the relevant probe. This is the most commonly used variable in array analysis. Background corrected signals were log2 transformed for statistical analysis for all samples. Signals below the "expression level" were removed to reduce false positive rates in samples. 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 to D2 control samples - Expression changes observed in cells in normal cultures Comparison of D0 control samples with D2 LMW-DS treatment - 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 data sets. A simple, non-stringent analysis of variance (p<0.05) was performed to look for expression patterns. Probes with no change across the three data sets were removed. Volcano plots were used to analyze the remaining probe sets for fold change and significance. To allow detection of expression patterns, in the first case, a change in expression of more than 20% of the probes (FC≧1.2 or FC≦0.84) was considered significant.

[0275] Quality parameters The seeding density was calculated from the cell number taken from the cell stock of Schwann cells. 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 for the low input RNA microarray from Agilent.

[0277] Analysis of the raw data showed that, as expected, significant differences existed between arrays. However, these differences (reflecting differences among the same control samples included on all arrays) were easily removed with normalization techniques. Median-centering of 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 that were not expressed in Schwann cells were removed prior to data analysis. The "less than expressed" level was set to 5 log2 transformed expression values. This left 15,842 unique analyzed probes in Schwann cell cultures. In the next step of the analysis, three sets of data (D0 control samples compared to D2 control samples; D0 control samples compared to D2 LMW-DS treated samples; D2 control samples compared to D2 LMW-DS) were analyzed to determine the effect of CM on the cells and the relative changes induced by LMW-DS.

[0279] 585 genes were differentially expressed in Schwann cell cultures when comparing D0 control samples to D2 control samples. 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); 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 interactions (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 ​​that represent the statistical significance associated with these genes having different pathways. The two p-values ​​represent the lower and upper limits 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 to 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); cell function and maintenance (9.53E-06 to 9.53E-06); and cell motility (1.43E-08 to 8.39E-04). -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 interactions (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 comparison of D2 control samples to D2 LMW-DS treated 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); Post-translational modification (2.56E-05~1.05E-03);Small molecule biochemistry (2.56E-05~3.7E-03);Cell signaling and interaction (5.05E-05~5.76E-03);Gene expression (7.18E-05~4.94E-03);Cell cycle (1.06E-04~5.95E-03);Cell development (1.06E-04~5.95E-03) );Cell function and maintenance (1.96E-04 to 5.95E-03);Cell 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);Cell damage (6.26E-04 to 6.26E-04);Lipid metabolism (6.26E -04~1.85E-03); molecular transport (6.26E-04~5.95E-03); protein synthesis (1.05E-03~1.93E-03); cellular response to therapeutic drugs (1.85E-03~1.85E-03); protein transport (2.66E-03~5.95E-03); and RNA post-transcriptional modification (4.32E-03~4.32E-03).

[0283] Mechanistic molecular network models allow for the simulation of the effects of differentially regulated molecules by LMW-DS and the assessment 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 2-day control cultures did not show any change at all in the LMW-DS-treated cultures for the same 2 days. One gene whose expression was 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 made this upregulation significantly stronger.

[0286] Expression analysis of HUVECs As previously described, genes not expressed in HUVEC were removed before any analysis was attempted. The "under expressed" level was set to 5 log2 transformed expression values. This left 15,239 unique probes analyzed in HUVEC cultures. The next step of the analysis was to analyze the three sets of data to determine the effect of CM on gene expression in 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 data sets. 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 data sets were removed, leaving a total of 12,313 probes (10,368 genes) for analysis.

[0287] Comparing the D0 to 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 modifications (4.86E-06 ~6.53E-04); cell movement (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 agents (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 to D2 LMW-DS treated samples. The molecular functions affected by these genes were 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); RNA ... 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 to 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); molecular transport (9.59E-04 to 2.57E-02); and cell cycle (1.22E-04 to 2.4E-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 Interactions (2.7E-03-2.4E-02);Cell Assembly and Organization organization (2.7E-03~2.57E-02); cell growth and proliferation (2.7E-03~2.4E-02); cell motility (2.7E-03~2.4E-02); energy production (2.7E-03~2.7E-03); nucleic acid metabolism (2.7E-03~1.07E-02); post-translational modification (2.7E-03~1.61E-02); gene expression (5.39E-03~2.36E-02); RNA post-transcriptional modification (5.39E-03~ 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 agents (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, it is possible to 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, whereas in treatment cultures LMW-DS was added to medium already containing growth factors.

[0293] Table 7 summarizes the results of gene expression changes in cultured HUVECs. 67 genes whose expression was altered (due to the effect of growth factors) in the control cultures for 2 days did not show any change at all in the LMW-DS-treated cultures for the same 2 days. 4 genes whose expression was increased in the control cultures containing growth factors were downregulated in the LMW-DS-treated cultures for the same 2 days. 11 genes that were downregulated by growth factors in the control cultures were upregulated in the LMW-DS-treated cultures for the same 2 days. 120 genes were significantly downregulated by growth factors, and this downregulation was stronger in the LMW-DS-treated cultures. 229 genes were upregulated in the control cultures, and the addition of LMW-DS made this upregulation significantly stronger. [Table 7]

[0294] We analyzed the effect of LMW-DS on several molecular pathways important for different disease states and therapeutic applications. To do this, 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 described previously, genes not expressed in motor neurons were removed before any analysis was attempted. The "under expressed" level was set to 5 log2-transformed expression values. This left 12,240 unique probes that met the expression threshold in at least three samples in the series. In a 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 what these cells need to differentiate. The stress factor present in these cultures is oxidative stress (normal in tissue culture conditions).

[0297] Comparing the D0 to 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 signaling and interactions (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); lipid metabolism (1.08E -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 Signaling (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 modifications (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 to 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); cell development (1.0E-06 to 1.0E-03); and cell motility (1.0E-06 to 1.0E-03). 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 modifications (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 activity (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); amino acid metabolism (7.75E-05 to 5.16E-03); ~4.68E-03);Small molecule biochemistry (7.75E-05~4.68E-03);Cell damage (8.23E-05~4.61E-03);Cell signaling and interactions (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 described previously, genes not expressed in motor neurons were removed before any analysis was attempted. The "under expressed" level was set to 5 log2-transformed expression values. This left 10,653 unique probes that met the expression threshold in at least three samples in the series. In a 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] The changes in gene expression under normal culture conditions mimic the normal developmental process of cortical neurons when they develop a cortical neuron phenotype from a set of dissociated cells. The growth factors in the normal medium are what these cells need to differentiate. The stress factor present in these cultures is oxidative stress (normal in tissue culture conditions).

