Drugs for treating coronavirus infections

Abstract

The present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the treatment of coronavirus infections or infectious diseases and infections caused by other HSPG-binding pathogens. The present invention also relates to the treatment of inflammatory diseases, such as ARDS and SIRS, that may occur in association with such infections.

Description

[Technical Field]

[0001] The present invention relates generally to agents for the treatment of infectious and inflammatory diseases and the long-term sequelae of these infectious diseases, and in particular to agents for the treatment of such coronavirus infections and diseases and inflammatory diseases that may be caused by coronavirus infections. [Background technology]

[0002] Coronaviruses (CoVs) are a group of related enveloped viruses that cause disease in mammals and birds. In humans, coronavirus infections can lead to respiratory tract infections, ranging from mild cases such as the common cold to other fatal cases such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19).

[0003] Coronaviruses have significantly varying risk factors. They can cause a cold, with key symptoms including fever and pharyngitis from enlarged tonsils. Coronaviruses can also cause pneumonia, which can be direct viral pneumonia or secondary bacterial pneumonia, and bronchitis, which can be direct viral bronchitis or secondary bacterial bronchitis. Coronaviruses can also be associated with long-term organ disease and functional decline, referred to as "long-COVID."

[0004] In December 2019, an outbreak of pneumonia was reported in Wuhan, China. On December 31, 2019, this outbreak was attributed to a novel strain of coronavirus, which was given the temporary name 2019-nCoV by the World Health Organization (WHO) and later renamed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses. The Wuhan strain was identified as a novel strain of betacoronavirus (β-CoV) from group 2B, with approximately 70% genetic similarity to SARS-CoV.

[0005] The lungs are typically the organ most affected by SARS-CoV-2. This is because the virus gains access to host cells through the most abundant enzyme in the lung, angiotensin-converting enzyme 2 (ACE2), in type II pneumocytes. The virus uses a specialized surface glycoprotein called the "spike" (peplomer) to bind to ACE2 and enter host cells. This binding between the spike protein and ACE2 is facilitated by heparan sulfate present on the host cell surface. As alveolar disease progresses, respiratory failure and death may occur.

[0006] Coronaviruses can also affect the gastrointestinal tract, as ACE2 is abundantly expressed in glandular and endothelial cells of the gastric, duodenal, and rectal epithelium and in enterocytes of the small intestine.

[0007] The infectious disease caused by SARS-CoV-2, i.e., COVID-19, has common symptoms in the form of fever, cough, and shortness of breath. Myalgia, sputum production, diarrhea, and pharyngitis are more rare. Most cases result in mild symptoms, but some progress to pneumonia, and in the most severely affected, especially in long-COVID patients, COVID-19 can rapidly progress to acute respiratory distress syndrome (ARDS), leading to respiratory failure, septic shock, hyperinflammation, oxidative stress, neurological damage, microthrombosis, fibrosis, and / or multiple organ failure.

[0008] Many efforts are being made worldwide to develop a vaccine against SARS-CoV-2. Currently, there is a lack of effective therapeutic agents for COVID-19 and SARS-CoV-2 infection and the long-term adverse consequences on damaged tissue. Therefore, there is a comprehensive need for effective therapeutic agents for the treatment of coronavirus infections, including COVID-19. Summary of the Invention

[0009] The overall aim is to provide a drug for the treatment of coronavirus infections or infectious diseases. The specific purpose is to provide a drug for the treatment of SARS-CoV-2 infection and COVID-19. These and other objects are achieved by the embodiments disclosed herein.

[0010] An embodiment of the present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the prevention, suppression and / or treatment of coronavirus infection or infectious disease.

[0011] Another aspect of the present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the prevention, suppression, or treatment of an inflammatory disease selected from the group consisting of acute respiratory distress syndrome (ARDS) and systemic inflammatory response syndrome (SIRS).

[0012] A further aspect of the present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the prevention, inhibition and / or treatment of infection or infectious disease caused by pathogens capable of binding to cell surface heparan sulfate proteoglycans (HSPGs).

[0013] The experimental data presented herein demonstrate that dextran sulfate, or a pharmaceutically acceptable salt thereof, can be used to treat coronavirus infections and other infectious diseases and their long-term consequences caused by pathogens capable of binding to HSPGs. Dextran sulfate, or a pharmaceutically acceptable salt thereof, can also stimulate the release of repair growth factors from tissue stores and suppress pro-inflammatory cytokines in selected immune cells. This suggests that dextran sulfate, or a pharmaceutically acceptable salt thereof, can prevent or at least significantly suppress the increase in circulating cytokine levels that causes inflammatory disease after coronavirus infection, ARDS, or SIRS. Dextran sulfate, in embodiments, also has the potential to degrade inflammatory scarring caused by inflammatory conditions and promote functional tissue regeneration. These effects are also important for patients with COVID-19 sequelae, as they normalize metabolism and improve muscle and liver function. Some embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows dextran sulfate competition for protein-protein interaction between amyloid beta and PrPC. [Figure 2] 1 shows plasma hepatocyte growth factor (HGF) levels in amyotrophic lateral sclerosis (ALS) patients before and 2 hours after administration of low molecular weight dextran sulfate (LMW-DS). [Figure 3] FIG. 1 shows LMW-DS-induced changes in brain glutamate levels. [Figure 4A-D] FIG. 1 shows LMW-DS altered levels of adenine nucleotides (ATP, ADP, AMP) and the ATP / ADP ratio as a measure of mitochondrial phosphorylation capacity. [Figure 5A-D] FIG. 1 shows LMW-DS altered levels of oxidized and reduced nicotine coenzyme. [Figure 6A-C] FIG. 1 shows LMW-DS altered levels of biomarkers indicative of oxidative stress. [Figure 7] FIG. 1 shows LMW-DS altered levels of nitrate as a measure of NO-mediated nitrosative stress. [Figure 8A-C] FIG. 1 shows LMW-DS altered levels of N-acetylaspartate (NAA) and its substrates. [Figure 9] Figure 1 shows the concentration of NAA measured in deproteinized brain homogenates of rats sacrificed 2 days after severe head trauma (sTBI) with or without a single administration of increasing doses of LWM-DS (1, 5, and 15 mg / kg body weight) administered 30 minutes after trauma induction. Controls were sham-operated animals. Values ​​are the means of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from sTBI 2 days, p<0.01. [Figure 10]Figure 1 shows the concentration of ATP measured in deproteinized brain homogenates of rats sacrificed 7 days after sTBI with or without administration of increasing doses of LWM-DS (single doses of 1, 5, and 15 mg / kg body weight and repeated doses of 15 mg / kg body weight). Controls are sham-operated animals. Values ​​are the mean of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from sTBI 2 days, p<0.01. [Figure 11] Figure 1 shows the concentration of ascorbic acid measured in deproteinized brain homogenates of rats sacrificed 7 days after sTBI with or without administration of increasing doses of LWM-DS (single doses of 1, 5, and 15 mg / kg body weight and repeated doses of 15 mg / kg body weight). Controls were sham-operated animals. Values ​​are the mean of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from sTBI 2 days, p<0.01. [Figure 12] Figure 1 shows glutathione (GSH) concentrations measured in deproteinized brain homogenates of rats sacrificed 7 days after sTBI with or without administration of increasing doses of LWM-DS (single doses of 1, 5, and 15 mg / kg body weight and repeated doses of 15 mg / kg body weight). Controls were sham-operated animals. Values ​​are means of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from sTBI 2 days, p<0.01. [Figure 13] Figure 1 shows the concentration of NAA measured in deproteinized brain homogenates of rats sacrificed 7 days after sTBI with or without administration of increasing doses of LWM-DS (single doses of 1, 5, and 15 mg / kg body weight and repeated doses of 15 mg / kg body weight). Controls were sham-operated animals. Values ​​are the mean of 12 animals. Standard deviations are represented by vertical bars. *Significantly different from control, p<0.01. **Significantly different from sTBI 2 days, p<0.01. [Figure 14] 1 shows the change in activated partial thromboplastin time (aPTT) in blood after administration of LMW-DS. [Figure 15]Human trabecular meshwork cells were stimulated with transforming growth factor beta 2 (TGFβ2) (1.0 ng / ml) in saline for 72 hours in the presence or absence of LMW-DS (4.0 μM), followed by immunofluorescence labeling for fibronectin (green) and nuclear staining with Hoechst (cyan). (15A) Representative images are maximum intensity projections from confocal Z-stacks, and (15B) histograms show quantification of fibronectin staining (mean ± SEM, n = 6), P < 0.05 (Wilcoxon test). [Figure 16] (16A) Schematic diagram showing the method for inducing the anterior segment fibrosis model. Twice-weekly intracameral injections of TGFβ1 induced fibrosis in the trabecular meshwork (TM), blocked AqH outflow, and elevated IOP. (16B) Line graph showing IOP measurements during the first 14 days of IC TGFβ1 treatment, followed by daily subcutaneous saline vehicle control or LMW-DS treatment with IC TGFβ1 for an additional 14 days. Normal IOP levels are indicated by the gray-shaded area. **P<0.01, ****P<0.0001 (2-way ANOVA). (16C+16D) Representative images of ocular tissue sections containing slopes of the anterior segment with associated histograms showing levels of immunoreactive laminin and fibronectin staining in the TM of saline- and LMW-DS-treated groups. **P<0.01, ***P<0.001 (t-test). (16E) Representative images and histograms showing the RGC marker BRN3a in retinal sections from saline and LMW-DS treatment groups. **P<0.01 (Mann-Whitney test), GCL-neuronal layer. (16F) Representative optical coherence tomography images and associated histograms showing segmented RNFL (arrows) in saline and LMW-DS treatment groups. ****P<0.0001 (t-test). Saline group n=5, LMW-DS group n=7. (16G) Schematic showing the potential mechanism of LMW-DS in POAG. [Figure 17]PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: (17A) LPS (0.01 ng / ml), (17B) peptidoglycan (30 ng / ml), (17C) pokeweed mitogen (1.0 μg / ml), (17D) PHA-L (1.0 μg / ml), (17E, 17F) CpG (0.2 μM or 1.0 μM) + IL-15 (15 ng / ml), or (17G, 17H) cytostim (10 μl / ml or 30 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200, or 600 μg / ml). IL-6 levels in the supernatants were quantified by ELISA. Data are presented as mean ± SEM obtained from 6 or 10–11 (LPS) donors. Data are plotted as percentage stimulation + vehicle. (-) indicates at least one donor was below the limit of detection. **Mann-Whitney U test comparison between vehicle + LPS and 600 μg / ml ILB™ + LPS, p=0.005. Mann-Whitney U test comparison between vehicle + PHA-L and 600 μg / ml ILB™ + PHA-L, p=0.048. [Figure 18] Monocytes purified from PBMCs were stimulated with either LPS (0.01 ng / ml) or peptidoglycan (30 ng / ml) in the absence of stimuli (medium) or with LPS (0.01 ng / ml) or peptidoglycan (30 ng / ml). Then, they were cultured for 24 hours in the presence or absence (vehicle) of LMW-DS (ILB™; 60 μg / ml, 200 μg / ml, or 600 μg / ml) (18A, 18D), dexamethasone (3.0 μM) (18B, 18E), or heparin (2.0 μg / ml, 6.0 μg / ml, or 20 μg / ml) (18C, 18F). IL-6 levels in cell culture supernatants were quantified by ELISA. Data are presented as mean ± SEM, n = 10. * indicates values ​​below the detection limit (5 pg / ml). + P < 0.05, +++ P < 0.001 for significant differences relative to stimulation (Mann-Whitney U test). [Figure 19]PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200, or 600 μg / ml). Interferon-gamma (IFNγ) levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, (*) indicates data from 6 donors. Compared to vehicle stimulation: # P<0.05, ## P<0.01, ### P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 20]PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200, or 600 μg / ml). Interleukin-8 / chemokine (C-X-C motif) ligand 8 (IL-8 / CXCL8) levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, (*) indicates data from 6 donors. Compared to vehicle stimulation: # P<0.05, ## P<0.01, ### P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 21]PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200, or 600 μg / ml). Tumor necrosis factor alpha (TNFα) levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, (*) indicates data from 6 donors. Compared to vehicle stimulation: # P<0.05, ## P<0.01, ### P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 22] PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200, or 600 μg / ml). IL-1β levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, (*) indicates data from 6 donors. Compared to vehicle stimulation: # P<0.05, ## P<0.01, ### P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 23] PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200, or 600 μg / ml). IL-10 levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, (*) indicates data from 6 donors. Compared to vehicle stimulation: # P<0.05, ## P<0.01, ### P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 24] (24A, 24B) Model of SARS-CoV-2 binding to the cell surface and (24C) inhibition of the interaction between the spike protein and ACE2 by LMW-DS. [Figure 25] Schematic representation of the beneficial effects and actions of LMW-DS in relation to COVID-19. [Figure 26] Serum NAA levels in patients after LMW-DS treatment. [Figure 27] Serum uric acid levels in patients after LMW-DS treatment. [Figure 28] Total serum oxypurines in patients after LMW-DS treatment. [Figure 29] Serum nitrate levels in patients after LMW-DS treatment. [Figure 30] Serum nitrate + nitrite levels in patients after LMW-DS treatment. [Figure 31] Serum MDA levels in patients after LMW-DS treatment. [Figure 32]Serum ALA levels in patients after LMW-DS treatment. [Figure 33] Serum CITR levels in patients after LMW-DS treatment. [Figure 34] Serum ORN / CITR levels in patients after LMW-DS treatment. [Figure 35] Serum α-tocopherol levels in patients after LMW-DS treatment. [Figure 36] Serum gamma-tocopherol levels in patients after LMW-DS treatment. [Figure 37] LMW-DS (ILB®) affects the SARS-CoV-2 spike protein that interacts with ACE-2, as assessed using the RayBiotech® Life ELISA assay. Data represent mean ± SEM (n=3). [Figure 38] Schematic representation of serum lactate levels in ALS patients after dextran sulfate treatment. [Figure 39] 1 shows a schematic representation of ALSAQ-40 ADL scores in ALS patients after dextran sulfate treatment. [Figure 40] 1 shows a schematic representation of serum myoglobin levels in ALS patients after dextran sulfate treatment. [Figure 41] 1 shows a schematic representation of serum creatine kinase levels in ALS patients after dextran sulfate treatment. [Figure 42] Schematic representation of serum hepatocyte growth factor (HGF) levels in ALS patients after dextran sulfate treatment. [Figure 43] 1 shows serum total bilirubin levels in ALS patients after dextran sulfate treatment. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates generally to agents for the treatment of infectious and inflammatory diseases and the long-term sequelae of these infectious diseases, and in particular to agents for the treatment of such coronavirus infections and diseases and inflammatory diseases that may be caused by coronavirus infections.

[0016] Coronaviruses (CoVs) belong to the Orthocoronavirinae subfamily of the family Coronaviridae, in the order Rerum Niribovirales, order Dovirales. They are enveloped viruses with a positive-sense single-stranded RNA genome and a helically symmetric nucleocapsid. Six species of human coronaviruses are known, and one species is subdivided into two distinct strains, resulting in seven strains of human coronaviruses overall. Four of these strains, human coronavirus OC43 (HCoV-OC43) of the genus β-CoV, human coronavirus HKU1 (HCoV-HKU1) of the genus β-CoV, human coronavirus 229E (HCoV-229E) of the genus α-CoV, and human coronavirus NL63 (HCoV-NL63) of the genus α-CoV, typically cause mild common cold symptoms. Three strains can cause severe illness in some cases; these three are Middle East Respiratory Syndrome-related coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV or SARS-CoV-1) and Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), all of which are β-CoV strains.

[0017] Coronavirus and coronavirus infection or infectious disease, as used herein, refers to any coronavirus and infection or infectious disease caused by such coronavirus in a mammal, preferably a human, subject, or patient. In certain embodiments, the coronavirus is selected from the group consisting of MERS-CoV, SARS-CoV, and SARS-CoV-2, and the coronavirus can cause a coronavirus infectious disease selected from the group consisting of MERS caused by MERS-CoV, SARS caused by SARS-CoV, and COVID-19 caused by SARS-CoV-2. In certain embodiments, the coronavirus is SARS-CoV-2, and the coronavirus infectious disease is COVID-19.

[0018] The present invention relates to the use of dextran sulfate in the prevention, suppression, and / or treatment of coronavirus infections and diseases and their consequences. As shown herein, dextran sulfate in embodiments has a multimodal mechanism of action relevant to the early infection, disease progression, and adverse tissue responses of those infected with SARS-CoV-2 (Figure 25). Dextran sulfate in embodiments also has beneficial effects on patients suffering from the long-term effects of COVID-19 symptoms, also known as long COVID and the post-COVID condition.

[0019] These multimodal mechanisms of action include disrupting host-pathogen protein-protein interactions during early infection, thereby inhibiting or suppressing coronavirus binding to target molecules such as ACE2 of SARS-CoV-2, thereby preventing or at least suppressing viral access to host cells.

[0020] As shown schematically in Figures 24A and 24B, similar to other betacoronaviruses, SARS-CoV-2 binding and internalization are mediated by spike glycoproteins (SPGs). SPGs bind not only to their receptor, angiotensin-converting enzyme 2 (ACE2), but also to glycosaminoglycans such as heparan sulfate (HS), which are found on the surface of most mammalian cells in the form of heparan sulfate proteoglycans (HSPGs). Binding of SPGs to tethered HS on the cell surface increases the local concentration of viral particles on the cell surface and promotes binding of SPGs to ACE2 (Figure 24B). Soluble, untethered embodiments of dextran sulfate can bind to SPGs as HS prior to receptor presentation, thereby inhibiting binding of SPGs to HSPGs and ACE2 on the cell surface and preventing, or at least significantly reducing, the local concentration, binding, and internalization of coronaviruses such as SARS-CoV-2 in infected subjects (Figure 24C). As a result, dextran sulfate embodiments not only prevent or at least inhibit coronavirus infection, but also, once a subject is infected with coronavirus, limit the spread and replication of the coronavirus throughout the subject's body by interfering with the interaction between the coronavirus and ACE2 and HSPGs on the cell surface.

[0021] Dextran sulfate embodiments may also reduce the transmission of coronaviruses, i.e., their spread in a population. More specifically, dextran sulfate embodiments may prevent coronaviruses from accessing human cells, thereby inhibiting viral replication and any subsequent viral shedding from infected cells. Such reduced viral shedding is not only beneficial to the subject by limiting spread throughout the subject's body, but also beneficial to the population, as infected subjects are less likely to spread coronaviruses to other subjects.

[0022] As a result, dextran sulfate has a therapeutic effect in stage I (early infection, Figure 25) by disrupting the interaction between the ACE2 receptor and SPGs by binding to SPGs on viral particles, as shown in Figures 24A-24C and 37.

[0023] Dextran sulfate of the embodiments also has effects on metabolic normalization and activation of tissue repair growth factors, such as hepatocyte growth factor (HGF) (Figures 2 and 42). These actions of dextran sulfate of the embodiments are important for suppressing the negative effects of coronavirus in infected subjects during the pneumonia stage (Stage II, Figure 25), in which the virus adversely affects the subject's respiratory tissues by causing pneumonia and stimulating growth factor-mediated scarless tissue repair. Dextran sulfate of the embodiments also has the ability to degrade and reverse existing scar formation, allowing for healing of damaged tissue. In addition, dextran sulfate of the embodiments can induce cellular metabolic normalization, such as improved mitochondrial function (Figures 4A-4D, 27-28; Tables 5-8, 15-18).

[0024] More specifically, dextran sulfate of the embodiments protects mitochondrial function and reduces oxidative stress, as seen in particular by improving the restoration of antioxidant status, protecting mitochondrial ATP energy replenishment by protecting ATP production and metabolism, and normalizing mitochondrial phosphorylation capacity, all of which are induced by dextran sulfate. As a result, dextran sulfate can normalize, protect, and preserve mitochondrial function in cells exposed to injury or disease, which is important for having optimally functioning cells that can fight infectious diseases.

[0025] As a result, the dextran sulfate of the embodiment has a therapeutic effect on stage II (pneumonia stage) of the infection by activating tissue repair growth factors and inducing metabolic normalization (Figure 25).

[0026] Dextran sulfate of the embodiments also has an effect on any disease following the hyperinflammatory phase associated with clinical symptoms, including acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), septic shock, disseminated intravascular coagulation (DIC), and even organ failure. Indeed, severely affected patients also exhibit a pronounced inflammatory response that can induce peripheral microthrombosis, resulting in DIC and the formation of microclots in affected blood vessels, including alveolar capillaries. Dextran sulfate of the embodiments has been shown to be anti-inflammatory, thus eliminating or at least reducing cytokine storm and hyperinflammation in subjects and the deleterious consequences of this hyperinflammation (Figures 17A, 17D, 18A, 19-23). ​​Dextran sulfate also has an anticoagulant effect, which may be useful in combating DIC and microclots observed in severely affected subjects (Figure 14).

[0027] The anti-inflammatory effect of dextran sulfate is selective in terms of its action on specific cells of the immune system, selectively reducing pro-inflammatory cytokines released by such cells. More specifically, the experimental data presented herein demonstrate that dextran sulfate specifically targets monocytes and T lymphocytes, resulting in a concentration-dependent, significant reduction in IL-6, IL-10, TNFα, and IFNγ by activated monocytes and in IL-6, IL-10, IL-1β, IL-8, TNFα, and IFNγ by activated T lymphocytes (Figures 17-23). ​​A significant advantage of the selective reduction of pro-inflammatory cytokines by dextran sulfate over, for example, dexamethasone and other steroids is that dextran sulfate does not affect all cytokine production by the immune system and does not reduce cytokine production to extremely low levels. In infectious diseases such as coronavirus infection, controlled immune system activation is necessary to combat the infection. In stark contrast to blocking, dextran sulfate embodiments can achieve such controlled activation by reducing selected pro-inflammatory cytokines from selected immune system cells, which reduces the risk of developing hyper-inflammatory conditions such as ARDS, SIRS, and septic shock, while still allowing the immune system to fight coronavirus infection.

[0028] The advantage of dextran sulfate over antibody-based anti-inflammatory drugs is that dextran sulfate does not target a single pro-inflammatory cytokine like antibodies, but reduces the levels of several important pro-inflammatory cytokines. Another advantage is that dextran sulfate has a relatively short half-life (C) in the body compared to antibodies. max 2-3 hours in humans), thereby allowing for a controlled anti-inflammatory effect over a well-defined time period that coincides with the period of risk of hyperinflammation after coronavirus infection.

[0029] Subjects recovering from COVID-19 may suffer from organ fibrosis caused by ARDS, SIRS, and microthrombosis. Such organ fibrosis may include not only pulmonary fibrosis but also other organs, such as renal fibrosis and cardiomyopathy. Dextran sulfate of the embodiment has an antithrombotic effect, which is useful for treating such organ fibrosis (Figures 15-16). Furthermore, dextran sulfate of the embodiment has been shown to have not only an antifibrotic effect but also a fibrolytic effect, i.e., to remove existing scars after organ fibrosis and facilitate scar-free tissue reconstruction.

[0030] Dextran sulfate of the embodiment has a therapeutic effect on stage III (hyperinflammatory stage) of infection by resolving all cytokine storms and hyperinflammation and having anti-fibrotic and anti-coagulant effects (Figure 25).

[0031] Emerging evidence suggests that post-COVID-19 symptoms, such as chronic fatigue syndrome and myalgic encephalomyelitis, are caused by metabolic dysregulation. More specifically, mitochondrial dysfunction and oxidative / nitrosative stress, including altered ATP production and increased oxidative / nitrosative stress, are observed in patients with post-COVID-19 symptoms. Dextran sulfate, according to embodiments, can attenuate the effects of such metabolic dysregulation and oxidative / nitrosative stress by normalizing metabolism and mitochondrial function (Figures 4A-4D, 27-28; Tables 5-8, 15-18).

[0032] Dextran sulfate of the embodiment was effective in restoring normal mitochondrial-associated energy metabolism and had a favorable effect on the concentration of purine triphosphate and pyrimidine nucleotides. In fact, dextran sulfate treatment could nearly normalize ATP levels and NAA concentrations in injured tissues compared with healthy subjects.

[0033] Dextran sulfate of the present embodiment led to a significant reduction in oxidative / nitrosative stress. In particular, the levels of ascorbic acid, the major water-soluble brain antioxidant, and glutathione (GSH), the major intracellular sulfhydryl group (SH) donor, were significantly improved. In addition, the levels of malondialdehyde (MDA), an end product of polyunsaturated fatty acids in membrane phospholipids and therefore used as a marker of reactive oxygen species (ROS)-mediated lipid peroxidation, were significantly reduced after dextran sulfate administration. In addition, the total nitrite / nitrate levels in the injured tissues were also significantly reduced (Figure 30). All of the above oxidative / nitrosative stresses showed an improvement in the restoration of antioxidant / anti-nitrosative status after dextran sulfate treatment (Figures 29-31, 33-36).

[0034] Dextran sulfate of the embodiment also has antithrombotic, antifibrotic, and fibrolytic effects, thereby providing therapeutic benefits in Stage IV (recovery phase, FIG. 25) of the present invention. Dextran sulfate of the embodiment further induces metabolic normalization by improving mitochondrial function in the patient's cells.

[0035] Dextran sulfate of the embodiment has further medical benefits in Stage IV (recovery phase, FIG. 25) by reducing muscle degeneration and improving muscle function (FIGS. 38-42).

[0036] Emerging data indicate that COVID-19, and in particular, long-term COVID-19, can cause liver damage. Experimental data presented herein demonstrate that dextran sulfate of embodiments has a favorable effect on liver function and can normalize disturbed liver function (FIG. 43), which is beneficial in stage IV disease (FIG. 25).

[0037] Dextran sulfate of the embodiments also has beneficial effects during the recovery phase (Figure 25) by inducing a significant release of hepatocyte growth factor (HGF), which plays an important role in organ regeneration and wound healing. High levels of plasma HGF induced by dextran sulfate of the embodiments (Figures 2, 42) are beneficial to COVID-19 patients by inducing tissue repair and regeneration and wound healing of organs and tissues adversely affected by SARS-CoV-2 infection and the inflammatory response induced by SARS-CoV-2 infection.

[0038] An embodiment of the present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the prevention, suppression and / or treatment of coronavirus infection or infectious disease.

[0039] In certain embodiments, the coronavirus infectious disease is selected from the group consisting of MERS, SARS, and COVID-19. In certain embodiments, the coronavirus infectious disease is COVID-19.

[0040] In certain embodiments, the coronavirus infection is selected from the group consisting of coronavirus infections caused by a coronavirus selected from the group consisting of MERS-CoV, SARS-CoV, and SARS-CoV-2. In certain embodiments, the coronavirus infection is caused by SARS-CoV-2.

[0041] An embodiment of the present invention also relates to the use of dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention, suppression and / or treatment of a coronavirus infection or infectious disease.

[0042] The present invention relates to a method for preventing, inhibiting and / or treating a coronavirus infection or infectious disease, the method comprising administering to a subject suffering from or at risk of suffering from a coronavirus infection or infectious disease an effective amount of dextran sulfate, or a pharmaceutically acceptable salt thereof.

[0043] As used herein, treating a coronavirus infection or infectious disease does not necessarily mean curative treatment of the coronavirus infection or infectious disease, but also encompasses suppressing or reducing short-term and long-term symptoms of the coronavirus infection or infectious disease. Thus, treatment also encompasses delaying the onset of the coronavirus infection or infectious disease, including preventing the onset of symptoms associated with the coronavirus infection or infectious disease or resolving existing symptoms.

[0044] Coronaviruses are not the only pathogens that use the cell surface glycosaminoglycan heparan sulfate (HSPG) as a receptor. In stark contrast, HSPGs also function as receptors for several viruses, as well as bacterial and parasitic pathogens, as shown in Table 1 below (Bartlett and Woo Park, Heparan Sulfate Proteoglycans in Infection, Glycans in Diseases and Therapeutics 2011: Chapter 2: 31-62, MSG Pavao (ed.)). [Table 1] TIFF0007788730000002.tif242162TIFF0007788730000003.tif218162

[0045] Thus, dextran sulfate embodiments can also be used to prevent, inhibit, and / or treat pathogen infections or infectious diseases caused by pathogens capable of binding to cell surface heparan sulfate proteoglycans (HSPGs), particularly those capable of binding to HSPGs to facilitate initial cellular binding and / or subsequent internalization.

[0046] Another aspect of the present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the prevention, inhibition, and / or treatment of infection or infectious disease caused by pathogens capable of binding to cell surface heparan sulfate proteoglycans (HSPGs).

[0047] In certain embodiments, pathogens can bind to HSPGs to facilitate initial cellular binding and / or subsequent internalization.

[0048] In certain embodiments, the pathogen is capable of binding to the heparan sulfate portion of the HSPG.

[0049] In certain embodiments, the pathogen is a bacterium, virus, prion, or parasite selected from Table 1. In certain embodiments, the pathogen is other than human immunodeficiency virus (HIV), e.g., other than HIV-1 and HIV-2.

[0050] In certain embodiments, the pathogen is a bacterium selected from the group consisting of Bacillus anthracis, Bacillus cereus, Borrelia burgdorferi, Bordetella pertussis, Chlamydia pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria meningitidis, Rickettsia tsutsugamushi, Pseudomonas gingivalis, Pseudomonas aeruginosa, Staphylococcus aureus, Group B Streptococcus, Streptococcus pyogenes, Streptococcus pneumoniae, and Salmonella enterocolitica.

[0051] Bacillus anthracis causes anthrax; Bacillus cereus causes nausea, vomiting, and diarrhea; Borrelia causes Lyme disease; Bordetella pertussis causes whooping cough; Chlamydia pneumoniae causes pneumonia; Chlamydia trachomatis causes chlamydia; Haemophilus influenzae causes bacteremia, pneumonia, epiglottitis, acute bacterial meningitis, cellulitis, osteomyelitis, and infectious arthritis; Helicobacter pylori causes gastritis and ulcers, but also a wide range of other diseases, such as idiopathic thrombocytopenic purpura, iron deficiency anemia, and atherosclerotic arthritis. Sclerosis, Alzheimer's disease, multiple sclerosis, coronary artery disease, periodontitis, Parkinson's disease, Guillain-Barré syndrome, rosacea, psoriasis, chronic urticaria, spot baldness, various autoimmune skin diseases, Henoch-Schönlein purpura, low blood levels of vitamin B12, autoimmune neutropenia, antiphospholipid syndrome, plasma cell hyperplasia, reactive arthritis, central chorioretinitis, open-angle glaucoma, blepharitis, diabetes, metabolic syndrome, various types of allergies, non-alcoholic fatty liver disease have been associated with liver disease, nonalcoholic steatohepatitis, liver fibrosis, and liver cancer; Listeria monocytogenes causes listeriosis; Mycobacterium tuberculosis causes tuberculosis; Neisseria gonorrhoeae causes gonorrhea; Neisseria meningitidis causes meningitis and other forms of meningococcal disease, including meningococcemia and life-threatening sepsis; Rickettsia tsutsugamushi causes scrub typhus; Porphyromonas gingivalis causes periodontal disease in the upper gastrointestinal tract, respiratory tract, and colon as well as in Alzheimer's disease and rheumatoid arthritis. Pseudomonas aeruginosa causes pneumonia and various septic conditions; Staphylococcus aureus causes membrane infections, including respiratory infections such as abscesses and sinusitis, and food poisoning; Group B Streptococcus causes neonatal infections, including neonatal sepsis, pneumonia, and meningitis; Streptococcus pyogenes causes membrane infections, neonatal infections, but also rheumatic fever, acute post-infectious glomerulonephritis, and PANDAS; Streptococcus pneumoniae causes pneumonia, and Yersinia enterocolitica causes Yersinia infections.

[0052] In certain embodiments, the pathogen is a virus selected from the group consisting of adeno-associated virus type 2 (AAV2), adenovirus, coronavirus, coxsackievirus, cytomegalovirus, dengue virus (DENV), foot-and-mouth disease virus (FMDV), herpes simplex virus 1 (HSV-1) and HSV-2, hepatitis B virus, hepatitis C virus, human gammaherpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus (KSHV)), human immunodeficiency virus 1 (HIV-1), human papillomavirus (HPV), human T-cell lymphotropic virus type 1 (HTLV1), Japanese encephalitis virus (JEV), pseudorabies virus, respiratory syncytial virus (RSV), rhinovirus, Sindbis virus (SINV), vaccinia virus (VACV), West Nile virus (WNV), and yellow fever virus.

[0053] In certain embodiments, the pathogen is a virus selected from the group consisting of AAV2, adenovirus, coronavirus, coxsackievirus, cytomegalovirus, DENV, FMDV, HSV-1 and HSV-2, hepatitis B virus, hepatitis C virus, HHV-8, HPV, HTLV1, JEV, pseudorabies virus, RSV, rhinovirus, SINV, VACV, WNV, and yellow fever virus.

[0054] Coxsackievirus causes aseptic meningitis; cytomegalovirus causes pneumonia; DENV causes dengue fever; FMDV causes foot-and-mouth disease; HSV causes herpes labialis, genital herpes, and contact infections; hepatitis B virus causes hepatitis B; hepatitis C virus causes hepatitis C, hepatocellular carcinoma, and lymphoma; HHV-8 causes Kaposi's sarcoma, primary effusion lymphoma, HHV-8-associated multicentric Castleman disease, and KSHV inflammatory cytokine syndrome. HPV causes precancerous lesions, genital warts, and laryngeal papillomatosis; HTLV-1 causes adult T-cell lymphoma (ATL), HTLV-1-associated myelopathy, uveitis, and Strongyloides stercoralis hyperinfection; JEV causes Japanese encephalitis; pseudorabies virus causes Aujeszky's disease; RSV causes respiratory tract infections, including bronchiolitis and pneumonia; rhinovirus causes the common cold; SINV causes Sindbis fever; WNV causes West Nile fever; and yellow fever virus causes yellow fever.

[0055] In certain embodiments, the pathogen is a parasite selected from the group consisting of Giardia lamblia, Leishmania spp., Encephalitozoon spp., Neospora caninum, Plasmodium spp., Toxoplasma gondii, and Trypanosoma cruzi.

[0056] Giardia lamblia causes giardiasis; Leishmania causes leishmaniasis; Encephalitozoon spp. causes microsporidiosis; Neospora caninum causes spontaneous abortion in infected livestock; Plasmodium spp. causes malaria; Toxoplasma gondii causes toxoplasmosis; and Trypanosoma cruzi causes Chagas disease in humans, dourine and slough in horses, and a brucellosis-like disease in cattle.