[0302] Comparing D0 to D2 control samples, 1101 genes were differentially expressed in motor neuron cultures. The molecular functions affected by these genes were 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 (4.28E-22 to 6.6E-04). Nucleotide transmission 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 .65E-07~5.18E-04;Cell damage (7.12E-06~4.54E-04);Proteolysis (1.62E-05~1.62E-05);Amino acid metabolism (2.11E-05~4.25E-04);Protein transport (3.4E-05~3.4E-05);Intracellular signal transduction (8.69E-05~3E-04);Post-translational modification (8.69E-05~2.15E-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 interactions (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); cell damage (9.23E-06 to 7. .65E-04;Amino acid metabolism (1.75E-05~1.64E-03);Cell cycle (1.75E-05~1.77E-03);Small molecule biochemistry (1.75E-05~1.79E-03);Protein synthesis (2.77E-05~1.5E-03);Protein transport (2.77E-05~1.9E-04);Intracellular signal transduction (7.65E-05~1.73E-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 are: 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 interactions (1.01E-04 to 1.01E-02); 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), marked as A in Figure 18, was inhibited, whereas complex IV (cytochrome c oxidase), marked as B in Figure 18, was activated. When LMW-DS was added to the cultures, complex III (cytochrome bc1), marked as 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 mitochondria of the inner membrane of all animals and all aerobic eukaryotes and most eubacteria. Mutations in complex III result in exercise intolerance as well as multisystem diseases. The bc1 complex contains 11 subunits, three respiratory subunits (cytochrome B, cytochrome C1, 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] In normal culture conditions, motor neurons appear to undergo significant oxidative stress, which leads to the activation of some apoptotic mechanisms, marked as F in FIG. 18, with activation of cytochrome C, AIF, caspases 3, 8 and 9. Furthermore, motor neurons are characterized by the production of amyloid beta, marked as E in FIG. 18, and the oxidation of fatty acids, marked as G in FIG. 18, which further exacerbates oxidative stress and FIAS1-mediated mitochondrial fragmentation in cells. Furthermore, complex V, marked as D in FIG. 18, is activated.

[0310] Addition of LMW-DS to the cultures ameliorates these negative effects by blocking the reaction pathway marked as F in FIG. 18 and preventing its negative effects on Aβ production and mitochondrial fragmentation and dysfunction and subsequent damage marked as E in FIG. 18, and by inhibiting fatty acid oxidation marked as G in FIG. 18, preventing and inhibiting apoptosis. LMW-DS also inhibits the reaction pathway involving TRAK1 and PINK1 marked as H in FIG. 18, thereby contributing to improved mitochondrial function. LMW-DS further reduced the levels of H2O2 marked as I in FIG. 18. An additional effect was the inhibition of HtrA2 marked as J in FIG. 18, which contributes to the inhibition of apoptosis.

[0311] Under normal culture conditions, cortical neurons are exposed to considerable oxidative stress that leads to Aβ production and Lewy body formation accompanied by activation of synuclein α and increased levels of ROS, marked as K in FIG. 18; apoptosis, marked as F in FIG. 18; mitochondrial fragmentation, marked as E in FIG. 18; and impaired mitochondrial function, marked as L in FIG. 18. Addition of LMW-DS to the cultures could prevent and reverse most of these deleterious effects, such as accumulation of Aβ and Lewy body pathology (marked as E, K in FIG. 18), mitochondrial dysfunction (marked as L in FIG. 18). Some apoptosis inducing mechanisms (marked as F in FIG. 18) remain active, likely due to their strong activation in the cultures.

[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 (also in 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 inhibited by baseline conditions in motor neuron cultures. Inhibition 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 protein complexes (CALM, Gβγ, GRM7, PICK1) marked as A in FIG. 19. More importantly, LMW-DS enhanced the activity and / or levels of glutamate transporters, especially SLC1A2 / 3, in Schwann cells, thereby resulting in the elimination 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 from manifesting.

[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 called glutamate-aspartate transporter (GLAST) or excitatory amino acid transporter 1 (EAAT1). SLC1A3 is primarily expressed in the plasma membrane, where it allows the removal of glutamate 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 the transport of glutamate 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 Muller glial cells. SLC1A3 is also expressed in many other tissues, including cardiac myocytes.

[0316] SLC1A2, member 2 of solute transporter family 1, 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 family member of the solute transporter family of proteins. This membrane-bound protein is the primary transporter that removes 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 deleterious, 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 concomitantly cell motility activated, whereas in HUVECs (1 molecule, ADAM11), adhesion was not affected but angiogenesis could be activated. [Table 10]

[0321] The effect of LMW-DS-induced differential gene expression in neurons was 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 alpha4beta1, 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, a finding that 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 HUVEC, 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 influenced the effects of several growth factors, either enhancing their activation or reducing their inhibition, present in the system. [Table 14]

[0330] In normal cultured cortical neurons, most pathway-dependent growth factors were significantly activated by normal medium. In most cases, this activation was not altered by LMW-DS. However, LMW-DS activated molecules that are downstream effectors of GDF7, and the effects of this growth factor were shown to be enhanced by LMW-DS. GDF7 is a potent differentiation factor for neurons, and the additional activation of these growth factors over the activation of BDNF and NT3 provides a good explanation for the enhanced differentiation of these cells in culture.

[0331] Consideration Normal culture conditions for HUVEC mimic the environment after 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 in this regard is that of angiogenesis after an ischemic condition such as stroke.

[0332] In Schwann cells, control cultures with high nutrient content and glucose recapitulate Schwann cell activation. LMW-DS-treated cultures mimicked the effect of LMW-DS added 24 hours after glial activation. The real-life scenario this recapitulates is glial activation after nervous system injury, such as after TBI.

[0333] Normal culture conditions, including high nutrient content and growth factors for both neurons, motor neurons and cortical neurons, mimic the environment during normal neuronal differentiation. The only negative effect in these cultures is the oxidative stress to which the cells are subjected. The real-life scenario to which this relates is a degenerative condition promoted by oxidative stress in the presence of sufficient growth and differentiation factors. This corresponds to early stages of neurodegenerative diseases or conditions in which oxidative stress plays a central role.