[0057] The present invention also relates to the use of dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention, inhibition and / or treatment of an infection or infectious disease caused by a pathogen capable of binding to cell surface HSPGs.

[0058] The present invention further relates to a method for preventing, inhibiting and / or treating an infection or infectious disease caused by a pathogen capable of binding to cell surface HSPGs, the method comprising administering an effective amount of dextran sulfate, or a pharmaceutically acceptable salt thereof, to a subject suffering from or at risk of suffering from an infection or infectious disease caused by a pathogen capable of binding to cell surface HSPGs.

[0059] Treatment of an infection or infectious disease caused by a pathogen capable of binding to cell surface HSPG does not necessarily mean curative treatment of the infection or infectious disease, but also encompasses inhibiting or reducing symptoms associated with the infection or infectious disease or elimination of existing symptoms. Thus, treatment also encompasses delaying the onset of the infection or infectious disease, including delaying the onset of symptoms associated with the infection or infectious disease.

[0060] ARDS is a form of respiratory failure characterized by the rapid onset of widespread inflammation throughout the lungs. ARDS can be caused by sepsis, pancreatitis, trauma, pneumonia, and aspiration. The underlying mechanisms involve diffuse damage to the cells that form the barrier of the lung's microscopic air sacs, surfactant dysfunction, immune system activation, and dysfunction of the body's regulation of blood clotting. In essence, ARDS impairs the lung's ability to exchange oxygen and carbon dioxide. Adult diagnosis is based on a PaO2 / FiO2 ratio (the ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen) of less than 300 mm Hg, despite positive end-expiratory pressure ventilation (PEEP) exceeding 5 cm H2O.

[0061] SIRS is an inflammatory condition that affects the entire body. It is the body's response to an infectious or non-infectious insult. SIRS is often a complication of failure of one or more organs or organ systems and can lead to acute kidney injury, shock, and multiple organ dysfunction syndrome.

[0062] SIRS is a severe condition associated with systemic inflammation, organ damage, and organ failure. It is a subset of cytokine storm, in which there is abnormal regulation of various cytokines. SIRS is also closely related to sepsis, in which patients meet the criteria for SIRS and have suspected or documented infection. Symptoms of SIRS include a temperature below 36°C or above 38°C, a heart rate above 90 beats / min, tachypnea (high respiratory rate) with a respiratory rate above 20 breaths / min, or an arterial partial pressure of carbon dioxide below 4.3 kPa (32 mmHg), 4000 cells / mm3, or 1000 cells / mm3. 3 (4x10 9 cells / L) or less than 12,000 cells / mm 3 (12x10 9 These include the presence of more than 10% immature neutrophils (band-shaped nuclei) or more than 10% white blood cells (>100 cells / L). If two or more of these criteria are met, with or without evidence of infection, a patient can be diagnosed with SIRS. Patients with SIRS and acute organ failure are referred to as having severe SIRS.

[0063] Dextran sulfate of the embodiments has been shown to be anti-inflammatory and therefore may resolve or at least reduce the cytokine storm and inflammation associated with ARDS and SIRS in subjects (Figures 17A, 17D, 18A, 19-23).

[0064] The anti-inflammatory effects of dextran sulfate are selective in terms of its action on specific cells of the immune system, selectively reducing pro-inflammatory cytokines released by such cells. More specifically, the experimental data presented herein demonstrate that dextran sulfate specifically targets monocytes and T lymphocytes, resulting in a concentration-dependent, significant reduction in IL-6, IL-10, TNFα, and IFNγ by activated monocytes and in IL-6, IL-10, IL-1β, IL-8, TNFα, and IFNγ by activated T lymphocytes (Figures 17-23). ​​A significant advantage of dextran sulfate's reduction of pro-inflammatory cytokines over, for example, dexamethasone and other steroids is that it does not completely block cytokines and does not affect all cytokine production by the immune system. In infectious diseases such as coronavirus infection, controlled immune system activation is necessary to combat the infection. Dextran sulfate embodiments can achieve such controlled activation by reducing, distinct from, the blockage of select pro-inflammatory cytokines from selected cells of the immune system, which reduces the risk of developing ARDS and SIRS.

[0065] A further aspect of the present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the prevention, suppression or treatment of an inflammatory disease selected from the group consisting of ARDS and SIRS.

[0066] In one embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof is for the prevention, suppression, or treatment of ARDS.

[0067] In another embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof is for the prevention, suppression or treatment of SIRS.

[0068] In certain embodiments, the inflammatory disease is caused by an infectious disease, such as a coronavirus infection or an infection with another HSPG-binding pathogen.

[0069] The present invention also relates to the use of dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention, suppression and / or treatment of an inflammatory disease selected from the group consisting of ARDS and SIRS.

[0070] The present invention further relates to a method for preventing, suppressing and / or treating an inflammatory disease selected from the group consisting of ARDS and SIRS, the method comprising administering an effective amount of dextran sulfate, or a pharmaceutically acceptable salt thereof, to a subject suffering from or at risk of suffering from an inflammatory disease selected from the group consisting of ARDS and SIRS.

[0071] As used herein, the treatment of inflammatory disease selected from the group consisting of ARDS and SIRS does not necessarily mean the curative treatment of inflammatory disease, but also includes the suppression or reduction of the short-term and long-term symptoms of inflammatory disease.Therefore, treatment also includes the delay in the occurrence of the symptoms associated with this inflammatory disease, and the delay in the occurrence of infectious disease or infectious disease, including the elimination of the long-term pathology such as pre-existing fibroproliferative condition, muscular disease and chronic fatigue condition after this disease.

[0072] The present invention also relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the prevention, suppression or treatment of the long-term effects of symptoms of COVID-19 caused by SARS-CoV-2 infection.

[0073] The long-term effects of COVID-19 symptoms include damage to target organs, particularly the heart, liver, and lungs. Imaging studies performed months after recovery from COVID-19 have shown persistent damage to the myocardium, even in individuals who experienced only mild COVID-19 symptoms. Furthermore, COVID-19 can cause long-term damage to the small air sacs (alveoli) in the lungs. The resulting scar tissue can lead to long-term respiratory problems. A high percentage of hospitalized COVID-19 patients have abnormal liver function. Common long-term symptoms are primarily fatigue, shortness of breath, cough, palpitations, and olfactory dysfunction. Other symptoms include chest pain, muscle and joint pain, weight loss, and gastrointestinal problems. Reported clinical measures of long-term effects in COVID-19 patients include pulmonary dysfunction and injury, cardiovascular effects such as myocardial inflammation, brain changes, and olfactory and gestational disorders (anosmia and hyposmia).

[0074] The present invention also relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention, suppression and / or treatment of the long-term effects of symptoms of COVID-19 caused by SARS-CoV-2 infection.

[0075] The present invention further relates to a method for preventing, suppressing and / or treating long-term effects of COVID-19 symptoms caused by SARS-CoV-2, comprising administering an effective amount of dextran sulfate, or a pharmaceutically acceptable salt thereof, to a subject suffering from or at risk of suffering from long-term effects of COVID-19 symptoms caused by SARS-CoV-2 infection.

[0076] In the following, the (average) molecular weight and sulfur content of dextran sulfate mentioned also apply to any pharmaceutically acceptable salt of dextran sulfate. Thus, the pharmaceutically acceptable salt of dextran sulfate preferably has the average molecular weight and sulfur content discussed in the following embodiments.

[0077] Dextran sulfate outside the preferred range of embodiments is believed to result in less efficacy and / or negative side effects on cells or subjects.

[0078] For example, dextran sulfates with molecular weights greater than 10,000 Da (10 kDa) typically have a lower efficacy-to-side effect profile than dextran sulfates with lower molecular weights. This means that the maximum dose of dextran sulfate that can be safely administered to a subject is lower for larger dextran sulfate molecules (>10,000 Da) than for dextran sulfate molecules with average molecular weights within the preferred range. As a result, when dextran sulfate is to be administered to a subject in vivo, such larger dextran sulfate molecules are less suitable for clinical use.

[0079] Dextran sulfate is a sulfated polysaccharide, particularly a sulfated glucan, i.e., a polysaccharide made from many glucose molecules. The average molecular weight defined herein indicates that individual sulfated polysaccharides may have molecular weights different from this average molecular weight, but the average molecular weight represents the average molecular weight of the 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.

[0080] 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.

[0081] Weight average molecular weight (M w ):

number

[0082] In one embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M of 10,000 Da or less. w In certain embodiments, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 2,000 Da to 10,000 Da. w It has.

[0083] In another embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 2,500 Da to 10,000 Da, preferably in the range of 3,000 Da to 10,000 Da. w In certain embodiments, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 3,500 Da to 9,500 Da, for example, in the range of 3,500 Da to 8,000 Da. w It has.

[0084] In another specific embodiment, the dextran sulfate or a pharmaceutically acceptable salt 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 6,500 Da or in the range of 4,500 Da to 5,500 Da. w It has.

[0085] Thus, in some embodiments, dextran sulfate or a pharmaceutically acceptable salt thereof has an M of 10,000 Da or less, 9,500 Da or less, 9,000 Da or less, 8,500 Da or less, 8,000 Da or less, 7,500 Da or less, 7,000 Da or less, 6,500 Da or less, 6,000 Da or less, or 5,500 Da or less. w It has.

[0086] In some embodiments, the dextran sulfate or pharmaceutically acceptable salt thereof has an M of 1,000 Da or more, 1,500 Da or more, 2,000 Da or more, 2,500 Da or more, 3,000 Da or more, 3,500 Da or more, 4,000 Da or more, or 4,500 Da or more. w Any of these embodiments may be used in combination with any of the above-presented M w and these may be combined with an upper limit of 10,000 Da or less.

[0087] In certain embodiments, the M of dextran sulfate or a pharmaceutically acceptable salt 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.

[0088] Number average molecular weight (M n ):

number

[0089] In one embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M as measured by NMR spectroscopy in the range of 1,850 to 3,500 Da. n It has.

[0090] In certain embodiments, dextran sulfate or a pharmaceutically acceptable salt thereof has an M as measured by NMR spectroscopy in the range of 1,850 Da to 2,500 Da, preferably in the range of 1,850 Da to 2,300 Da, for example in the range of 1,850 Da to 2,000 Da. n It has.

[0091] Thus, in some embodiments, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M of 3,500 Da or less, 3,250 Da or less, 3,000 Da or less, 2,750 Da or less, 2,500 Da or less, 2,250 Da or less, or 2,000 Da or less. n Furthermore, dextran sulfate or a pharmaceutically acceptable salt thereof has an M of 1,850 Da or more. n It has.

[0092] In one embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of sulfates per glucose unit in the range of 2.5 to 3.0.

[0093] In certain embodiments, dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of sulfates per glucose unit in the range of 2.5 to 2.8, preferably in the range of 2.6 to 2.7.

[0094] In one embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of glucose units in the range of 4.0 to 6.0.

[0095] In certain embodiments, dextran sulfate or a pharmaceutically acceptable salt 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.

[0096] In one embodiment, dextran sulfate or a pharmaceutically acceptable salt thereof has an Mn as measured by NMR spectroscopy in the range of 1,850 to 3,500 Da, an average number of sulfates per glucose unit in the range of 2.5 to 3.0, and an average sulfation at the C2 position in the glucose units of the dextran sulfate of at least 90%.

[0097] In one embodiment, the dextran sulfate has an average glucose unit number of about 5.1, an average sulfate number per glucose unit in the range of 2.6 to 2.7, and an M in the range of 1,850 Da to 2,000 Da. n It has.

[0098] In one embodiment, the pharmaceutically acceptable salt of dextran sulfate is sodium dextran sulfate. In a specific embodiment, the sodium dextran sulfate has an average glucose unit count of about 5.1, an average sulfate count per glucose unit in the range of 2.6 to 2.7, and a Na concentration in the range of 2,100 Da to 2,300 Da. + M containing counter ions n It has.

[0099] In one embodiment, the dextran sulfate has an average glucose unit count of 5.1, an average sulfate count per glucose unit of 2.7, and a Na concentration of about 1,900-1,950 Da as measured by NMR spectroscopy. + Excluded average M n and Na by NMR spectroscopy measurement of approximately 2,200-2,250 Da. + Content average M n It has.

[0100] Dextran sulfate according to embodiments can be provided as a pharmaceutically acceptable salt of dextran sulfate, for example, the sodium salt or potassium salt.

[0101] Presently preferred dextran sulfates according to the claimed embodiments are disclosed in WO 2016 / 076780.

[0102] The subject is preferably a mammalian subject, more preferably a primate, and especially a human subject. However, dextran sulfate or a pharmaceutically acceptable salt thereof can also be used for veterinary purposes. Non-limiting examples of animal subjects include primates, cats, dogs, pigs, horses, mice, and rats.

[0103] Dextran sulfate or a pharmaceutically acceptable salt thereof is preferably administered to a subject by injection, particularly 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 in addition, dextran sulfate or a pharmaceutically acceptable derivative thereof may be injected, for example, directly into a tissue or organ or other site in the subject's body where the desired effect is to occur.

[0104] The dextran sulfate or a pharmaceutically acceptable salt thereof of the embodiment is preferably formulated as an aqueous injection solution using a selected solvent or excipient. The solvent is conveniently an aqueous solvent, particularly a buffer solution. Non-limiting examples of such buffer solutions include citrate buffers, such as citrate monohydrate (CAM) buffer, or phosphate buffers. For example, the dextran sulfate of the embodiment can be dissolved in saline, such as 0.9% NaCl saline, and then optionally buffered with 75 mM CAM and adjusted to a pH of about 5.9 using sodium hydroxide. Non-buffered solutions are also possible, including aqueous injection solutions such as saline, i.e., NaCl (aqueous). Furthermore, if a buffer solution is desired, buffer systems other than CAM can be used.

[0105] Embodiments are not limited to injection, and other routes of administration can alternatively be used, including oral, nasal, buccal, rectal, transdermal, transbronchial, or topical. The active compound dextran sulfate is then formulated with suitable excipients or carriers, which are selected based on the particular route of administration.

[0106] Suitable dosage ranges for dextran sulfate or a pharmaceutically acceptable salt thereof can vary depending on the application, e.g., in vitro versus in vivo, the size and weight of the subject, the condition being treated, and other considerations. For human subjects in particular, possible dosage ranges would be 1 μg / kg to 100 mg / kg body weight, preferably 10 μg / kg to 50 mg / kg body weight.

[0107] In a preferred embodiment, dextran sulfate or a pharmaceutically acceptable salt thereof is formulated to be administered at a dosage of 0.05 to 50 mg / kg body weight of a subject, preferably 0.05 or 0.1 to 40 mg / kg body weight of a subject, and more preferably 0.05 or 0.1 to 30 mg / kg body weight of a subject, or 0.1 to 25 mg / kg body weight of a subject, or 0.1 to 15 mg / kg body weight of a subject, or 0.1 to 10 mg / kg body weight of a subject. Preferred dosages are selected from the range of 0.25 to 5 mg / kg body weight, preferably 0.5 to 2.5 mg / kg body weight, and more preferably 0.75 to 2 mg / kg body weight of a subject.

[0108] Dextran sulfate or a pharmaceutically acceptable salt thereof can be administered in a single dose, such as in the form of a single bolus injection, which can be very rapidly infused into a subject, but is conveniently infused over a period of time, such that the dextran sulfate solution is infused into the patient over several minutes, such as 5-10 minutes.

[0109] Alternatively, dextran sulfate or a pharmaceutically acceptable salt thereof can be administered multiple times, ie, at least twice, during the treatment period.

[0110] Dextran sulfate or a pharmaceutically acceptable salt thereof can be administered sequentially, simultaneously, or in the form of a composition containing dextran sulfate or a pharmaceutically acceptable salt thereof and at least one other active agent. The at least one active agent can be selected from any agent useful for any of the above diseases, disorders, or conditions. The at least one active agent can also be in the form of cells for cell therapy, such as stem cells, including, but not limited to, embryonic stem cells (ESCs) and mesenchymal stromal cells (MSCs).

[0111] Example In the following examples, the sodium salt of dextran sulfate, designated herein as low molecular weight dextran sulfate (LMW-DS), was used (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780). Example 1 Binding to host tissues is a critical step for coronavirus entry and dissemination, therefore, disruption of host-pathogen protein-protein interactions may be an effective way to inhibit coronavirus entry.

[0112] This study investigated the effect of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780) on oligomeric β-amyloid and pathogenic protein PrP. C We investigated its ability to inhibit protein-protein interactions between α- and β-glucan, and revealed its potential to inhibit protein-protein interactions.

[0113] material and method Chemicals and antibodies Streptavidin-HRP was obtained from BioLegend; β-amyloid-(1-42)-biotin was obtained from Innovagen; normal human cellular prion protein (PrP C) from Merck; TMB from eBioscience; 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) from Sigma; anti-amyloid beta antibody clone 6E10 from BioLegend; anti-mouse HRP from Cell Signaling; dextran sulfate sodium salt (DSSS) with an average MW >500,000 Da from Sigma; dextran (MW 450,000-650,000 Da) from Sigma; Maxisorp plates from Sigma.

[0114] Preparation of amyloid beta oligomers The oligomerization of β-amyloid was optimized based on previous methods (Stine et al., Methods Mol. Biol. 2011, 670:13-32; Aimi et al., J. Neurochem. 2015, 134:611-617). Briefly, amyloid β was dissolved in HFIP to a final concentration of 1.0 mM, subjected to protective sonication, and the HFIP was carefully evaporated. The resulting peptide film was stored in a sealed container at -20°C. Prior to use, the peptide film was slowly dissolved in DMSO to a final concentration of 5.0 mM and subjected to protective sonication for 10 minutes. To prepare oligomers, the DMSO solution was diluted in ice-cold DMEM medium to a final concentration of 100 μM and incubated at 37°C for 16 hours (β-amyloid-biotin). To prepare monomers, the DMSO solution was diluted in ice-cold 18 M Ohm water to a final concentration of 100 μM and used immediately.

[0115] Identification of amyloid beta monomers and oligomers Preparations optimized for the generation of amyloid-β monomers or oligomers were solubilized in 5% SDS-containing gel sample buffer without reducing agents. Proteins were measured on a 15% Bis-Tris gel using MES running buffer without reducing agents. The gels were transferred to PVDF, blocked in 10% nonfat milk, and then incubated with anti-amyloid-β antibody overnight at 4°C. Then, the gels were developed with anti-mouse HRP followed by ECL and exposed to film.

[0116] Oligomeric amyloid beta and PrP C ELISA method to quantify protein-protein interactions between PrP was diluted to 10x coating volume in carbonate coating buffer (100 μl; 500 ng PrP per well). C (final volume of 1000 μl). 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 β-biotin peptide preparation (final concentration 200 nM) was carefully mixed with the test compound and added to each well. The plate was incubated for 60 minutes at room temperature, washed, treated with streptavidin-HRP, and after further washing, developed with TMB (the reaction was stopped with 2N H2SO4). The absorbance was read at 450 nm within 30 minutes. All conditions were performed in triplicate. C Amyloid β-biotin binding to β was calculated as described by Aimi et al., J Neurochem. 2015, 134:611-617.

[0117] Curve fitting Quantitative pharmacological analysis was performed by iterative curve fitting to a floating four-parameter logistic equation.

[0118] result DSSS competed for the protein-protein interaction between oligomeric Aβ and PrP in a concentration-dependent manner, similar to LMW-DS (Fig. 1; Table 2). Quantitative pharmacological analysis showed that, despite clear differences in competitive binding and Hill coefficients at head-to-head levels, LMW-DS exhibited a similar overall affinity to DSSS, suggesting a different interaction between the two compounds (Fig. 1; Table 2). In contrast to DSSS and LMW-DS, dextran inhibited the binding of oligomeric Aβ and PrP. C The protein-protein interaction between the two proteins could not be clearly competed. [Table 2]

[0119] Consideration High molecular weight dextran sulfate (DSSS) binds oligomeric amyloid beta and PrP C reported that protein-protein competition between amyloid-β and amyloid-β occurs at effective concentrations in the low μg / ml range (Aimi et al., J Neurochem. 2015, 134:611-617). In this study, optimization of the method resulted in a significantly higher proportion of oligomeric amyloid-β compared to the Aimi et al. study. Optimization of the protein-protein interaction ELISA resulted in a greater degree of specific protein-protein interaction; the larger dynamic range of competition facilitated quantitative pharmacological analysis of the interaction with competing compounds. Thus, this study represents an improvement over the study reported by Aimi et al.

[0120] DSSS and LMW-DS express oligomeric amyloid beta and PrP C and showed comparable affinity for protein-protein interactions with IC values ​​of 0.62 ± 0.07 and 0.42 ± 0.16 μg / mL, respectively. 50 Hill analysis of the nature of the competition showed that LMW-DS exhibited a shallower competition curve compared to the relatively high Hill coefficients associated with DSSS, providing evidence for different pharmacological actions between DSSS and LMW-DS.

[0121] Therefore, LMW-DS can be used to compete for the protein-protein interaction between oligomeric amyloid beta and PrP, thereby preventing or at least inhibiting the protein-protein interaction. This effect observed with LMW-DS has potential in coronavirus infections and coronavirus infectious diseases involving protein-protein interactions. Therefore, LMW-DS has the potential to disrupt the interaction between the SARS-CoV-2 trimer spike protein and ACE2. This is further demonstrated in Example 13.

[0122] Example 2 This example investigated the potential of LMW-DS to enhance the release and activation of tissue repair growth factors in the blood of ALS patients.

[0123] material and method This was a single-center, single-arm, open-label study evaluating the safety, tolerability, and efficacy of subcutaneously (sc) administered LMW-DS in patients with ALS. There were 10 planned clinic visits: one two-part screening visit (Visit 1a and Visit 1b), five investigational medication (IMP) administration visits (Visits 2 through 6), and three follow-up visits (Visits 7 through 9). Each individual patient's study participation, including the screening and follow-up visits, was planned to last approximately 4 months.

[0124] The active pharmaceutical ingredient of IMP was low-molecular-weight dextran sulfate (LMW-DS) (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780). The drug was used as a solution for subcutaneous injection and consisted of 20 mg / mL LMW-DS and 9 mg / mL NaCl. Each glass vial contained 10 mL. The administered dose depended on the patient's weight at Visit 2, prior to the first LMW-DS administration. LMW-DS was injected subcutaneously into alternating sides of the abdomen, thigh, or buttocks (in that order of preference). Five 1 mg / kg injections were administered with a one-week dosing interval. Changes in immunoreactive HGF were measured from blood samples collected at each visit using a commercially available ELISA (R&D HGF ELISA).

[0125] result LMW-DS induced a significant increase in plasma HGF levels in ALS patients, with a maximum peak at 2 hours after LMW-DS administration (Fig. 2, Table 3), and this level decreased but was still significantly higher at 6 hours after LMW-DS administration (Table 3). [Table 3]

[0126] Consideration HGF, sometimes called scatter factor (SF), is a paracrine cell growth, motility, and morphogenesis factor that has been shown to play a major role in embryonic organ development, as well as in adult organ regeneration and wound healing.

[0127] High levels of plasma HGF induced by LMW-DS may benefit COVID-19 patients by inducing tissue repair and regeneration and wound healing in organs and tissues adversely affected by SARS-CoV-2 infection and the inflammatory response induced by SARS-CoV-2 infection, including ARDS, SIRS, and / or organ fibrosis.

[0128] Example 3 The effects of daily subcutaneous injections of LMW-DS on glutamate excitotoxicity and mitochondrial function in rats after severe traumatic brain injury (sTBI) were assessed by high-performance liquid chromatography (HPLC) analysis of frozen brain samples. The results suggest that LMW-DS interferes with mitochondrial function, improving energy metabolism and reducing glutamate excitotoxicity.

[0129] material and method sTBI induction and drug administration protocol The experimental protocol used in this study was approved by the Ethics Committee of the Catholic University of Rome in accordance with international standards and guidelines for animal care. Male Wistar rats weighing 300–350 g body weight (bw) were kept in a controlled environment and provided with standard laboratory chow and water ad libitum. The rats were divided into three groups: 1) n = 6 animals received sTBI, received drug administration 30 min later, and were sacrificed 2 days after TBI (acute phase 1). 2) n = 6 animals received sTBI, drug administration 30 min later, and sacrificed 7 days after TBI (acute phase 2). 3) n = 6 animals received sTBI, received drug treatment 3 days after TBI, and were sacrificed 7 days after TBI (chronic phase).

[0130] Animals received an anesthetic mixture of 35 mg / kg body weight ketamine and 0.25 mg / kg body weight midazolam via intraperitoneal injection. According to the "weight-drop" impact acceleration model (Marmarou et al., J Neurosurg. 1994;80:291-300), sTBI was induced by dropping a 450 g weight from a height of 2 m onto the head of rats protected by a metal disk previously fixed on the skull. Rats that suffered skull fractures, seizures, epistaxis, or did not survive the impact were excluded from the study. At the end of each treatment period, rats were re-anesthetized and then immediately sacrificed.

[0131] Drug treatment was administered by subcutaneous injection of 0.5 ml of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780; 15 mg / kg) according to the protocol outlined above.

[0132] Brain tissue processing In all animals, an in vivo osteotomy craniotomy was performed during 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., Neurosurgery. 2005;56:582-589; Vagnozzi et al., Neurosurgery. 2007;61:390-395; Amorini et al., J Cell Mol Med. 2017;21:530-542). Briefly, whole brain homogenates were homogenized with 7 ml of ice-cold, nitrogen-saturated precipitation solution (CHCN + 10 mM KHPO, pH 7.40 (3:1; v:v)) using an Ultra-Turrax set (Janke & The centrifugation was performed at 24,000 rpm (Kunkel, Staufen, Germany). After centrifugation at 20,690 x g for 10 min at 4°C, the clear supernatant was saved, and the pellet was supplemented with 3 ml of precipitation solution and homogenized again as described above. A second centrifugation was performed (20,690 x g for 10 min at 4°C), the pellet was saved, and the supernatant was combined with the previous one and extracted by vigorous shaking with twice the volume of HPLC-grade CHCl3, followed by centrifugation as described above. The upper aqueous phase, containing water-soluble low-molecular-weight compounds, was collected and subjected to two further chloroform washes (this procedure allows for 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, which was stored at -80°C until assayed.

[0133] HPLC analysis of purine-pyrimidine metabolites An aliquot of each deproteinized tissue sample was filtered through a 0.45 μm HV Millipore filter and loaded (200 μl) onto a Hypersil C-18, 250 x 4.6 mm, 5 μm particle size column (Thermo Fisher Scientific, Rodano, Milan, Italy) with its own guard column. The column was then coupled to a HPLC system consisting of a Surveyor System (Thermo Fisher Scientific, Rodano, Milan, Italy) equipped with a high-sensitivity diode array detector (equipped with a 5 cm optical path flow cell) set at a wavelength of 200-300 nm. Data acquisition and analysis were performed on a PC using the ChromQuest software package provided by the HPLC manufacturer.

[0134] Metabolites belonging to the purine-pyrimidine profile (described below) and related to tissue energy status, mitochondrial function, and oxidative-nitrosative stress were separated in a single chromatographic run according to a slightly modified, established ion-pair HPLC method (Lazzarino et al., Anal. Biochem. 2003;322:51-59; Tavazzi et al., Clin. Biochem. 2005;38:997-1008). Assignment and calculation of compounds of interest in chromatographic runs of tissue extracts were performed by comparing the retention times, absorption spectra, and areas of the peaks at appropriate wavelengths (206, 234, and 260 nm) with those of peaks in chromatographic runs of freshly prepared ultrapure standard mixtures of known concentrations.

[0135] 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 form (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).

[0136] HPLC analysis of free amino acids and amino group-containing compounds Simultaneous determination of primary free amino acids (FAAs) and amino-group-containing compounds (AGCCs) (described below) was performed using precolumn derivatization of samples with a mixture of orthophthalaldehyde (OPA) and 3-mercaptopropionic acid (MPA), as described in detail elsewhere (Amorini et al., J Cell Mol Med. 2017;21:530-542; Amorini et al., Mol Cell Biochem. 2012;359:205-216). Briefly, a derivatization mixture consisting of 25 mmol / L OPA, 1% MPA, and 237.5 mmol / L sodium borate, pH 9.8, was prepared daily and introduced into the autosampler. Automated 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 glutamate, a 20-fold dilution with HPLC-grade HO was performed prior to the derivatization procedure in deproteinized brain extracts and subsequent injection. Separation of OPA-AA and OPA-AGCC was carried out using two mobile phases (mobile phase A = 24 mmol / L CH3COONa + 24 mmol / L Na2HPO4 + 1% tetrahydrofuran + 0.1% trifluoroacetic acid, pH 6.5; mobile phase B = 40% CH3OH + 30% CH3CN + 30% HO) at a flow rate of 1.2 ml / min using an appropriate step gradient (Amorini et al., J Cell Mol Med. 2017;21:530-542; Amorini et al., Mol Cell Biochem. 2012;359:205-216).

[0137] 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.

[0138] 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).

[0139] statistical analysis Normal data distribution was tested using the Kolmogorov-Smirnov test. Differences between groups were estimated by two-way analysis of variance for repeated measures. Fisher's constrained least squares method was used as a post-hoc test. Only two-sided p values ​​less than 0.05 were considered statistically significant.

[0140] result The most obvious result of brain levels of 24 standard and nonstandard amino acids and primary amino group-containing compounds was that LMW-DS treatment significantly suppressed the sTBI-induced increase in glutamate (GLU) (Figure 3), thus ensuring a reduction in excitotoxicity resulting from excess of this compound.

[0141] However, this effect was only observed when the drug was administered early after injury (30 min after sTBI), and there was no effect on this excitotoxicity marker when LMW-DS was injected 3 days after sTBI. It is also worth emphasizing that LMW-DS had a significant beneficial effect on compounds involved in the so-called methyl cycle (Met, L-Cystat, SAH; see Table 4). [Table 4] TIFF0007788730000009.tif180162Table 3 lists the compounds in μmol / g (ww).

[0142] As can be seen in Table 5, LMW-DS positively affected various compounds related to energy metabolism and mitochondrial function. Of particular interest were the adenine nucleotide concentration and the ATP / ADP ratio as measures of mitochondrial phosphorylation capacity (Figures 4A-4D). [Table 5] TIFF0007788730000011.tif242162TIFF0007788730000012.tif242161TIFF0007788730000013.tif242162TIFF0007788730000014.tif50162Table 4 lists the compounds in nmol / g(ww). Significant changes in oxidized and reduced nicotine coenzymes were also observed (Figures 5A-5D).

[0143] Parameters related to oxidative stress were also measured, and a significant reduction in oxidative stress was detected after administration of LMW-DS. In particular, ascorbic acid, the major water-soluble brain antioxidant, and GSH, the major intracellular SH donor, were measured. The results, as shown in Table 5 and Figures 6A-6C, showed a significant improvement in their levels after administration of LMW-DS.

[0144] Additionally, MDA, a polyunsaturated fatty acid end product of membrane phospholipids and therefore a marker of ROS-mediated lipid peroxidation, was also measured. MDA levels were significantly reduced after administration of LMW-DS. After treatment with LMW-DS, all of the above oxidative stress markers demonstrated improved restoration of antioxidant status (Figures 6A-6C).

[0145] Indices representing NO-mediated nitrosative stress (nitrite and nitrate) were also analyzed. LMW-DS administration significantly reduced nitrate concentrations in both acute and chronic sTBI (Figure 7).

[0146] NAA is a brain-specific metabolite and a useful biochemical marker for monitoring deterioration or recovery after TBI. NAA is synthesized in neurons from aspartate and acetyl-CoA by aspartate N-acetyltransferase. To ensure NAA turnover, this molecule must travel between intracellular compartments to reach oligodendrocytes, where it is degraded to acetate and aspartate by aspartoacylase (ASPA). To ensure the availability of the substrate aspartate, upregulation of the catabolic enzymes ASPA and NAA decreases, and acetyl-CoA is an indicator of a metabolically impaired state. In this study, NAA and its substrate were measured after sTBI, and significant improvement in levels was observed after LMW-DS administration (Figures 8A-8C).

[0147] These effects on energy metabolites were particularly evident when animals received LMW-DS treatment early (30 min) after injury. It is important to note that the overall beneficial effects of LMW-DS were observed when animals were sacrificed 2 days after sTBI or 7 days after sTBI. In this group of animals, the overall recovery of metabolism related to AGCC and energy metabolites was more evident, suggesting a sustained positive effect of LMW-DS treatment on cerebral metabolism.

[0148] Consideration The data presented herein suggest that early administration of LMW-DS reverses adverse changes in metabolic homeostasis by reducing the level of glutamate excitotoxicity and protecting mitochondrial function.

[0149] More specifically, LMW-DS protects mitochondrial function and reduces oxidative stress, as evidenced by, among other things, improved restoration of antioxidant status, protection of mitochondrial ATP energy replenishment by protecting ATP production and metabolism, and normalization of mitochondrial phosphorylation capacity, all of which are induced by LMW-DS. As a result, LMW-DS can protect and preserve mitochondrial function in cells exposed to damage or disease, which is important for having functional cells capable of fighting infectious diseases. Metabolic normalization induced by LMW-DS is beneficial for COVID-19 patients during the pneumonia stage (Stage II) and recovery stage (Stage IV) (Figure 25).

[0150] Example 4 The aim of this study was to evaluate the potential neuroprotective effects of LMW-DS against biochemical, molecular, and histoanatomical damage produced by an experimental model of severe closed head diffuse traumatic brain injury (sTBI) in rats. In this study, 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.

[0151] material and method sTBI induction and drug administration protocol Male Wistar rats (n=160) weighing 300-350 g were used in this study. They were fed standard laboratory chow and water ad libitum in a controlled environment.

[0152] Animals received an intramuscular injection of an anesthetic mixture containing 35 mg / kg of ketamine and 0.25 mg / kg of midazolam. Diffuse sTBI was induced according to the "weight-drop" impact acceleration model established by Marmarou et al. J. Neurosurg. 1994, 80:291-300. This model produces diffuse axonal injury and can reproduce the physical and mechanical characteristics of human diffuse TBI.