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

[0335] Thus, results from the HUVEC cell model indicate that LMW-DS can protect against cell damage and promote the development of new blood vessels in damaged or diseased tissues following a stroke, and results from Schwann cells indicate that LMW-DS can protect against cell loss in a diseased or damaged 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 diseased and injured nervous systems, which is relevant, for example, in neurodegenerative diseases and TBI.

[0337] Of particular importance is that LMW-DS increased glutamate uptake in glial cells, as shown by Schwann cells. However, LMW-DS did not alter the production of glutamate by neurons. This is important because glutamate is necessary for LTP, i.e., learning and memory. Therefore, it is beneficial that LMW-DS did not alter the production of glutamate by neurons, since glutamate is necessary for normal neurotransmission in the above-mentioned processes. However, the increased levels of glutamate released from damaged or dying cells are efficiently taken up by surrounding glial cells due to the effect of LMW-DS. Thus, activation of glutamate transporters caused by LMW-DS in glial cells effectively removed glutamate released by damaged or dying neurons from the neural gap. This, in turn, prevented glutamate from exerting 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 movement, 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. In HUVECs, cell adhesion was not affected. 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 (scarring) signals in injured tissue.

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

[0343] The effects demonstrated by the gene expression data support 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 later phases when early repair attempts are already diminished.

[0348] Thus, 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, in any neurodegenerative condition, LMW-DS be administered at an early stage of the disease or condition, when the tissue has sufficient repair potential.

[0349] By protecting cell metabolism, LMW-DS can 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 can support these damaged tissues and restore some lost function while enhancing remaining endogenous repair mechanisms.

[0350] The anti-scarring effect of LMW-DS indicates its potential use in the treatment of fibroproliferative (scar-forming) conditions, including, for example, glaucoma, proliferative vitreoretinopathy, SAH, traumatic brain and spinal cord injuries, invasive surgery, post-operative adhesions, rotator cuff injuries, burns, reconstructive surgery, ulcerative conditions (diabetes), etc. Experimental results support a role for LMW-DS in both preventing the development of fibroproliferative (scar-forming) conditions and in degrading established fibrotic scars in such fibroproliferative (scar-forming) 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) leads to retinal ganglion cell death (30-40%). From the start of the experiment, 15 mg / kg LMW-DS was administered by daily subcutaneous injection to assess RGC protection compared to controls. 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 days 0 to 28; Immunohistochemistry to count 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 in groups 1 and 2 at day 28 by laminin and fibronectin; 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 8-10 week-old male Sprague-Dawley rats (Charles River, Kent, UK), weighing 175-200 g, housed under a 12-h light-dark cycle with food and water ad libitum, were used for these experiments. Surgeries were performed at the Biomedical Services Unit of the University of Birmingham in accordance with the Home Office guidelines laid down 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 with 2-5% isoflurane / 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 tunnel created with a homemade, disposable, sterile glass micropipette (Harvard Apparatus, Kent, UK) using a 15° disposable blade, allowing repeated twice-weekly (twice a week) IC injections (every Sunday and Thursday) of 3.5 μl of active human recombinant TGF-β1 (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 perfused transcardially 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. Eyes dissected for IHC were post-fixed by immersion in 4% PFA in PBS for 2 h 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 h each at 4°C, and then embedded in optimal cutting temperature embedding medium (Thermo Shandon, Runcorn, UK) in peeling mould containers (Agar Scientific, Essex, UK). Optimal cutting temperature: 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), left at 37°C for 2 h, and stored at -20°C.

[0357] Immunohistochemistry Frozen sections were left to thaw for 30 min, then washed 3x5 min in PBS, followed by permeabilization with 0.1% Triton X-100 (Sigma) for 20 min. Sections were blocked for 30 min 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), followed by washing 3x5 min in PBS and incubation with secondary antibodies (Table 11) for 1 h at room temperature (RT; 20-25°C). Sections were then washed 3x5 min in PBS and mounted with 4',6-diamidino-2-phenylindole (DAPI)-containing Vectorshield mounting medium (Vector Laboratories). All control tissue sections incubated with secondary antibodies alone were 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 by the same pre-defined quadrant size for all eyes / treatments within the TM, ECM deposition was quantified within this defined TM quadrant, and the % immunofluorescent pixels above a normalized background threshold were calculated using ImageJ software (National Institutes of Health, USA). For each antibody, a threshold level of luminance in the region of the TM was established using intact, untreated eye sections to define the reference level for study group analysis of pixel intensity. Images were assigned randomized numbers to ensure blinding of treatment groups during quantification by assessors.

[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 from 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 assigned randomized numbers to ensure blinding of 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 ANOVA for two-group comparisons ± SEM and are presented in the text or displayed 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 (FIG. 20) and fibronectin (FIG. 21) angles.

[0362] Consideration LMW-DS treatment induced degradation of existing TM scar elements, as laminin and fibronectin levels were significantly less in the angles of dextran sulfate-treated rats. Thus, this anti-scarring effect of LMW-DS indicates that the drug can be used to degrade existing scars, thereby allowing, 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 represents a major burden to the health care 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 adversely affect physiological neurotransmission and, in addition, form neurotoxic complexes. Part of the deleterious effects of oligomeric β-amyloid protein are due to the formation of cellular prion protein (PrP C), thus pharmacological strategies to inhibit this protein-protein interaction have potential as disease-modifying therapeutics.

[0365] This study aims to evaluate the efficacy and safety of oligomeric β-amyloid and PrP in LMW-DS to determine their disease-modifying potential as therapeutic agents to treat AD. C The ability of the antibody 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 average MW >500,000 Da from Sigma; dextran (MW 450,000-650,000 Da) from Sigma; Maxisorp plates from Sigma.

[0367] Preparation of amyloid β oligomers 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 HFIP was carefully evaporated. The resulting peptide film was stored at -20°C in a sealed container. Before use, the peptide film was slowly dissolved in DMSO to a final concentration of 5.0 mM and subjected to protective sonication for 10 min. 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 h (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 Optimized preparations for the generation of Aβ monomers or oligomers were solubilized in 5% SDS-containing gel sample buffer without reducing agents. Proteins were measured on 15% Bis-Tris gels using MES running buffer without reducing agents. Gels were transferred to PVDF, blocked in 10% nonfat milk, and then incubated with anti-Aβ antibody overnight at 4°C, 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 to 10x coating volume in carbonate coating buffer (100 μl; 500 ng PrP per well) CA final volume of 1000 µl of 1000 µl of 100% PBS was applied to a Maxisorp plate. The plate was then sealed and placed at 4°C overnight. The coated plate was carefully washed in PBS-Tween 20 and blocked with 2% BSA in PBS. The plate was washed and 100 µl of oligomeric amyloid beta-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 min at room temperature, washed, treated with streptavidin-HRP and developed with TMB after further washing (reaction stopped with 2N H2SO4). Absorbance was read at 450 nm within 30 min.