[0153] Severe TBI was induced by dropping a 450 g weight from a height of 2 m onto the head of a rat protected by a helmet (a metal disk previously fixed on the skull with dental cement) to distribute the mechanical force evenly to the brain. The rat was placed in a prone position on a special polyurethane foam bed inserted into a special container; this foam dissipates most of the potential energy (from the mechanical force) and prevents any rebound of the animal after the impact, which could result in spinal cord injury.

[0154] 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, the rats were re-anesthetized and then immediately sacrificed. These time points correspond to the worst biochemical damage (2 days) or, in the case of a minimally damaged brain, full metabolic recovery (7 days).

[0155] Drug treatment was administered by subcutaneous injection of 0.5 ml of LMW-DS (ILB® Tikomed, Viken, Sweden, WO 2016 / 076780) at three different concentrations (1, 5, and 15 mg / kg body weight) according to the protocol outlined below. Sham-operated animals underwent the same anesthesia procedure except for TBI and served as a control group.

[0156] Experimental design To test the efficacy of three different concentrations of LMW-DS at two different time points after TBI, the rats used in this study were divided into four groups. As specified below, in each group, animals were subjected to a specific treatment for metabolic analysis, and other animals were used for histomorphometric testing, according to the procedures described below.

[0157] 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

[0158] group 2 Following the induction of sTBI and without pharmacological treatment, rats were divided into the following subgroups: 1. Twelve animals received sTBI and were sacrificed 2 days after TBI. 2. 12 animals received sTBI and were sacrificed 7 days after TBI. Four additional rats for each subgroup were used for histomorphometric studies. Total rats in this group: n=32.

[0159] group 3 Rats underwent sTBI induction, received a single dose of LMW-DS 30 minutes after TBI, and were sacrificed 2 days after TBI. The rats were divided into the following subgroups: 1. Twelve animals underwent sTBI induction and were treated with 5 mg / kg body weight of LMW-DS. 2. 12 animals that underwent sTBI induction and were treated with 5 mg / kg body weight of LMW-DS. 3. 12 animals that underwent sTBI induction and were treated with 15 mg / kg body weight of LMW-DS. Four additional rats for each subgroup were used for histomorphometric studies. Total rats in this group: n=48.

[0160] group 4 Rats underwent sTBI induction, received a single dose of LMW-DS 30 minutes after TBI, and were sacrificed 7 days after TBI. The rats were divided into the following subgroups: 1. Twelve animals underwent sTBI induction and were treated with 5 mg / kg body weight of LMW-DS. 2. 12 animals that underwent sTBI induction and were treated with 5 mg / kg body weight of LMW-DS. 3. 12 animals that underwent sTBI induction and were treated with 15 mg / kg body weight of LMW-DS. Four additional rats for each subgroup were used for histomorphometric studies. Total rats in this group: n=48.

[0161] group 5 Rats (n = 12) underwent sTBI and received repeated administration of the maximum dose of LMW-DS (15 mg / kg body weight) at 30 min, 3 days, and 5 days after TBI. They were then sacrificed 7 days after TBI. Four additional rats were used for histomorphometric studies. Total rats in this group: n = 16.

[0162] Brain tissue processing for biochemical and gene expression analyses To minimize metabolite loss, an in vivo osteotomy craniotomy was performed on all animals during anesthesia. The rat skull was carefully removed to expose the brain, which was then rapidly incised along the sagittal groove to separate the two hemispheres. The hemisphere reserved for biochemical analysis was freeze-clamped with aluminum tongs pre-cooled in liquid nitrogen and immersed in liquid nitrogen. A freeze-clamp procedure was implemented to accelerate tissue freezing, thereby minimizing the potential for metabolite loss.

[0163] The remaining hemisphere, reserved for molecular biology analysis, was placed in 5-10 volumes of RNAlater® solution (Invitrogen Life Technologies), an RNA stabilizing solution that stabilizes RNA and protects it from degradation. The brain samples were stored overnight at 4°C to allow the solution to completely penetrate the tissue.

[0164] Tissue homogenization for metabolite analysis was performed as follows: After wet weight (ww) determination, the frozen hemispheres were placed in 7 ml of ice-cold, nitrogen-saturated precipitation solution consisting of CH3CN + 10 mM KH2PO4, pH 7.40 (3:1; v:v) (1:10 w / v), and homogenization was performed using an Ultra-Turrax homogenizer set (Janke & Kunkel, Staufen, Germany) at 24,000 rpm / min. After centrifugation at 20,690 x g for 10 min at 4°C, the clear supernatant was saved, and the pellet was supplemented with an aliquot of 10 mM KH2PO4, homogenized again as described above, and stored overnight at -20°C to ensure complete recovery of the aqueous phase from the tissue. A second centrifugation was performed (20,690 × g, 10 min at 4 °C), and the supernatant was combined with the previous one and extracted by vigorous stirring with twice the volume of HPLC-grade CHCl3 and centrifuged as above. The upper aqueous phase (containing water-soluble low molecular weight compounds) was collected and subjected to two further chloroform washes (this procedure allows the removal of all organic solvents and any lipid-soluble compounds from the buffered tissue extract), and the volume was adjusted with 10 mM KH2PO4, pH 7.40, to obtain a final aqueous 10% tissue homogenate, which was stored at -80 °C until assayed.

[0165] HPLC analysis of energy metabolites, antioxidants and oxidative / nitrosative stress biomarkers An aliquot of each deproteinized tissue sample was filtered through a 0.45 μm HV Millipore filter and loaded (200 μl) onto a Hypersil C-18, 250 x 4.6 mm, 5 μm particle size column (Thermo Fisher Scientific, Rodano, Milan, Italy) with its own guard column. The column was then coupled to a HPLC system consisting of a Surveyor System (Thermo Fisher Scientific, Rodano, Milan, Italy) equipped with a high-sensitivity diode array detector (equipped with a 5 cm optical path flow cell) set at a wavelength of 200-300 nm. Data acquisition and analysis were performed on a PC using the ChromQuest software package provided by the HPLC manufacturer.

[0166] Metabolites related to tissue energy status, mitochondrial function, antioxidants, and metabolites specific to oxidative / nitrosative stress (described below) were separated in a single chromatographic run according to a slightly modified, existing ion-pair HPLC method (Lazzarino et al., Anal Biochem. 2003;322:51-59; Tavazzi et al., Clin Biochem. 2005;38:997-1008). Assignment and calculation of compounds of interest in chromatographic runs of tissue extracts were performed by comparing the retention times, absorption spectra, and areas of peaks with those of chromatographic runs of freshly prepared ultrapure standard mixtures of known concentrations using appropriate wavelengths (206, 234, and 260 nm).

[0167] 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-acetylglucosamine (UDP-GlcNac), UDP-N-acetylgalactosamine (UDP-GalNac), AMP, GDP-glucose, UDP, GDP, NADP + , ADP-ribose, CTP, ADP, UTP, GTP, NADH, ATP, NADPH, malonyl-CoA, coenzyme A (CoA-SH), acetyl-CoA, N-acetylaspartate (NAA).

[0168] HPLC analysis of free amino acids and amino group-containing compounds Simultaneous determination of primary free amino acids (FAAs) and amino-group-containing compounds (AGCCs) (described below) was performed using precolumn derivatization of the sample with a mixture of OPA and MPA as described by Amorini et al., J Cell Mol Med. 2017;21(3):530-542; Amorino et al., Mol Cell Biochem. 2012;359:205-216. Briefly, a derivatization mixture consisting of 25 mmol / L OPA, 1% MPA, and 237.5 mmol / L sodium borate, pH 9.8, was prepared daily and introduced into the autosampler. Automated precolumn derivatization of samples (15 μl) with OPA-MPA was performed at 24 °C, and 25 μl of the derivatized mixture was loaded onto an HPLC column (Hypersil C-18, 250 × 4.6 mm, 5 μm particle size, thermostated at 21 °C) for subsequent chromatographic separation. To accurately quantify glutamate, deproteinized brain extracts were diluted 20-fold with HPLC-grade HO before the derivatization procedure and subsequent injection. Separation of OPA-AA and OPA-AGCC was achieved using two mobile phases (mobile phase A = 24 mmol / L CH3COONa + 24 mmol / L Na2HPO4 + 1% tetrahydrofuran + 0.1% trifluoroacetic acid, pH 6.5; mobile phase B = 40% CH3OH + 30% CH3CN + 30% HO) at a flow rate of 1.2 mL / min using an appropriate step gradient.

[0169] 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.

[0170] 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).

[0171] Brain tissue processing for histomorphometric analysis After appropriate anesthesia, rats were transcardially perfused as described by Di Pietro et al., Sci Rep. 2017, 7(1):9189. Briefly, a thoracotomy was performed, and heparin solution was administered into the portal vein to avoid blood clotting throughout the procedure. A right atriotomy was then performed, and a perfusion needle was advanced into the ascending aorta. Perfusion was performed with 100 ml of phosphate buffered saline (PBS) pH 7.4 to flush out the blood, followed by a further perfusion with 100 ml of 4% paraformaldehyde (PFA) pH 7.4 in PBS. After rapid removal from the skull, each brain was postfixed by immersion in 4% PFA in PBS at 4°C for 2 hours. Cryoprotection was achieved by immersing whole brains in PBS enriched with increasing sucrose solutions (10%, 20%, and 30%) for 24 hours, followed by embedding in optimal cutting temperature embedding medium (OCT) (Thermo Shandon, Runcorn, UK) in peel-mold containers (Agar Scientific, Essex, UK). Brains immersed in OCT were rapidly frozen in crushed dry ice and then stored at -80°C.

[0172] 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.

[0173] result Summary of biochemical data recorded 2 days after sTBI Effects of increasing doses of LMW-DS on measured cerebral energy metabolism Table 6 summarizes the values ​​for phosphorylated high-energy purine and pyrimidine compounds. It is particularly clear that sTBI caused a depletion of triphosphate nucleotides (ATP, GTP, UTP, and CTP), accompanied by an increase in ADP and N-acetylated derivatives of UDP-glucose (UDP-GlcNac) and UDP-galactose (UDP-GalNac).

[0174] At this time point after injury, treatment with LMW-DS was only partially effective in improving cellular energy metabolism, with significantly elevated levels of high-energy phosphates (ATP, GTP, and CTP) recorded at all three drug doses tested. No effect was observed on UTP and ADP concentrations. It is worth recalling that 48 hours after TBI in rats is a critical time point for cerebral metabolism and coincides with the greatest changes in mitochondrial function, including alterations in mitochondrial quality control. In this experimental TBI model, this time point can be considered a kind of "turning point" at which recovery or non-recovery of cerebral metabolism is determined. [Table 6] TIFF0007788730000016.tif75162 In Tables 6-25, bold indicates significant differences relative to control (p<0.05); bold underline indicates significant differences relative to TBI (p<0.05); and bold italics indicates significant differences relative to both control and TBI (p<0.05).

[0175] Effects of increasing doses of LMW-DS on nicotine coenzyme Oxidized NAD + and NADP + The values ​​for nicotine coenzymes (NADH and NADPH) and reduced (NADH and NADPH) are summarized in Table 7. Table 7 also shows the calculated NAD + We report the dimensionless value of the NADH / NADH ratio, which is suitable for assessing how dependent metabolism is on glycolysis or mitochondrial oxidative phosphorylation.

[0176] As previously observed herein, sTBI increases NAD + , NADPH + and NAD +At this point, treatment with LMW-DS was effective only at the highest dose tested (15 mg / kg body weight), resulting in significant protection of the nicotine coenzyme pool and avoiding a metabolic switch towards glycolysis, thereby indirectly suggesting an overall better mitochondrial function. [Table 7]

[0177] Effect of increasing doses of LMW-DS on CoA-SH derivatives Table 8 reports data on free CoA-SH and CoA-SH derivatives. Acetyl-CoA, in particular, is a key compound for mitochondrial metabolism, enabling the correct functioning of the tricarboxylic acid cycle (TCA cycle), thereby ensuring a continuous supply of electrons to the electron transport chain (ETC). TCA is the primary cell cycle pathway for the generation of reduced coenzymes (NADH and FADH2), which fuel the ETC and oxidative metabolism by transferring their electrons to mitochondrial complexes I and II, respectively. All compounds, especially acetyl-CoA, were significantly affected by sTBI. Partial recovery of this compound was observed at 5 or 15 mg / kg body weight. LWM-DS was administered to animals 30 minutes after injury. [Table 8]

[0178] Effects of increasing doses of LMW-DS on antioxidant and oxidative / nitrosative stress biomarkers Table 9 shows the major water-soluble brain antioxidants (ascorbic acid and GSH) and the effects of oxidative (MDA) and nitrosative stress (-NO2 - and -NO3 - ) biomarker concentrations are shown. Malondialdehyde (MDA) originates from the breakdown of unsaturated fatty acids in membrane phospholipids as a result of ROS-mediated lipid peroxidation. Nitrite (-NO2 - ) and nitrate (-NO3 -) is a stable end product of nitric oxide (NO) metabolism, which is produced in excess by inducible nitric oxide synthase (iNOS) under pathological conditions and generates reactive nitrogen species (RNS) through reaction with ROS.

[0179] Two days after impact, a 25-45% decrease in both water-soluble antioxidants occurred in sTBI-induced rats. A consequent increase in oxidative / nitrosative stress signatures was also noted. Administration of LWM-DS significantly restored both ascorbic acid and reduced glutathione (GSH) concentrations, and reductions in brain tissue nitrite and nitrate were evident. These effects were more pronounced when 15 mg / kg body weight was used. [Table 9]

[0180] Effects of increasing doses of LMW-DS on dephosphorylated purines and pyrimidines Most of the compounds reported in Table 10 originate from the degradation pathways of purine and pyrimidine nucleotides and are indirectly linked to cellular energy metabolism. In rats with sTBI, all of these compounds, except CDP-choline, had higher brain concentrations, and most of these were favorably affected by drug administration. [Table 10] TIFF0007788730000021.tif12161

[0181] Effect of increasing doses of LMW-DS on N-acetylaspartic acid (NAA) NAA is the most abundant N-acetylated amino acid in mammalian brains, and its concentration is roughly equivalent to that of the neurotransmitter glutamate in humans. Although the biological role of NAA has not yet been fully elucidated, we have previously shown in both preclinical and clinical studies that TBI reduces NAA concentrations and that its time course after head injury mirrors that of ATP. Notably, we found that sTBI induces irreversible changes in NAA homeostasis, that NAA is a good surrogate marker of cerebral energy metabolism, and that the decline and recovery of NAA levels in athletes after concussion is much slower than symptom resolution. Therefore, NAA has particular relevance in TBI testing.

[0182] Two days after impact, a 40% decrease in whole-brain NAA was observed in sTBI rats (Figure 9). LMW-DS had a beneficial effect on NAA concentrations when administered at 5 or 15 mg / kg body weight. Although significantly lower than controls, NAA in rats receiving either of the two drug doses was significantly higher than that found in sTBI rats, with the highest NAA levels found in rats receiving the highest dose of LMW-DS.

[0183] Effects of increasing doses of LMW-DS on free amino acids involved in neurotransmission The compounds listed in Table 11 are amino acids involved in neurotransmission directly (GLU, GABA) or indirectly (GLN, ASP, ASN, GLY, SER, THR, ALA). GLU, in particular, is the major excitatory amino acid, and its effects are counteracted by GABA. The excitotoxicity of GLU is regulated by SER, GLY, THR, and ALA, which is related to the function of the GLU-GLN cycle in neurons and astrocytes. As shown in previous studies, we found that most of these amino acids were increased in sTBI rats 2 days after injury. Treatment of animals with a single dose of LMW-DS was partially effective when the drug was injected subcutaneously at 5 or 15 mg / kg body weight. In most cases, the values ​​of these various compounds were significantly better than those observed in untreated sTBI animals, but not better than control values. [Table 11]

[0184] Effects of increasing doses of LMW-DS on free amino acids involved in the methyl cycle The free amino acids reported in Table 12 are involved in the so-called methyl cycle, which regulates the homeostasis of compounds acting as methyl donors in cellular metabolism, or in the formation of cysteine, the only amino acid with a free -SH group. Severe head trauma caused significant changes in the key players in this important metabolic pathway. Restoration of methionine was achieved by LWM-DS at all doses tested. Drug treatment was partially effective in normalizing the other amino acids. Supplementary information for changes in L-cystathionine (L-Cystat) is provided in the corresponding table 7 days after impact. [Table 12]

[0185] Effects of increasing doses of LMW-DS on free amino acids involved in the production of nitric oxide (NO) Table 13 shows the concentrations of free amino acids directly involved in the production of NO in the reaction catalyzed by nitric oxide synthase (NOS), a family of enzymes that exists in three isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The last isoform (iNOS) is involved in nitrosative stress. Nitric oxide is produced through a complex reaction in which arginine (ARG) undergoes partial oxidation to donate a nitrogen atom, generating citrulline (CITR) and NO. Two days after sTBI, animals showed a concomitant decrease in Arg and an increase in CITR, consistent with data showing an increase in the stable NO end products nitrite and nitrate (Table 8). Administration of LMW-DS was particularly effective when a dose of 15 mg / kg body weight was used. [Table 13]

[0186] Effect of increasing doses of LMW-DS on long-chain free amino acids The free amino acids reported in Table 14 are useful sources of carbon skeletons for the generation of α-keto acids that cells use to replenish the TCA cycle. Of these compounds, only isoleucine (ILE) was significantly affected by sTBI and restored in drug-treated rats. [Table 14]

[0187] Effect of increasing doses of LMW-DS on free amino acids and aromatic free amino acids acting as osmolytes The results summarized in Table 15 clearly demonstrate that sTBI results in increased concentrations of all these free amino acids. In particular, the increase in taurine (TAU) may suggest an attempt to counteract the effects of cellular edema by increasing the levels of one of the most important brain osmolytes. Separately, the increase in aromatic amino acids may suggest a reduction in the biosynthesis of the neurotransmitters serotonin (formed from tryptophan) and dopamine (produced by biotransformation first from phenylalanine and then from tyrosine). No significant effect of LMW-DS administration was observed at this time point after impact. [Table 15]

[0188] Summary of biochemical data recorded 7 days after sTBI Effects of increasing doses of LMW-DS on measured cerebral energy metabolism Table 16 summarizes the values ​​for phosphorylated high-energy purine and pyrimidine compounds. It is particularly clear that no recovery of depletion of triphosphate nucleotides (ATP, GTP, UTP, and CTP) was observed 7 days after sTBI. A concomitant increase in AMP and ADP was accompanied by significant changes in the concentrations of UDP derivatives (UDP-Glc, UDP-Gal, UDP-GlcNac, and UDP-GalNac). It should be emphasized that, in general, longer times after injury are often characterized by exacerbated biochemical, metabolic, and molecular changes induced by sTBI.