[0370] All conditions were performed in triplicate. C Amyloid β-biotin binding to 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 successfully oligomerized with higher apparent efficiency 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 still no signal was evident. The lack of signal, and the known theoretical maximum binding capacity of the Maxisorp plate (600-650 ng / cm) are reasons for this. 2 ) showed that the coating level was not optimal. C A range of coating levels was evaluated; 250 ng PrP per well C In the 500 ng PrP per well, a relatively small signal of oligomeric amyloid beta was discernible. C A larger, more reproducible signal was evident at a coating level of 1.0 μg / well. This coating amount is consistent with published literature (Beringe et al., Brain. 2003, 126:2065-2073: using 500 ng / well; Nakato et al., J Immunol. 2012, 189:1540-1544: using 250 ng / well; Souan et al., Eur J Immunol. 2001, 31:2338-2346: using 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 protein-protein interactions between PrP and DSSS in a concentration-dependent manner (Figure 23; Table 12). Quantitative pharmacological analysis showed that, despite clear differences in competitive binding and Hill coefficients at the head-to-head level, LMW-DS exhibited a similar overall affinity to DSSS, 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 Aβ and PrP C It was not possible to clearly compete for protein-protein interactions between [Table 16]

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

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

[0377] Thus, LMW-DS is a marker for oligomeric amyloid beta and PrP C This effect seen in LMW-DS is due to the fact that oligomeric amyloid beta and PrP compete for protein-protein interactions between these proteins, and can therefore be used to prevent, or at least inhibit, this protein-protein interaction. C These compounds have potential for treating 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 effect of LMW-DS against biochemical, molecular and histoanatomical damage produced by an experimental model of closed head diffuse severe TBI (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 Induction of sTBI 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] The animals received an anesthetic mixture of 35 mg / kg body weight ketamine and 0.25 mg / kg body weight midazolam by intramuscular injection. Diffuse sTBI was induced according to the "falling weight" impact acceleration model set out by Marmarou et al. J.Neurosurg.1994,80:291-300. This model produces diffuse axonal injury and is able to reproduce the physical and mechanical characteristics of human diffuse TBI.

[0381] Severe TBI was induced by the fall of a 450 g weight from a height of 2 m onto the head of the 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 bed of special polyurethane foam inserted into a special container, this foam dissipates most of the potential energy (originating from the mechanical force) and prevents any rebound of the animal after the impact that could cause 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 anesthetized again and then immediately sacrificed. These time points correspond to worst biochemical damage (2 days) or, in the case of minimally damaged brains, 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 carry out the study of 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, in which, as specified hereafter, animals were subjected to a specific treatment for metabolic analysis and other animals were for histomorphometric studies, according to the procedure 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 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. Twelve animals received sTBI and were sacrificed 7 days after TBI. Four additional rats for each subgroup were used for histomorphometric studies.Total of rats in this group: n=32. group 3 Rats were subjected to induction of sTBI, received a single dose of LMW-DS 30 min 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. Twelve animals received sTBI and were treated with 15 mg / kg body weight of LMW-DS. Four additional rats for each subgroup were used for histomorphometric studies.Total of rats in this group: n=48. group 4 Rats were subjected to induction of sTBI, received a single dose of LMW-DS 30 min after TBI, and 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. Twelve animals received sTBI and were treated with 15 mg / kg body weight of LMW-DS. Four additional rats for each subgroup were used for histomorphometric studies.Total of rats in this group: n=48. group 5 Rats (n=12) underwent induction of sTBI, 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, and were 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 bone resection craniotomy was performed in all animals during anesthesia. The rat skull was carefully removed to expose the brain, which was quickly incised along the sagittal groove to separate the two hemispheres. The hemisphere dedicated to biochemical analysis was freeze-clamped with aluminum tongs pre-cooled in liquid nitrogen and immersed in liquid nitrogen. A freeze-clamp procedure was introduced to accelerate tissue freezing, thereby minimizing potential metabolite loss.

[0387] The remaining hemisphere, dedicated to molecular biology analysis, was placed in 5-10 volumes of RNAlater® solution (Invitrogen Life Technologies), an RNA stabilizing solution that stabilizes RNA and protects it from degradation. Brain samples were stored overnight at 4°C to allow the solution to completely permeate 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 at 24,000 rpm / min using an Ultra-Turrax homogenizer set (Janke & Kunkel, Staufen, Germany). After centrifugation at 20,690xg for 10 min at 4°C, the clear supernatant was saved and the pellet was supplemented with an aliquot of 10 ml KH2PO4, homogenized again as above, and stored at -20°C overnight to ensure complete recovery of the aqueous phase from the tissue. A second centrifugation was performed (20,690xg, 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 the 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), the volume was adjusted with 10 mM KH2PO4, pH 7.40 to obtain a final aqueous 10% tissue homogenate and 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, 250x4.6 mm, 5 μm particle size column (Thermo Fisher Scientific, Rodano, Milan, Italy) with its own guard column and coupled to an HPLC instrument consisting of a Surveyor System (Thermo Fisher Scientific, Rodano, Milan, Italy) 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 an existing ion-pair HPLC method with slight modifications (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 was performed by comparing the retention times, absorption spectra and areas of peaks with those of chromatographic runs of freshly prepared ultrapure standard mixtures with 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, and N-acetylaspartic acid (NAA).

[0392] HPLC analysis of free amino acids and amino group-containing compounds Simultaneous determination of primary free amino acids (FAA) and amino group-containing compounds (AGCC) (described below) was performed using pre-column derivatization of samples 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, 237.5 mmol / l sodium borate, pH 9.8 was prepared daily and introduced 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, 250x4.6mm, 5 μm particle size, thermostated at 21°C) for subsequent chromatographic separation. To accurately quantify glutamate, deproteinized brain extracts were diluted 20-fold with HPLC grade H2O before the derivatization procedure and subsequent injection. Separation of OPA-AA and OPA-AGCC was performed with 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% H2O) 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 concentration.

[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 perfused transcardially as described by Di Pietro et al., Sci Rep. 2017, 7(1):9189. Briefly, a thoracotomy was performed and a heparin solution was administered into the portal vein to avoid blood clotting during all procedures. A right atrium incision was then performed and the 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 a further perfusion with 4% paraformaldehyde (PFA) pH 7.4 in PBS solution. After rapid removal from the skull, each brain was postfixed by immersion in 100 ml of 4% PFA in PBS solution at 4°C for 2 h. Cryoprotection was obtained by immersion of whole brains in PBS enriched with increasing sucrose solutions (10%, 20% and 30%) for 24 h, followed by embedding in optimal cutting temperature embedding medium (OCT) (Thermo Shandon, Runcorn, UK) in peeling mould 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 Values ​​for phosphorylated high-energy purine and pyrimidine compounds are summarized in Table 13. It is particularly evident that depletion of triphosphate nucleotides (ATP, GTP, UTP, and CTP) was caused by sTBI, 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 higher values ​​of high-energy phosphates (ATP, GTP, and CTP) recorded at all three drug doses tested. No effect was observed on the concentrations of UTP and ADP. It is worth recalling that 48 hours after TBI in rats is a critical time point for cerebral metabolism, coinciding with the greatest changes in mitochondrial function, including alterations in mitochondrial quality control. In this experimental model of TBI, this time point can be considered as a kind of "turning point" at which recovery or non-recovery of cerebral metabolism is determined. [Table 17] TIFF2025077046000024.tif16162

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

[0400] Effects of increasing doses of LMW-DS on nicotine coenzyme Oxidized (NAD + and NAD + The values ​​for nicotine coenzymes in their reduced (NADH and NADPH) and reduced forms are summarized in Table 14. Table 14 also shows the calculated NAD + We report dimensionless values ​​of the NADH / 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 + , NAD + 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 to allow correct functional operation of the tricarboxylic acid cycle (TCA cycle), thereby ensuring a continuous supply of electrons to the electron transport chain (ETC). TCA is the main cell cycle for the generation of reduced coenzymes (NADH and FADH2), which are the fuel for the ETC and oxidative metabolism by transferring their electrons to mitochondrial complexes I and II, respectively. All compounds, especially acetyl-CoA, are significantly affected by sTBI. Partial recovery of this compound was observed at 5 or 15 mg / kg body weight. LWM-DS was administered to the animals 30 min 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 decomposition of unsaturated fatty acids in membrane phospholipids as a result of ROS-mediated lipid peroxidation. Nitrite (-NO2 - ) and nitrates (-NO3- ) is a stable end product of nitric oxide (NO) metabolism that 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 signature was noted as well. Administration of LWM-DS significantly restored both ascorbic acid and reduced glutathione (GSH) concentrations, and a decrease in brain tissue nitrite and nitrate was 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 subjected to sTBI, all 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 nearly equal to that of the neurotransmitter glutamate in humans. Although the biological role of NAA has not yet been fully elucidated, we have clearly shown in both preclinical and clinical trials 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 decrease and recovery of NAA levels in athletes after concussion is much slower than the clearance of symptoms. Thus, NAA has special relevance to the study of TBI.

[0407] A 40% decrease was observed in whole brain NAA 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 administered either one of the two drug doses was significantly higher than that found in sTBI rats, with the greatest 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 directly (GLU, GABA) or indirectly (GLN, ASP, ASN, GLY, SER, THR, ALA) involved in neurotransmission. In particular, GLU is the major excitatory amino acid, the action of which is 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 involving 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 the untreated sTBI animal group, but not better than those of the controls. [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 main actors of 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. Complementary explanations for the changes in L-cystathionine (L-Cystat) are shown 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 generation of NO in the reaction catalyzed by nitric oxide synthase (NOS), a family of enzymes present in three isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The last isoform (iNOS) is the one involved in nitrosative stress. Nitric oxide is generated through a complex reaction in which arginine (ARG) undergoes partial oxidation to provide a nitrogen atom to generate citrulline (CITR) and NO. Animals 2 days after sTBI showed a concomitant decrease in Arg and 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 sources of carbon skeletons available 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 show that sTBI results in an increase in the 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 the 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). In general, it should be emphasized that 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 led to a more clear general improvement of cerebral energy metabolism when the drug dose was higher than 1 mg / kg body weight. Even in rats receiving repeated doses of LWM-DS at 15 mg / kg body weight, differences from controls were recorded, but significantly higher values ​​of nucleotide triphosphates were found in drug-treated animals. Of particular relevance is the progressive recovery of the calculated dimensionless value of the ATP / ADP ratio (which is considered a good indicator of mitochondrial phosphorylation capacity), which was continuously increased by increasing doses of drug administered to sTBI animals. [Table 27] TIFF2025077046000035.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 drug administration at any dose tested led to a significant increase in the most important high energy phosphates.

[0416] Effects of increasing doses of LMW-DS on nicotine coenzyme Oxidized (NAD + and NAD + The values ​​for nicotine coenzymes in the form of NADH and NADPH are summarized in Table 23. Table 23 also shows the calculated NAD + We report dimensionless values ​​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 +Extremely large decreases in the / NADH ratio were recorded in sTBI rats 7 days after injury. Treatment with LMW-DS at all doses, except for the lowest, resulted in significant improvements in the concentration of nicotine coenzyme. In particular, single and repeated doses of 15 mg / kg body weight of LMW-DS significantly improved the NAD + Normalize your levels and get the right 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 administration of 5 or 15 mg / kg body weight (both doses as single and repeated doses) 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. No recovery of concentrations of both water-soluble antioxidants occurred in sTBI-induced rats 7 days after impact. Traces of extremely high levels of oxidative / nitrosative stress were recorded as well. The effects of single and repeated administration of LWM-DS were particularly beneficial in restoring concentrations of both ascorbic acid and reduced glutathione (GSH), with a clear reduction in brain tissue nitrite and nitrate. These effects were also evident 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 degradation pathways of purine and pyrimidine nucleotides 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 LMW-DS administration of increasing doses 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 more abundant in sTBI rats 7 days after injury when 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, either in a single or repeated dose. 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 the impact when compared to the corresponding values ​​of the controls. An increase in MET was observed in animals that received the maximum dose of LWM-DS (both single or repeated administration). As already observed 2 days after the injury, these drug levels led to a significant increase in L-cystathionine (L-Cystat). Since this compound is an intermediate in the production of cysteine ​​(CYS), it can be assumed 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 with 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 increase in CITR, consistent with data showing 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 show 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 (assessed by the Glasgow Coma Scale), three different types of TBI can be identified: mild TBI (mTBI), moderate TBI and severe TBI (sTBI). The ratio of occurrence of mTBI:sTBI has been calculated to be about 22:1. Unfortunately, the consequences of TBI are often disabling and sometimes lead to permanent or temporary impairment of cognitive, physical and psychosocial functioning, with an associated reduced or altered state of consciousness. Thus, patients are affected in several important aspects, mainly their ability to function properly and maintain social relationships, mainly their ability to support themselves.