[0189] At this time point after injury, treatment with LWM-DS resulted in a more pronounced overall improvement in cerebral energy metabolism when the drug dose was higher than 1 mg / kg body weight. Differences from controls were recorded even in rats receiving repeated doses of 15 mg / kg body weight of LWM-DS, with significantly higher values ​​of nucleotide triphosphates observed in the drug-treated animals. Particularly relevant is the progressive recovery of the calculated dimensionless value of the ATP / ADP ratio (considered a good indicator of mitochondrial phosphorylation capacity), which continuously increased with increasing doses of drug administered to sTBI animals. [Table 16] TIFF0007788730000028.tif98162 To better demonstrate that drug effects are related to drug dose, we graphically report the results for ATP in Figure 10. It can be observed that the increase in ATP is somehow related to dose and that drug administration at all doses tested resulted in a significant increase in the most important high-energy phosphates.

[0190] Effects of increasing doses of LMW-DS on nicotine coenzyme Oxidized NAD + and NADP + The values ​​for nicotine coenzymes (NADH and NADPH) and reduced (NADH and NADPH) are summarized in Table 17. Table 17 also shows the calculated NAD+ We report the dimensionless value of the NADH / NADH ratio, which is suitable for assessing how heavily dependent metabolism is on glycolysis or mitochondrial oxidative phosphorylation.

[0191] As previously observed, nicotine coenzyme and NAD + A significant decrease in the NAD / NADH ratio was observed in sTBI rats 7 days after injury. Treatment with LMW-DS resulted in significant improvements in nicotine coenzyme levels, except for the lowest dose. In particular, single and repeated doses of 15 mg / kg body weight of LMW-DS significantly improved NAD as measured in control animals. + Normalize levels and ensure proper NAD + / NADH ratio could be restored. [Table 17]

[0192] Effect of increasing doses of LMW-DS on CoA-SH derivatives Table 18 reports data on free CoA-SH and CoA-SH derivatives. A significant positive effect of 5 or 15 mg / kg body weight (both doses as single and repeated administration) was detected for both CoA-SH and acetyl-CoA, suggesting much more favorable metabolic conditions for the functional operation of the TCA cycle. [Table 18]

[0193] Effects of increasing doses of LMW-DS on antioxidant and oxidative / nitrosative stress biomarkers Table 19 shows the major water-soluble brain antioxidants (ascorbic acid and GSH) and the effects of oxidative (MDA) and nitrosative stress (-NO2 - and -NO3 -) biomarker concentrations. 7 days after impact, no recovery of the concentrations of both water-soluble antioxidants occurred in sTBI-induced rats. Signs of extremely high levels of oxidative / nitrosative stress were also recorded. The effects of single and repeated administration of LWM-DS were particularly beneficial in restoring the concentrations of both ascorbic acid and reduced glutathione (GSH), with a clear reduction in brain tissue nitrite and nitrate. These effects were also significant when 5 mg / kg body weight was used. [Table 19] For a better understanding of drug effect relative to drug dose, we report the results for ascorbic acid and GSH graphically in Figures 11 and 12.

[0194] Effects of increasing doses of LMW-DS on dephosphorylated purines and pyrimidines Further deterioration in most of the compounds reported in Table 20, originating from the purine and pyrimidine nucleotide degradation pathway and indirectly linked to cellular energy metabolism, was observed 7 days after injury in rats subjected to sTBI. Most of these compounds were favorably affected by drug administration. [Table 20] TIFF0007788730000033.tif58162

[0195] 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 13). Interestingly, a dose-dependent increase in NAA was detected in rats receiving a single, increasing dose of LMW-DS or repeated administration of the highest dose tested.

[0196] Effects of increasing doses of LMW-DS on free amino acids involved in neurotransmission The compounds listed in Table 21 are amino acids directly (GLU, GABA) or indirectly (GLN, ASP, AASN, GLY, SER, THR, ALA) involved in neurotransmission. Most of these amino acids were still higher in sTBI rats 7 days after injury compared to controls. Administration of LMW-DS was effective, especially when the drug was administered subcutaneously at 15 mg / kg body weight in single or repeated doses. Of particular relevance is the normalization of Glu, thus allowing LMW-DS to eliminate the cause of excitotoxicity caused by excessive Glu release after sTBI. [Table 21]

[0197] Effects of increasing doses of LMW-DS on free amino acids involved in the methyl cycle As shown in Table 22, the levels of free amino acids involved in the so-called methyl cycle or the formation of cysteine ​​were still different in sTBI rats 7 days after impact when compared with the corresponding values ​​in controls. An increase in MET was observed in animals receiving the maximum dose of LWM-DS (both single and repeated administration). As already observed 2 days after injury, these drug levels resulted in a significant increase in L-cystathionine (L-Cystat). Since this compound is an intermediate in the production of cysteine ​​(CYS), it is reasonable to assume that an increase in L-Cystat could result in an increase in CYS. It is worth recalling that the measurement of CYS requires a specific additional HPLC assay with an additional derivatization using F-MOC, a fluorescent compound that reacts with secondary amines and CYS. [Table 22]

[0198] Effects of increasing doses of LMW-DS on free amino acids involved in the production of nitric oxide (NO) Table 23 shows the concentrations of free amino acids directly involved in the production of NO. Seven days after sTBI, animals showed a concomitant decrease in ARG and an increase in CITR, consistent with data showing the stable NO end products nitrite and nitrate (Table 8). Administration of LMW-DS was particularly effective when doses of 5 or 15 mg / kg body weight (single and repeated) were used. [Table 23]

[0199] 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 24, which are useful sources of carbon skeletons for the production of α-keto acids that cells use to replenish the TCA cycle, and the drugs of interest, were virtually normal 7 days after sTBI. [Table 24]

[0200] Effect of increasing doses of LMW-DS on free amino acids and aromatic free amino acids acting as osmolytes The results summarized in Table 25 clearly demonstrate that sTBI results 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 25]

[0201] Consideration Studies conducted to evaluate the effects of increasing doses of LMW-DS on brain metabolites in rats subjected to the induction of sTBI at different times after injury in a large panel demonstrated that administration of this compound resulted in a general recovery of brain metabolism.

[0202] LMW-DS was effective in restoring the severely imbalanced mitochondrial-associated energy metabolism in untreated sTBI animals, with favorable effects on the concentrations of purine triphosphate and pyrimidine nucleotides. Notably, ATP levels 7 days after impact were only 16% lower than control values, while a 35% decrease was observed in sTBI rats (Table 16 and Figure 10). Of note, NAA concentrations in LMW-DS-treated animals at the same time point were only 16% lower than control values, while sTBI animals showed 48% lower levels of this compound. This finding again strongly confirms the strict link between NAA homeostasis and proper mitochondrial energy metabolism and emphasizes the importance of pharmacological interventions that can favorably affect mitochondrial function.

[0203] The overall recovery of brain metabolism obtained by LMW-DS administration was also accompanied by the metabolism of nicotine coenzymes and free CoA-SH and CoA-SH derivatives, indicating that drug-treated animals, despite suffering from sTBI, had subnormal coenzymes that ensured proper redox reactions and allowed good functioning of the TCA cycle.

[0204] The above-mentioned improvement in brain metabolism was certainly responsible for another significant drug effect, namely the disappearance of GLU excitotoxicity. Furthermore, the drug affected sulfur-containing amino acids. This effect was probably related to the drug molecule containing the S atom. The increased bioavailability of this atom resulted in a net increase in the biosynthesis of these amino acids, one of which (MET) is crucial for methylation reactions and the so-called methyl cycle.

[0205] Further positive effects recorded in this study were an increase in antioxidants and a decrease in biochemical signatures of oxidative / nitrosative stress in sTBI rats treated with LMW-DS. Since dysfunctional mitochondria are the major intracellular source of both ROS and RNS, this phenomenon may even be closely related to the normalization of mitochondrial function. It is plausible that the effect of LMW-DS was more evident 7 days after sTBI than 2 days after. This strongly suggests that the overall recovery of cerebral metabolism by drug administration is not a transient phenomenon.

[0206] This experiment confirmed the favorable effects of LMW-DS in protecting mitochondrial function and reducing oxidative stress, as seen in particular in the restoration of antioxidant status, the protection of mitochondrial ATP energy replenishment by protecting ATP production and metabolism, and the normalization of mitochondrial phosphorylation capacity, all induced by LMW-DS. As a result, LMW-DS can protect and preserve mitochondrial function in cells exposed to damage or disease, which is important for having functional cells capable of fighting infectious diseases. Metabolic normalization induced by LMW-DS is beneficial for COVID-19 patients during the pneumonia stage (stage II) and recovery stage (stage IV) (Figure 25).

[0207] Example 5 This example investigated the potential of LMW-DS as an anticoagulant and antithrombotic agent in ALS patients.

[0208] material and method This was a single-center, single-arm, open-label study evaluating the safety, tolerability, and efficacy of subcutaneously (sc) administered LMW-DS in patients with ALS. There were 10 planned clinic visits: one two-part screening visit (Visit 1a and Visit 1b), five IMP administration visits (Visits 2 through 6), and three follow-up visits (Visits 7 through 9). Each individual patient's study participation, including the screening and follow-up visits, was planned to last approximately 4 months.

[0209] The active pharmaceutical ingredient of IMP was low molecular weight dextran sulfate (LMW-DS) (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780). LMW-DS was prepared as a solution for subcutaneous injection and consisted of 20 mg / mL LMW-DS and 9 mg / mL NaCl. Each glass vial contained 10 mL. The administered dose depended on the patient's weight at Visit 2, prior to the first LMW-DS administration. LMW-DS was injected subcutaneously into alternating sides of the abdomen, thigh, or buttocks (in that order of preference). Five 1 mg / kg injections were administered with a one-week dosing interval. Changes in activated partial thromboplastin time (APTT) were recorded from blood samples collected at each visit.

[0210] result The mean baseline APTT before LMW-DS injection was 26.5 s. After each LMW-DS treatment, a transient increase was observed, reaching a maximum 2–2.5 h after administration, with relative changes from baseline of +33.5% and +26.5%, respectively. APTT values ​​returned to pre-administration levels 6 h after administration. Consideration Acute tissue inflammation increases coagulation factor VIII, leading to a decrease in aPTT in the blood and microthrombosis in the tissue. The experimental data presented in this example demonstrate that LMW-DS reduces aPTT in the blood, thereby functioning as an anticoagulant and antithrombotic agent. Such anticoagulant and antithrombotic effects are beneficial for COVID-19 subjects during the hyperinflammatory phase (Stage III) (Figure 25).

[0211] Example 6 In this study, LMW-DS was 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 26) enable characterization of test agents across a broad set of systems that model various human disease states in an unbiased manner. BioMAP® systems are constructed with one or more primary cell types derived from healthy human donors, with stimuli such as cytokines or growth factors added to capture relevant signaling networks that naturally occur in human tissues or pathological conditions. Vascular biology is modeled in both Th1 (3C system) and Th2 (4H system) inflammatory environments, as well as a Th1 inflammatory state specific to arterial smooth muscle cells (CASM3C system). Additional systems recapitulate aspects of the systemic immune response, including monocyte-promoted Th1 inflammation (LPS system) or T cell stimulation (SAg system), chronic Th1 inflammation driven by macrophage activation (Mphg system), and T cell-dependent activation of B cells occurring in germinal centers (BT system). The BE3C system (Th1) and BF4T system (Th2) represent pulmonary airway inflammation, while the MyoF system models myofibroblast-mediated lung tissue remodeling. Finally, skin biology is addressed with the KF3CT system, which models Th1 skin inflammation, and the HDF3CGF system, which models wound healing.

[0212] Each test drug generates a signature BioMAP® profile created from changes in protein biomarker readouts within the individual system environment. Biomarker readouts (7–17 per system) are selected for therapeutic and biological relevance, predict disease outcomes or specific drug effects, and are validated using reagents with known mechanisms of action (MoA). Each readout is quantitatively measured using immunologically based methods to detect proteins, such as ELISA, or functional assays that measure proliferation and viability. BioMAP® readouts are diverse and include cell surface receptors, cytokines, chemokines, matrix molecules, and enzymes. Altogether, the BioMAP® Diversity PLUS panel contains 148 biomarker readouts that capture biological changes within the physiological context of a specific BioMAP® system.

[0213] material and method Four concentrations of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780; 150 nM, 440 nM, 1.3 μM, 4 μM) were tested at Eurofins using a BioMAP® Diversity PLUS panel.

[0214] Diversity PLUS Method Human primary cells in the BioMAP system were used at an early passage (passage 4 or earlier) to minimize adaptation to cell culture conditions and maintain physiological signaling responses. All cells were derived from a pool of multiple donors (n = 2–6), purchased commercially, and handled according to the manufacturer's recommended conditions. Prior to addition to the Mphg system, CD14 + Human blood derived monocytes are differentiated into macrophages in vitro. Abbreviations are used as follows: human umbilical vein endothelial cells (HUVEC), peripheral blood mononuclear cells (PBMC), human neonatal dermal fibroblasts (HDFn), B cell receptor (BCR), T cell receptor (TCR), and toll-like receptor (TLR).

[0215] 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 ligand)], BF4T system [bronchial epithelial cells and HDFn + (TNFα and IL-4)], BE3C system [coronary artery smooth muscle 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)].

[0216] 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). The BT system uses CD19 cells co-cultured with PBMCs. +These consist of B cells stimulated with a BCR activator and low levels of TCR stimulation. Test agents prepared in DMSO (small molecule; final concentration ≤0.1%) or PBS (biologicals) are added at the indicated concentrations 1 hour before stimulation and allowed to remain in culture for 24 hours or the following time periods: 48 hours, MyoF system; 72 hours, BT system (soluble reading); 168 hours, BT system (secreted IgG). Each plate includes a drug control appropriate for each system (e.g., 1.1 μM legacy control test agent colchicine), a negative control (e.g., unstimulated conditions), and a vehicle control (e.g., 0.1% DMSO). Cell-associated and membrane-targeted biomarker levels 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 to subconfluence and measured at the optimized time points for each system (48 hours: 3C and CASM3C systems; 72 hours: BT and HDF3CGF systems; 96 hours: SAg system). Cytotoxicity against adherent cells is measured by SRB (24 hours: 3C, 4H, LPS, SAg, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, and / Mphg systems; 48 hours: MyoF system) and alamarBlue staining of suspension cells (24 hours: SAg system; 42 hours: BT system) at the indicated time points.

[0217] Data analysis Biomarker measurements in test drug-treated samples were divided by the mean value of the control samples (at least six vehicle controls from the same plate) to generate a ratio, followed by log 10 The significance prediction envelope is calculated using the previously collected vehicle control data with 95% confidence intervals.

[0218] Profile Analysis Two or more consecutive concentration changes in the same direction relative to the vehicle control were outside the significance envelope, with an effect size (|log 10 Biomarker activity is annotated if there is at least one concentration with a ratio |>0.1). If these activities are increased in some systems but decreased in others, the primary activity of the biomarker is called modulated. If total protein is reduced by more than 50% (log of SRB), 10 If the ratio or alamarBlue level <-0.3), the cytotoxicity status is recorded and indicated by a thin black arrow above the x-axis. If cytotoxicity is detected in three or more systems, the compound is considered to have broad cytotoxicity. Concentrations of test agents with detectable broad cytotoxicity are excluded from biomarker activity annotation and downstream benchmarking, similarity searching, and cluster analysis. Antiproliferative effects are observed when the SRB or alamarBlue log from cells seeded at lower densities is higher. 10 Defined by a ratio value <-0.1 and indicated by a gray arrow above the X-axis, cytotoxic and antiproliferative arrows only require one concentration to meet the indicated threshold for profile annotation.

[0219] Benchmark Analysis If the readings for both profiles are outside the significance envelope and have an effect size greater than 20% in the same direction, the common biomarker readings are annotated. If one profile has a reading outside the significance envelope with an effect size >20% and the other profile has a reading inside the envelope or in the opposite direction, the differentiation biomarker is annotated. Unless otherwise specified, the highest non-cytotoxic concentrations of both the test agent and the benchmark agent are included in the benchmark overlay analysis.

[0220] Similarity analysis If the readouts for both profiles are outside the significance envelope and have an effect size greater than 20% in the same direction, common biomarker readouts are annotated. Test agent concentrations with three or more systems with detectable cytotoxicity are excluded from the similarity analysis. Test agent concentrations with one to two systems with detectable cytotoxicity are included in the similarity search analysis along with a database overlay of the highest test agent concentration match. This is followed by an additional overlay of the next highest test agent concentration that does not contain a system with detectable cytotoxicity and its respective database match. To determine the degree of similarity between BioMAP® profiles of compounds performed on the Diversity PLUS panel, we developed a custom similarity measure (BioMAP Z-Standard), a combinatorial method with improved performance in mechanistic classification of reference agents compared to other measures tested (including Pearson and Spearman correlation coefficients). This method more efficiently accounts for variations in the number of data points, systems, active biomarker readouts, and amplitude of biomarker readout changes characteristic of BioMAP® profiles. A Pearson correlation coefficient (r) is first generated to measure the linear association between the two profiles based on the similarity in the direction and magnitude of the relationship. Because Pearson correlation can be affected by the magnitude of either biomarker activity, a per-system weighted average Tanimoto measure is used as a filter to account for the under-representation of less robust systems. The Tanimoto measure does not consider the amplitude of biomarker activity, but rather addresses whether the identity and number of readings are common to the weights on a per-system basis. The real-valued Tanimoto measure first normalizes each profile to a unit vector (e.g.,

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[0221] Cluster analysis Cluster analysis (functional similarity map) uses the results of pairwise correlation analysis to project the "similarity" of drug profiles from multidimensional space to two dimensions. The functional clustering of drug profiles generated during this analysis uses Pearson correlation values ​​for pairwise comparisons of profiles for each concentration of each drug, and then subjects the pairwise correlation data to multidimensional scaling. Profiles that are similar with a Pearson correlation coefficient (r) ≥ 0.7 are connected by lines. Drugs that do not cluster with each other are interpreted as mechanistically distinct. This analysis is performed for projects containing three or more test drugs. Cytotoxic concentrations are excluded from the cluster analysis.

[0222] Mechanism heat map analysis Mechanism heat map analysis provides visualization of 19 consensus mechanisms, allowing comparison of biomarker activity across test compounds and all compound concentrations and consensus mechanisms. The composite consensus profile used in mechanism heat map analysis is an average representative BioMAP® profile of multiple compounds from structurally distinct chemical classes. Profiles were calculated by averaging each biomarker endpoint value for all selected profiles (multiple drugs at various concentrations) to construct a consensus mechanism profile. Biomarker activity is colored in the heat map for consensus mechanisms and compounds if they have expression outside the significance envelope compared to the vehicle control. Red represents increased protein expression, blue indicates decreased expression, and white indicates unchanged or levels within the filtering criteria. Darker shades of color represent larger changes in biomarker activity compared to the vehicle control. Mechanism heat maps were generated using R and the gplot package for R.

[0223] Assay Acceptance Criteria The BioMAP® assay includes multiparameter datasets generated by the BioMAP® platform for drugs tested in the systems that make up the Diversity PLUS panel. The assay includes a drug control appropriate for each system (e.g., the legacy control test drug colchicine), a negative control (e.g., unstimulated conditions), and a vehicle control (e.g., DMSO). The BioMAP® assay is plate-based, and data acceptance criteria depend on both plate performance (%CV of vehicle control wells) and system performance across the system's historical controls. 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 system biomarker readouts in the positive control reference dataset, and the Pearson value between each profile and the mean of the remaining profiles in the dataset is calculated. The total number of Pearson values ​​used to determine the 1% false-negative cutoff is the total number of profiles present in the reference dataset. The Pearson value at one percentile of all calculated values ​​is the 1% false-negative Pearson cutoff. If the Pearson value between the negative control or drug control profile of the experimental plate and the average of the historical control profiles in the reference dataset exceeds this 1% false negative Pearson cutoff, the system passes the test. If each individual system passes the Pearson test and 95% of all project plates have a %CV<20%, the overall assay is accepted.

[0224] result The BioMAP® Diversity PLUS panel included 12 individual BioMAP human primary cell-based co-culture systems, as shown in Table 26. [Table 26] TIFF0007788730000046.tif182162 Two or more consecutive concentrations that changed in the same direction relative to the vehicle control were outside the 95% significance envelope, with an effect size (|log 10 Biomarker 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 its activity was increased in some systems but decreased in others.

[0225] LMW-DS was active in 25 annotated readouts. LMW-DS was not cytotoxic to any of the human primary cells at the concentrations tested in this study. LMW-DS-mediated changes in key biomarker activity included decreased vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein-1 (MCP1), soluble tumor necrosis factor alpha (sTNFα), interferon-inducible T-cell alpha chemoattractant (I-TAC), monokine-induced by gamma interferon (MIG), and inflammation-related activity in the form of interferon gamma-inducible protein 10 (IP-10) and increased eotaxin 3 (Eot3), and interleukin 8 (IL-8). LMW-DS also had immunomodulatory activity in the form of reduced secretion of immunoglobulin G (sIgG) and macrophage colony-stimulating factor (M-CSF) and increased soluble IL-17A (sIL-17A) and cluster of differentiation 69 (CD69). LMW-DS also exhibited tissue remodeling activity in the form of increased matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator receptor (uPAR), and epidermal growth factor receptor (EGFR), and hemostasis-related activity in the form of increased thrombomodulin (TM). Table 27 summarizes the effects of LMW-DS on 12 different human primary cell types in the BioMAP® Diversity PLUS panel. [Table 27] The BioMAP® Reference Database contains BioMAP® profiles for over 4,500 bioactive agents (biologics, approved drugs, chemicals, and experimental agents) and can be used to classify and identify the most similar profiles.

[0226] In an unsupervised search for mathematically similar compound profiles in the BioMAP® Reference Database, LMW-DS (4M) was most similar to Clexane (30 μg / ml) (Pearson's correlation coefficient, r=0.701). Clexane (enoxaparin sodium) is an anticoagulant low-molecular-weight heparin used to treat deep vein thrombosis (DVT). There are five common activities annotated in the following systems: BT (sIgG, sIL-17A), CASM3C (MIG), and HDF3CGF (VCAM-1, IP-10).

[0227] Consideration In the study, LMW-DS was characterized by profiling with the BioMAP® Diversity PLUS panel of human primary cell-based assays that model the complex tissue and disease biology of organs (vasculature, immune system, skin, lung) as well as general tissue biology. The BioMAP® Diversity PLUS panel assessed the biological impact of LMW-DS under conditions that maintain the complex crosstalk and feedback mechanisms associated with in vivo outcomes.

[0228] LMW-DS was active but not cytotoxic at the concentrations tested in this study. LMW-DS was moderately and selectively antiproliferative against human primary endothelial cells only at the highest concentration (4 μM). The LMW-DS profile had 25 annotated readouts and demonstrated modulation of immune and inflammation-related readouts and matrix-related biomarkers. Specific activity included decreased inflammation-related sTNFα, VCAM-1, IP-10 (CXCL10), MIG (CXCL9), I-TAC (CXCL11), and MCP-1, and increased IL-8. Moderate increases in eotaxin-3 were observed only at lower concentrations in the BF4T system. Immunomodulatory activity included decreased sIgG and IL-17A and IL-17F in the BT system, but no antiproliferative effects on B cells. Decreased M-CSF and increased CD69, sIL-17A, and IL-17F were also identified. LMW-DS also regulated 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, whereas increased expression of the chemotactic factor IL-8 was observed in multiple systems. Collectively, these data suggest that LMW-DS is involved in regulating immune activation and / or immune recovery responses in inflammation and wound healing biology.

[0229] Modulation of inflammatory markers indicates the utility of LMW-DS in the treatment of multiple chronic and acute inflammatory conditions and diseases that have an inflammatory component.

[0230] Following injury, select components of the innate / pro-inflammatory and acquired immune responses are initially upregulated to maintain defense against foreign pathogens, remove tissue debris present at the injury site, and orchestrate the tissue remodeling, cell proliferation, and angiogenesis processes associated with the wound response. However, for proper wound healing to proceed, this initial inflammatory response must be modulated or halted to allow matrix reconstruction, recellularization, and tissue remodeling. Such immune recovery activity, induced by LMW-DS, includes activation of MMP-1, PAR-1, and uPAR, demonstrating its potential utility in treating COVID-19-injured tissues that would otherwise undergo harmful fibrosis.

[0231] LMW-DS modulated many biomarker activities in the HDF3CGF system, but only IL-8 in the MyoF system. While both systems contain fibroblasts, HDF3CGF models wound healing and the matrix remodeling associated with such wound healing, whereas MyoF models fibrosis through collagen deposition. Therefore, the results indicate that LMW-DS possessed immunomodulatory and tissue remodeling activities but did not induce undesirable collagen fibrosis, which can result in harmful fibrotic deposition.

[0232] In conclusion, LMW-DS appears to normalize and resolve inflammation present in tissues following trauma or disease, and these results are therefore consistent with the effects of LMW-DS observed in the previous examples.

[0233] Thus, LMW-DS modulates the secretion of pro-inflammatory cytokines and chemokines, thereby suppressing inflammatory signaling in disease-compromised tissues. These findings have direct relevance for suppressing harmful inflammatory responses, including ARDS, SIRS, and septic shock, observed in the most severely affected COVID-19 subjects. Furthermore, the tissue remodeling effects of LMW-DS may be beneficial for targeting pulmonary fibrosis, renal fibrosis, and cardiomyopathy in COVID-19 subjects.

[0234] Example 7 Analysis of gene expression changes induced by LMW-DS was investigated in several cell lines.

[0235] 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 a final concentration of 0.01 mg / ml. After 48 hours, cells were harvested from the treated flasks. Thus, the collected data represent (a) untreated cells (day 0 control and day 2 control) and (b) cells treated with LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780) for 48 hours (day 2 LMW-DS treatment).

[0236] Coating of tissue culture dishes for all cells 25cm 2 Flasks were coated by adding 2 ml of a 50 μg / ml solution of poly-d-lysine in Hank's balanced salt solution (HBSS) per flask and incubated overnight at 37°C in the dark. Flasks were washed with cell culture water and air-dried in the dark for 30 minutes. Flasks were coated by adding 1 ml of a 25 μg / ml solution of laminin in phosphate-buffered saline (PBS) per flask and incubated for 2 hours at 37°C in the dark. Prior to cell seeding, the laminin flasks were washed three times with PBS.

[0237] Human umbilical vein endothelial cells (HUVECs) Medium 200 + Large Vessel Endothelial Supplement (M200 + LVES) supplement (1:50) was prepared and pre-warmed to 37°C. Cells were thawed in a 37°C water bath for no more than 2 minutes and gently transferred to a 50 ml tube containing 20 ml of Dulbecco's Modified Eagle's Medium Nutrient Mixture F12 (DMEM-F12). The cell suspension was mixed by carefully inverting the tube twice. The cells were centrifuged at 400 x g for 10 minutes. The supernatant was removed, and the cells were resuspended in 10 ml of medium (M200 + LVES supplement).

[0238] Cells were counted using a Cellometer. 1,000,000 cells / flask was used for 25 cm 2 1000 cells were seeded into flasks (n=8) and medium was added to a total volume of 5 ml per flask. Cells were incubated at 37°C under 5% CO2. Cells were allowed to settle for 24 hours before LMW-DS treatment.

[0239] 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. The cells from the 12 vials were gently transferred to a tube containing 10 ml of high glucose DMEM medium and centrifuged at 400 relative centrifugal force (RCF) for 10 minutes. The pellet was resuspended in medium. The cells from the 12 vials were mixed and applied to 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.

[0240] Mouse cortical neurons (Lonza) The medium was prepared by adding 10 ml of B-27 serum-free supplement and 2.5 ml of GlutaMAX™-I supplement to 500 ml of Neurobasal medium. The medium was preheated to 37°C. Cells from 12 vials were sequentially thawed 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.

[0241] The cells were centrifuged at 200xg for 5 minutes. The supernatant was removed (down to the final 0.5 ml) and the cells were gently resuspended by trituration. The cells from the 12 vials were mixed and plated onto a pre-coated 25cm 2 The cells were evenly distributed among flasks (n=8). The cells were incubated at 37°C under 5% CO2 for 24 hours.

[0242] Mouse motor neuron (Aruna) The media was prepared according to Table 28. [Table 28] The medium (see Table 28) was pre-warmed to 37°C. The cells were thawed in a 37°C water bath for no more than 2 minutes. 9 ml of medium was gently added dropwise. The cell suspension was mixed by carefully inverting the tube twice. The cells were counted using a Cellometer. The cells were centrifuged at 200 x g for 5 minutes. The supernatant was removed (down to the last 0.5 ml) and the cells were gently resuspended by trituration. The cells from 8 vials were mixed and plated onto a pre-coated 25 cm 2 The cells were evenly distributed among flasks (n=8). Cells were incubated at 37°C under 5% CO2 for 24 hours before treatment.

[0243] Drug Treatment LMW-DS was prepared at a stock concentration of 20 mg / ml and kept in a temperature-monitored refrigerator at 4°C. Fresh 100X LMW-DS stock (1.0 mg / ml) was prepared in sterile DMEM-F12. The concentrated drug stock was filter-sterilized and added to the respective media (19.6 ml of CM and 0.4 ml of LMW-DS stock solution). A control was made using 19.6 ml of CM and 0.4 ml of DMEM-F12. LMW-DS and CM were added to their respective flasks (5 ml each), resulting in a concentration of 0.01 mg / ml LMW-DS in each dish containing a total of 10 ml of CM.

[0244] Culture harvest and cell lysis The CM was aspirated into a clean, labeled 15 ml Falcon tube. The flask (without medium) was placed in a -80°C freezer for 30 minutes. The CM in the Falcon tube was centrifuged at 3000 x g for 5 minutes. The supernatant was removed, and the small pellet was resuspended in 2.5 ml of Trizol:water (4:1) solution at room temperature (RT, approximately 22°C).

[0245] The frozen flasks were removed from the freezer one by one, and Trizol-water was transferred from the appropriate tube into the flask. The flasks were left at room temperature for 5 minutes, after which the contents were aspirated back into 15 ml Falcon tubes (after thoroughly rinsing the bottom of the flask with the solution). The flasks were examined under a microscope to ensure complete removal of cells. The collected lysates in the 15 ml Falcon tubes were placed in a -80°C freezer.

[0246] RNA extraction The homogenate-containing Falcon tube was removed from the freezer and stored at room temperature for 5 minutes to allow complete disintegration of nucleoprotein complexes. Two 1 ml aliquots of lysate were removed from each sample, and 200 μl of chloroform was added to each (0.2 ml of chloroform per ml of Trizol reagent was used during the cell lysis step), and the tubes were vigorously shaken. The samples were stored at room temperature for 2–3 min and then centrifuged at 12,000 × g for 15 min at 4°C. The mixture separated into three layers: a bottom red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remained in the upper aqueous phase, DNA in the white interphase (interphase), and protein in the pink bottom (organic) phase. Three-quarters of the upper aqueous phase was transferred to a new, clean Eppendorf tube. RNA was precipitated from the aqueous phase by adding an equal volume of 100% ethanol. The precipitated RNA was immobilized on a Spin Cartridge, washed twice, and dried. RNA was eluted with 50 μl of warm RNase-free water. The quantity and quality of purified RNA were determined by NanoDrop. RNA was stored at -80°C before being shipped to Source Bioscience for array analysis.

[0247] Expression data analysis plan Expression data were downloaded into separate files for each cell line. "Background-corrected" expression is data from the array's "gProcessedSignal," which is the result of subtracting background signal from the actual signal of the associated probe. This is the most commonly used variable in array analysis. Background-corrected signals were log2-transformed for statistical analysis across all samples. To reduce the false positive rate in a sample, signals below the "expression level" were removed. The "below expression" level was set to 5 of the log2-transformed expression level.

[0248] statistical analysis To reduce variability in the results, it was decided to perform median centering on all arrays prior to analysis based on the expression pattern of the control probes for each array. Data were grouped by cell type, and each cell type was analyzed using the following algorithm: Comparison of D0 control samples with D2 control samples - Expression changes observed in cells in normal cultures Comparison of D0 control samples with D2 LMW-DS-treated samples - Expression changes observed in cells in LMW-DS-treated cultures Comparison of D2 LMW-DS-treated samples with D2 control samples - Differential expression induced by LMW-DS in culture

[0249] A preliminary analysis was performed to screen out genes that were not differentially expressed between any combination of the three datasets. A simple, non-stringent ANOVA (p<0.05) was performed to look for expression patterns. Probes that did not change across the three datasets were removed. A volcano plot was used to analyze the remaining probe sets for fold change and significance. To allow for the detection of expression patterns, in the first case, a change in probe expression of more than 20% (fold change (FC) ≥ 1.2 or FC ≤ 0.84) was considered significant.

[0250] Quality Parameters The seeding density was calculated from the number of cells taken from the Schwann cell stock. HUVECS were seeded at their optimal density. Additional quality control from the array service provider indicated that the RNA was of high quality (no degradation) and the quantity was within the parameters of the low input RNA microarray from Agilent.