[0429] TBI is considered to be a complex pathological process consisting of a primary insult (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, triggering 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 usually tested for their ability to interfere with the biochemical and molecular changes that occur to brain tissue metabolism, as well as 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) as well as 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, since mitochondrial NAA biosynthesis has a high indirect energy consumption, changes in NAA brain concentrations are closely related to changes 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 carried out to evaluate the effect 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 restoration 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. In particular, ATP levels 7 days after impact were only 16% lower than control values, whereas a 35% decrease was observed in sTBI rats (Table 21 and Figure 25). Of note, NAA concentrations in LMW-DS-treated animals at the same time point were only 16% lower than control values, whereas sTBI animals showed 48% lower values ​​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 functional behavior.

[0434] The general restoration 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, meaning that drug-treated animals, despite suffering from sTBI, had subnormal coenzymes ensuring proper redox reactions and allowing good functional operation of the TCA cycle.

[0435] The abovementioned improvement in brain metabolism was certainly responsible for another remarkable drug effect, namely the disappearance of GLU excitotoxicity. Furthermore, the drug affected sulfur-containing amino acids. Presumably, this effect is related to the drug molecule containing an S atom. The increase in the 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 the increase in antioxidants and the reduction of biochemical signatures of oxidative / nitrosative stress in sTBI rats administered LMW-DS. Since dysfunctional mitochondria are the main intracellular source of both ROS and RNS, this phenomenon would even be highly relevant to the normalization of mitochondrial function. It is plausible that the effect of LMW-DS was more evident 7 days after sTBI than 2 days after sTBI. This strongly suggests that the general 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, and even repeated doses of 15 mg / kg body weight are often similar to a single dose of the same dose. That is, repeated administration of the drug was not always advantageous.

[0437] This contradictory result may be explained by the following: 1) it is well known that sTBI induces the breakdown 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 of point 2) is correct, the administration performed 30 min after injury may have been performed while the BBB was still open / altered; 4) if the hypotheses of points 2) and 3) are correct, the administration early after injury may have been performed while the BBB was still open / altered, resulting in the incorporation of the compound into the brain compartment. the passage would be facilitated and the drug could induce its beneficial effects on brain metabolism and function, including BBB normalization; 5) if what we reported in point 4) is correct, it would mean that the administration of 15 mg / kg body weight LMW-DS 30 min after sTBI, in addition to initiating brain metabolic normalization, would further lead to BBB closure, thereby limiting the possibility of obtaining additional effects by a repeated drug administration protocol, meaning that 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 is characterized by profiling 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) allow characterization of test agents in an unbiased manner across a broad set of systems that model 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 disease states. 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 the context of systemic immune responses, 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 the BF4T system (Th2) represent pulmonary airway inflammation, while the MyoF system models myofibroblast-pulmonary 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 agent generates a signature BioMAP® profile created from changes in protein biomarker readouts in the individual system environment. Biomarker readouts (7-17 per system) are selected for their therapeutic and biological relevance, predict disease outcomes or specific drug effects, and are validated with reagents with known mechanisms of action (MoA). Each readout is quantitatively measured by immune-based methods that detect proteins, e.g., ELISA, or functional assays that measure proliferation and viability. BioMAP® readouts are diverse and include cell surface receptors, cytokines, chemokines, matrix molecules, and enzymes. In total, the BioMAP® Diversity PLUS panel contains 148 biomarker readouts that capture biological changes that occur 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 Use human primary cells in the BioMAP system 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. Prior to addition to the / Mphg system, they were CD14 + Human blood derived monocytes are differentiated in vitro into macrophages. 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 PBMC+(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 [HUVEC and M1 macrophages+zymosan (TLR2 ligand)].

[0443] The systems are derived from single cell types or co-culture 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 co-cultured with PBMCs and stimulated with BCR activators and low levels of TCR stimulation. Test agents prepared in DMSO (small molecule; final concentration ≦0.1%) or PBS (biologics) are added at the indicated concentrations 1 hour prior to stimulation and remain in culture for 24 hours or the following times indicated (48 hours, MyoF system; 72 hours, BT system (soluble readings); 168 hours, BT system (secreted IgG)). Each plate includes a drug control appropriate for each system (e.g., legacy control test agent colchicine at 1.1 μM), a negative control (e.g., unstimulated conditions) and a vehicle control (e.g., 0.1% DMSO). Biomarker levels of cell-associated and cell 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 optimization time points for each system (48 h: 3C and CASM3C systems; 72 h: BT and HDF3CGF systems; 96 h: 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 systems; 48 h: MyoF system) and by alamarBlue staining of suspension cells (24 h: SAg system; 42 h: BT system).

[0444] 4. Data Analysis Biomarker measurements in test drug treated samples were divided by the average of the control samples (at least six vehicle controls from the same plate) to generate a ratio, followed by log10 Transform. Significance prediction envelopes are calculated using 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 the biomarker has 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 the total protein level is decreased by more than 50% (log 2 of SRB), the biomarker activity is annotated if the biomarker has at least one concentration with a ratio |>0.1. 10 Ratio or alamarBlue level <-0.3), cytotoxicity status is recorded and indicated by thin black arrows above the x-axis. Compounds are considered to have broad cytotoxicity if cytotoxicity is detected in three or more systems. 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 not observed when SRB or alamarBlue log 100 from cells seeded at lower densities are compared. 10 Defined by a ratio value <-0.1 and indicated by a grey arrow above the X-axis. Cytotoxic and anti-proliferative arrows only require one concentration to meet the indicated threshold for profile annotation.

[0446] Benchmark Analysis Common biomarker reads are annotated if reads for both profiles are outside the significance envelope with effect sizes >20% in the same direction. Differentiation biomarkers are annotated if one profile has reads outside the significance envelope with effect sizes >20% and the other profile has reads inside the envelope or in the opposite direction. Unless otherwise specified, the highest non-cytotoxic concentrations of both test and benchmark agents 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 of more than 20% in the same direction, the common biomarker readouts are annotated. Concentrations of test agents with 3 or more systems with detectable cytotoxicity are excluded from the similarity analysis. Concentrations of test agents with 1-2 systems with detectable cytotoxicity are included in the similarity search analysis with an overlay of the database match with the highest concentration of the test agent. This is followed by an additional overlay of the next highest concentration of the test agent that does not contain a system with detectable cytotoxicity and the respective database match. To determine the degree of similarity between BioMAP® profiles of compounds performed in 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's and Spearman's correlation coefficients). This method more efficiently accounts for the variability in the number of data points, systems, activity biomarker readouts, and amplitude of biomarker readout changes that are 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 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 scales each profile with 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 "closeness" of drug profiles from a multidimensional space into two dimensions. The functional clustering of drug profiles generated during this analysis uses Pearson correlation values ​​for pairwise comparison 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 heatmap analysis provides visualization of 19 consensus mechanisms that allow comparison of biomarker activity across test compounds and all compound concentrations and consensus mechanisms. The composite consensus profile used in mechanism heatmap 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) and constructing a consensus mechanism profile. Biomarker activity is colored in the heatmap for consensus mechanisms and compounds if they have expression compared to vehicle control outside the significance envelope. Red represents increased protein expression, blue represents decreased expression, and white indicates unchanged or levels within the filtering conditions. Darker shades of color represent larger changes in biomarker activity compared to vehicle control. Mechanism heatmaps were created using R and the gplot package for R.

[0450] Assay Acceptance Criteria BioMAP® assays include multi-parameter data sets 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., 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 historical controls for that system. QA / QC Pearson testing 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 the system biomarker readings in the positive control reference dataset, and the Pearson value between each profile and the average 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 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] TIFF2025077046000052.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 of more than 20% (|log 10 Biomarker activities were annotated if they had at least one concentration with a ratio |>0.1). The primary activity of a biomarker was called modulated if these activities were 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 reduced 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 showed 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 effect of LMW-DS on 12 different human primary cells in the BioMAP® Diversity PLUS panel. [Table 38]

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

[0455] In an unsupervised search of mathematically similar compound profiles in the BioMAP® Reference Database, LMW-DS (4M) is 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 cross-talk and feedback mechanisms associated with in vivo outcomes.

[0457] LMW-DS was active and not cytotoxic at the concentrations tested in this study. LMW-DS was moderately and selectively anti-proliferative towards human primary endothelial cells only at the highest concentration (4 μM). The LMW-DS profile had 25 annotated readouts and showed modulation of immune and inflammation related readouts as well as matrix related biomarkers. Specific activity 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 activity included reduced sIgG and IL17A and IL17F in the BT system, but no anti-proliferative effect on B cells. Reduced M-CSF and increased CD69 were identified as well. 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 chemotactic factor IL8 was confirmed in multiple systems. Collectively, these data indicate 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 treating multiple chronic and acute inflammatory conditions and diseases that contain an inflammatory component, such as ALS.

[0459] After injury, initially, select components of the innate / pro-inflammatory and acquired 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 is induced by LMW-DS and includes activation of MMP-1, PAR-1, and uPAR, demonstrating induced immune resolution that has utility in treating tissues damaged by trauma, including neurotrauma, that would otherwise have resulted in deleterious fibrosis formation.

[0460] LMW-DS modulated many biomarker activities in the HDF3CGF system, but only IL8 in the MyoF system. Although both systems contain fibroblasts, HDF3CGF models wound healing and matrix remodeling associated with such wound healing, whereas MyoF is more of a fibrosis model of collagen deposition. Thus, the results indicate that LMW-DS had immunomodulatory and tissue remodeling activities, but did not induce undesirable collagen fibrosis that could result in harmful fibrotic deposition.

[0461] In conclusion, LMW-DS appears to normalize and resolve inflammation present in tissues following trauma or disease, and therefore these results are 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 survey of differentially regulated genes followed by functional analysis.

[0463] material and method Induction of sTBI 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 fed standard laboratory chow and water ad libitum in a controlled environment. The animals received an anesthetic mixture of 35 mg / kg body weight ketamine and 0.25 mg / kg body weight midazolam by intraperitoneal injection. Severe traumatic brain injury (sTBI) was induced by dropping a 450 g weight from a height of 2 m onto the head of the rat, which was protected 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 suffered 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 anesthetized again and then immediately sacrificed.

[0464] Test Compounds 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 2 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 subcutaneous injections of 0.5 ml 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 An in vivo osteotomy craniotomy was performed under anesthesia in all animals. The rat skull was carefully removed and the brain was exposed, 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 effect 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 performing different replicate statistics provided information about 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] Comparison 7 days after TBI showed that out of 2741 genes deregulated by TBI (7 days), only 124 (4.5%), 169 (6.1%) and 85 (3.1%) remained deregulated when 1 mg / kg, 5 mg / kg and 15 mg / kg LMW-DS were administered, respectively, compared to healthy control animals. With three repeated doses of 15 mg / kg LMW-DS, the number of genes remaining deregulated compared to healthy control animals was 116 (4.25%) genes.

[0481] Pathway analysis and mechanistic investigations Pathway analysis of differentially regulated genes was performed using the Ingenuity pathway analysis package. Analysis was performed specifically on pathways and molecular processes and diseases related to scar formation and fibrosis, including dementia, Alzheimer's disease, ALS, TBI and stroke, as well as neurodegenerative diseases including glaucoma and normal pressure hydrocephalus (NPH) following 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 the pathways, the number of molecular processes and diseases significantly affected within 2 days of TBI was moderate. However, the effects of TBI were almost abolished by LMW-DS administered 30 minutes after injury (Tables 36 and 37). [Table 39] TIFF2025077046000055.tif232162TIFF2025077046000056.tif232162TIFF2025077046000057.tif32162 * Unclear effect [Table 40] TIFF2025077046000059.tif231162TIFF2025077046000060.tif231162TIFF2025077046000061.tif60162 * Unclear effect [Table 41] TIFF2025077046000063.tif230162TIFF2025077046000064.tif229162TIFF2025077046 000065.tif230162TIFF2025077046000066.tif231162TIFF2025077046000067.tif53162 * Unclear effect [Table 42] TIFF2025077046000069.tif228162TIFF2025077046000070.tif229162TIFF2025077046000071.tif229162 TIFF2025077046000072.tif228162TIFF2025077046000073.tif228162TIFF2025077046000074.tif130162 * Unclear effect

[0483] Within 7 days, the effects induced by TBI were significant, with many genes being deregulated. Thus, the 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). As with 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 almost abolished by LMW-DS administered 30 minutes after injury (Tables 40 and 41). [Table 43] TIFF2025077046000076.tif228162TIFF2025077046000077.tif229162TIFF202 5077046000078.tif228162TIFF2025077046000079.tif231162TIFF20250770460 00080.tif229162TIFF2025077046000081.tif228162TIFF2025077046000082.t if228162TIFF2025077046000083.tif229162TIFF2025077046000084.tif206162 * Unclear effect [Table 44] TIFF2025077046000086.tif231162TIFF2025077046000087.tif232162TIFF2025077046000088.tif220162TIFF2025077046000089.t if214162TIFF2025077046000090.tif230162TIFF2025077046000091.tif231162TIFF2025077046000092.tif233162TIFF20250770460 00093.tif229162TIFF2025077046000094.tif231162TIFF2025077046000095.tif225162TIFF2025077046000096.tif230162TIFF202 5077046000097.tif229162TIFF2025077046000098.tif228162TIFF2025077046000099.tif231161TIFF2025077046000100.tif145162 [Table 45] TIFF2025077046000102.tif230162TIFF2025077046000103.tif227162TIFF2025077046000104.tif231162TIFF202 5077046000105.tif232162TIFF2025077046000106.tif230162TIFF2025077046000107.tif233162TIFF2025077046 000108.tif232162TIFF2025077046000109.tif225162TIFF2025077046000110.tif234162TIFF2025077046000111. tif233162TIFF2025077046000112.tif230162TIFF2025077046000113.tif230162TIFF2025077046000114.tif46162 [Table 46] TIFF2025077046000116.tif232162TIFF2025077046000117.tif227162TIFF2025077046000118.tif232162TIFF202 5077046000119.tif229162TIFF2025077046000120.tif233162TIFF2025077046000121.tif232162TIFF20250770460 00122.tif232162TIFF2025077046000123.tif226162TIFF2025077046000124.tif233162TIFF2025077046000125.t if229162TIFF2025077046000126.tif230162TIFF2025077046000127.tif227162TIFF2025077046000128.tif227162 * Unclear effect

[0484] Consideration LMW-DS was able to attenuate and restore the effects of TBI in 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 related to neurodegenerative diseases as well as fibrosis and scar formation. Results revealed that LMW-DS could beneficially affect these pathways even when they were severely perturbed.

[0485] It can 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, different part measures in different embodiments can be combined into other configurations, where technically possible.

Claims

1. A pharmaceutical composition comprising dextran sulfate or a pharma- ceutical acceptable salt thereof for the treatment of glutamate excitotoxicity, wherein the dextran sulfate or a pharma- ceutical acceptable derivative 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 ).

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

3. 3. A pharmaceutical composition according to 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. A pharmaceutical composition comprising dextran sulfate or a pharma- ceutical acceptable salt thereof for the treatment of neuroinflammation, wherein the dextran sulfate or a pharma- ceutical acceptable derivative 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 ).

9. 1. A pharmaceutical composition comprising dextran sulfate or a pharma- ceutical acceptable salt thereof for resolving established scars in a subject suffering from fibrosis or a fibrotic disease, disorder or condition, said dextran sulfate or a pharma- ceutical acceptable derivative thereof having 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 ).

10. 10. The pharmaceutical composition of claim 9, wherein the fibrotic disease, disorder or condition is selected from the group consisting of glaucoma, proliferative vitreoretinopathy, traumatic brain or spinal cord injury, cerebral subarachnoid hemorrhage, invasive surgery, postoperative adhesions, rotator cuff injuries, burns, reconstructive surgery, pulmonary fibrosis, idiopathic pulmonary fibrosis, progressive massive fibrosis, radiation-induced lung injury after cancer treatment, liver cirrhosis, biliary atresia, atrial fibrosis, endomyocardial fibrosis, old myocardial infarction, glial scar, pancreatitis, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloids, mediastinal fibrosis; myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis; retroperitoneal fibrosis, scleroderma or systemic sclerosis.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the dextran sulfate or a pharma- ceutically acceptable derivative thereof has an average sulfur content in the range of 15 to 20%.

12. The dextran sulfate or a pharma- ceutically acceptable derivative thereof has a number average molecular weight (M) in the range of 1,850 to 3,500 Da, preferably in the range of 1,850 to 2,500 Da, as measured by nuclear magnetic resonance (NMR) spectroscopy. ｎ 12. The pharmaceutical composition according to claim 1, wherein

13. The dextran sulfate or a pharma- ceutically acceptable derivative thereof has an M in the range of 1,850 to 2,000 Da as measured by NMR spectroscopy. ｎ The pharmaceutical composition of claim 12, having the formula:

14. 14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the dextran sulfate or a pharma- ceutically acceptable derivative thereof has an average number of sulfates per glucose unit in the range of 2.5 to 3.0, preferably in the range of 2.5 to 2.8, and more preferably in the range of 2.6 to 2.

7.

15. 15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the dextran sulfate or a pharma- ceutically acceptable derivative thereof has an average of 5.1 glucose units and an average number of sulfates per glucose unit of 2.6 to 2.

7.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the dextran sulfate or a pharma- ceutically acceptable salt thereof is a sodium salt of dextran sulfate.

17. 1. Use of dextran sulfate or a pharma- ceutical acceptable salt thereof in the manufacture of a medicament for the treatment of glutamate excitotoxicity, wherein the dextran sulfate or a pharma- ceutical acceptable derivative 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 ) and use.

18. 1. Use of dextran sulfate or a pharma- ceutical acceptable salt thereof in the manufacture of a medicament for the treatment of neuroinflammation, wherein the dextran sulfate or a pharma- ceutical acceptable derivative 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 ) and use.

19. 1. Use of dextran sulfate, or a pharma- ceutical acceptable salt thereof, in the manufacture of a medicament for resolving established scars in a subject suffering from fibrosis or a fibrotic disease, disorder or condition, wherein the dextran sulfate or a pharma- ceutical acceptable derivative 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 ) and use.

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