[0251] Analysis of the raw data indicated that, as expected, significant differences existed between arrays. These differences (reflecting differences among the same control samples included on all arrays) were, however, easily removed with normalization techniques. Median-centering the data to remove selected inter-array variations did not affect the overall differences expected to be observed between controls representing different concentrations of RNA.

[0252] Expression analysis of Schwann cells As previously described, genes not expressed in Schwann cells were removed prior to data analysis. The "under-expressed" level was set to 5 log2-transformed expression levels. This left 15,842 unique analytical probes in Schwann cell cultures. In the next step of the analysis, three sets of data (comparison of D0 control samples to D2 control samples; comparison of D0 control samples to D2 LMW-DS-treated samples; comparison of D2 control samples to D2 LMW-DS-treated samples) were analyzed to determine the effects of CM on cells and the relative changes induced by LMW-DS.

[0253] Comparing D0 to D2 control samples, 585 genes were differentially expressed in Schwann cell cultures. The molecular functions affected by these genes included 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), and cell proliferation. related to molecular transport (1.18E-04 to 2.29E-03); protein transport (1.62E-04 to 1.6E-03); glucose metabolism (3.22E-04 to 1.78E-03); gene expression (3.98E-04 to 2.2E-03); intracellular signaling (4.39E-04 to 2.25E-03); cell-cell signaling and interaction (5.05E-04 to 2.48E-03); cell damage (7.69E-04 to 1.58E-03); cell cycle (1.12E-03 to 1.8E-03); amino acid metabolism (1.6E-03 to 1.6E-03); and nucleic acid metabolism (1.6E-03 to 1.6E-03).

[0254] The values ​​shown above are p-values ​​representing the statistical significance associated with these genes in different pathways. The two p-values ​​represent the lower and upper bounds of the observed statistical significance (p<0.05 is significant).

[0255] LMW-DS induced differential expression of 1244 genes in Schwann cell cultures, as assessed by comparing D0 control samples with D2 LMW-DS-treated samples. The molecular functions affected by these genes included cell morphology (1.43E-08 to 8.39E-04); cell motility (1.4E-07 to 9.6E-04); post-translational modifications (3.93E-07 to 6.71E-05); protein synthesis (3.93E-07 to 1.08E-04); protein transport (3.93E-07 to 1.26E-06); cell death and survival (2.13E-06 to 8.65E-04); cell assembly and organization (7.46E-06 to 8.24E-04); DNA replication, recombination, and repair (7.46E-06 to 7.46E-06); and cell function and maintenance (9.53E-06 to 9.53E-06). -06~6.46E-04); gene expression (1.27E-05~4.92E-04); cell development (1.29E-05~9.06E-04); cell growth and proliferation (1.29E-05~9.06E-04); cell-cell signaling and interaction (1.97E-05~8.81E-04); amino acid metabolism (4.22E-05~8.24E-04); small molecule biochemistry (4.22E-05~8.24E-04); lipid metabolism (4.81E-05~3.64E-04); molecular transport (3.64E-04~3.64E-04); and cell cycle (4.53E-04~4.86E-04).

[0256] LMW-DS induced differential expression of 700 genes in Schwann cell cultures, as assessed by comparing D2 LMW-DS-treated samples with D2 control samples. The molecular functions affected by these genes included cell morphology (1.49E-07 to 5.62E-03); cell assembly and organization (1.49E-07 to 5.95E-03); cell motility (7.24E-07 to 6.06E-03); cell death and survival (9.41E-06 to 5.95E-03); amino acid metabolism (2.56E-05 to 3.7E-03); and cell morphology (1.49E-07 to 5.62E-03). Post-translational modifications (2.56E-05 to 1.05E-03); small molecule biochemistry (2.56E-05 to 3.7E-03); cell signaling and interactions (5.05E-05 to 5.76E-03); gene expression (7.18E-05 to 4.94E-03); cell cycle (1.06E-04 to 5.95E-03); cell development (1.06E-04 to 5.95E-03) ); Cellular function and maintenance (1.96E-04 to 5.95E-03); Cellular growth and proliferation (2.35E-04 to 5.95E-03); DNA replication, recombination, and repair (2.75E-04 to 5.95E-03); Intracellular signal transduction (5.92E-04 to 2.54E-03); Cellular damage (6.26E-04 to 6.26E-04); Lipid metabolism (6.26E -04 to 1.85E-03); molecular transport (6.26E-04 to 5.95E-03); protein synthesis (1.05E-03 to 1.93E-03); cellular response to therapeutic drugs (1.85E-03 to 1.85E-03); protein transport (2.66E-03 to 5.95E-03); and RNA post-transcriptional modification (4.32E-03 to 4.32E-03).

[0257] Mechanistic molecular network models simulate the effects of differentially regulated molecules by LMW-DS and allow for the evaluation of the functional consequences of these changes. In silico models show that LMW-DS suppresses neuronal cell death, apoptosis, and protein synthesis, and activates angiogenesis, cell migration, cell viability, cell survival, cell motility, cell proliferation, cell differentiation, cell homeostasis, cell cycle progression, cell transformation, and RNA expression. Table 29 summarizes the results of changes in gene expression in cultured Schwann cells. [Table 29] Twenty-one genes whose expression changed in the control cultures for 2 days showed no change whatsoever in the LMW-DS-treated cultures for the same 2 days. One gene whose expression increased in the control cultures was downregulated in the LMW-DS-treated cultures for the same 2 days. Thirteen genes that were downregulated in the control cultures were upregulated in the LMW-DS-treated cultures for the same 2 days. 122 genes were significantly downregulated by growth factors in the medium, and this downregulation was even stronger than in the LMW-DS-treated cultures. 441 genes were upregulated in the control cultures, and the addition of LMW-DS significantly strengthened this upregulation.

[0258] Expression analysis of HUVECs As previously described, genes not expressed in HUVECs were removed before any analysis was attempted. The "under-expressed" level was set to 5 log2-transformed expression levels. This left 15,239 unique probes for analysis in HUVEC cultures. In the next step of the analysis, the three sets of data were analyzed to determine the effects of CM on gene expression in the cells and the differences induced by LMW-DS. A preliminary analysis was performed to select and remove genes that were not differentially expressed between any combination of the three data sets. A simple, non-stringent ANOVA (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.

[0259] Comparing the D0 control samples to the D2 control samples, 1551 genes were differentially expressed in HUVEC cultures. The molecular functions affected by these genes are cell assembly and organization (2.55E-15 to 1.29E-03); cell function and maintenance (2.55E-15 to 1.29E-03); cell cycle (1.98E-11 to 1.32E-03); cell morphology (3.18E-10 to 1.29E-03); gene expression (1.05E-08 to 2.01E-04); cell development (1.66E-07 to 1.37E-03); cell growth and proliferation (1.66E-07 to 1.37E-03); DNA replication, recombination, and repair (2.04E-07 to 9.84E-04); cell death and survival (2.09E-07 to 1.3E-03); and RNA post-transcriptional modification (4.86E-06 ~6.53E-04); cell motility (9.9E-06~1.18E-03); post-translational modifications (1.92E-05~1.34E-03); cell-cell signaling and interactions (2.19E-05~9.1E-04); protein synthesis (5.49E-05~1.14E-03); cell damage (8.16E-05~8.16E-05); molecular transport (6.27E-04~6.27E-04); protein transport (6.27E-04~6.27E-04); intracellular signaling (8.86E-04~8.86E-04); cellular response to therapeutic drugs (9.84E-04~9.84E-04); and protein degradation (1.14E-03~1.14E-03).

[0260] LMW-DS induced differential expression of 1779 genes in HUVEC cultures, as assessed by comparing D0 control samples with D2 LMW-DS-treated samples. The molecular functions affected by these genes included cell assembly and organization (4.14E-17 to 9.7E-04); cell function and maintenance (4.14E-17 to 8.05E-04); cell cycle (5.83E-14 to 9.85E-04); cell morphology (1.69E-10 to 7.48E-04); gene expression (7.99E-09 to 8.62E-04); cell death and survival (2E-08 to 8.4E-04); cell development (1.28E-07 to 8.88E-04); cell growth and proliferation (1.28E-07 to 8.88E-04); DNA replication, recombination, and repair (3.07E-07 to 9.7E-04); and RNA replication, recombination, and repair (3.07E-07 to 9.7E-04). post-transcriptional modifications (1.13E-06 to 6.31E-04); cell motility (1.42E-06 to 8.34E-04); post-translational modifications (3.4E-05 to 9.17E-04); cell-cell signaling and interactions (6.97E-05 to 9.56E-04); molecular transport (7.43E-05 to 9.7E-04); protein transport (7.43E-05 to 7.43E-05); RNA transport (1.57E-04 to 5.72E-04); protein synthesis (1.92E-04 to 9.02E-04); cell damage (2.47E-04 to 6.28E-04); and intracellular signaling (4.64E-04 to 9.02E-04).

[0261] LMW-DS induced differential expression of 76 genes in HUVEC cultures, as assessed by comparing D2 control samples with D2 LMW-DS-treated samples. The molecular functions affected by these genes were DNA replication, recombination, and repair (9.62E-05 to 2.57E-02); cell cycle (1.22E-04 to 2.4E-02); cell development (1.59E-04 to 2.67E-02); cell morphology (4.64E-04 to 2.42E-02); cell function and maintenance (4.64E-04 to 2.57E-02); lipid metabolism (9.49E-04 to 1.07E-02); and molecular transport (9.62E-05 to 2.57E-02). .49E-04-1.61E-02); Small Molecule Biochemistry (9.49E-04-1.87E-02); Cell Damage (1.6E-03-2.62E-02); Cell Death and Survival (2.06E-03-2.67E-02); Amino Acid Metabolism (2.7E-03-2.7E-03); Glucose Metabolism (2.7E-03-1.07E-02); Cell Signaling and Interaction (2.7E-03-2.4E-02); Cell Assembly and Organization Origenation (2.7E-03 to 2.57E-02); Cell growth and proliferation (2.7E-03 to 2.4E-02); Cell motility (2.7E-03 to 2.4E-02); Energy production (2.7E-03 to 2.7E-03); Nucleic acid metabolism (2.7E-03 to 1.07E-02); Post-translational modification (2.7E-03 to 1.61E-02); Gene expression (5.39E-03 to 2.36E-02); RNA post-transcriptional modification (5.39E-03 to 2.4E-02); drug metabolism (8.07E-03 to 1.61E-02); vitamin and mineral metabolism (8.07E-03 to 8.07E-03); protein synthesis (1.07E-02 to 1.07E-02); RNA transport (1.07E-02 to 1.07E-02); cellular response to therapeutic drugs (1.24E-02 to 1.24E-02); and free radical scavenging activity (1.43E-02 to 1.43E-02).

[0262] 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.

[0263] Using mechanistic molecular network models, we can simulate the effects of differentially regulated genes by LMW-DS and explore the functional consequences of these changes. In silico models show that LMW-DS suppresses neuronal cell death, apoptosis, and protein synthesis, and activates angiogenesis, cell migration, cell viability, cell survival, cell motility, cell proliferation, cell differentiation, cell homeostasis, cell cycle progression, cell transformation, and RNA expression. HUVEC control cultures contained growth factors. In treated cultures, LMW-DS was added to medium already containing growth factors.

[0264] Table 30 summarizes the results of gene expression changes in cultured HUVECs. 67 genes whose expression changed (due to the effects of growth factors) in control cultures for 2 days showed no change whatsoever in LMW-DS-treated cultures for the same 2 days. Four genes whose expression increased in control cultures containing growth factors were downregulated in LMW-DS-treated cultures for the same 2 days. Eleven genes that were downregulated by growth factors in control cultures were upregulated in LMW-DS-treated cultures for the same 2 days. 120 genes were significantly downregulated by growth factors, and this downregulation was even stronger than in LMW-DS-treated cultures. 229 genes were upregulated in control cultures, and the addition of LMW-DS significantly strengthened this upregulation. [Table 30] We analyzed the effects of LMW-DS on several molecular pathways important for different disease states and therapeutic applications. For this analysis, we compared the effect of LMW-DS addition on gene expression to that observed in cells in CM, and predicted functional effects based on the observed changes in expression patterns.

[0265] Expression analysis of motor neurons As previously described, genes not expressed in motor neurons were removed before any analysis was attempted. The "below expression" level was set to 5 log2-transformed expression levels. This left 12,240 unique probes that met the expression threshold in at least three samples in the series. In the next step, the three sets of data were analyzed to determine the effects of CM on cells and the differences induced by LMW-DS.

[0266] The changes in gene expression under normal culture conditions mimic the normal developmental process of motor neurons when they develop a motor neuron phenotype from a set of dissociated cells. The growth factors in the normal medium are necessary for these cells to differentiate. The stressor present in these cultures is oxidative stress (normal in tissue culture conditions).

[0267] Comparing the D0 control samples to the D2 control samples, 485 genes were differentially expressed in the motor neuron cultures. The molecular functions affected by these genes were: cell death and survival (1.99E-17 to 1.98E-04); cell motility (1.14E-16 to 1.91E-04); cell assembly and organization (1.22E-16 to 1.93E-04); cell function and maintenance (1.22E-16 to 1.95E-04); cell morphology (6.46E-16 to 1.74E-04); cell-cell signaling and interaction (3.16E-12 to 1.95E-04); cell development (1.59E-10 to 1.93E-04); cell growth and proliferation (1.59E-10 to 1.9E-04); molecular transport (4.27E-10 to 1.89E-04); protein synthesis (9.85E-09 to 5.03E-05); and lipid metabolism (1.08E-04). -08~1.61E-04); Small Molecule Biochemistry (1.08E-08~1.89E-04); Gene Expression (8.45E-08~3.8E-05); Cell Cycle (4.55E-07~1.09E-04); Free Radical Scavenging (7.12E-07~1.65E-04); Intracellular Signal Transduction (1.23E-05~1.89E-04); Vitamins and Minerals related to ral metabolism (1.23E-05 to 1.89E-04); protein degradation (3.07E-05 to 1.31E-04); glucose metabolism (3.32E-05 to 1.61E-04); drug metabolism (4.16E-05 to 4.16E-05); post-translational modification (7.1E-05 to 1.31E-04); and protein folding (7.1E-05 to 7.1E-05).

[0268] LMW-DS induced differential expression of 315 genes in motor neurons, as assessed by comparing D0 control samples with D2 LMW-DS-treated samples. The molecular functions affected by these genes were cell death and survival (6.54E-08 to 9.06E-03); cell motility (8.21E-08 to 5.42E-03); cell assembly and organization (8.36E-08 to 9.01E-03); cell function and maintenance (8.36E-08 to 9.01E-03); cell morphology (2.9E-06 to 8.75E-03); and cell development. Biogenesis (1.04E-05 to 9.01E-03); Cell growth and proliferation (1.04E-05 to 7.83E-03); DNA replication, recombination and repair (2.79E-05 to 8.01E-03); Cell-cell signaling and interaction (8.18E-05 to 7.11E-03); Post-translational modification (1.32E-04 to 7.56E-03); Protein degradation (1.32E-04 to 4. 35E-03); protein synthesis (1.32E-04~5.09E-03); gene expression (1.9E-04~9.01E-03); cell damage (3.58E-04~9.01E-03); cell cycle (6.08E-04~9.01E-03); free radical scavenging (7.41E-04~7.31E-03); amino acid metabolism (7.67E-04~6.61E-03); small related to 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).

[0269] LMW-DS induced differential expression of 425 genes in motor neurons as assessed by comparison of D0 control samples to D2 LMW-DS treated samples. The molecular functions affected by these genes are cell death and survival (2.87E-08 to 6.27E-03); cell motility (4.73E-07 to 6.47E-03); cell morphology (4.95E-07 to 7.47E-03); cell development (1.02E-06 to 7.13E-03); cell growth and proliferation (1.02E-06 to 7.48E-03); cell assembly and organization (7.03E-06 to 7.47E-03); cell function and maintenance (7.03E-06 to 7.47E-03); gene expression (1.95E-05 to 6.18E-03); cell cycle (2.88E-05 to 7.48E-03); DNA replication, recombination, and repair (3.39E-05 to 5.16E-03); and amino acid metabolism (7.75E-05 ~4.68E-03); Small Molecule Biochemistry (7.75E-05~4.68E-03); Cellular Damage (8.23E-05~4.61E-03); Cellular Signaling and 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); Translation These findings are related to post-translational modification (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 31]

[0270] Expression analysis of cortical neurons As previously described, genes not expressed in motor neurons were removed before any analysis was attempted. The "under-expressed" level was set at 5 log2-transformed expression levels. This left 10,653 unique probes that met the expression threshold in at least three samples in the series. In the next step, the three sets of data were analyzed to determine the effects of CM on cells and the differences induced by LMW-DS.

[0271] Under normal culture conditions, changes in gene expression mimic the normal development of cortical neurons when they develop a cortical neuron phenotype from a set of dissociated cells. The growth factors in the normal medium are necessary for these cells to differentiate. The stressor present in these cultures is oxidative stress (normal in tissue culture conditions).

[0272] Comparing the D0 control samples to the D2 control samples, 1101 genes were differentially expressed in the motor neuron cultures. The molecular functions affected by these genes included cell assembly and organization (3.57E-25 to 6.65E-04); cell function and maintenance (3.57E-25 to 6.65E-04); cell morphology (4.28E-22 to 6.36E-04); cell development (4.28E-22 to 6.53E-04); cell growth and proliferation (4.28E-22 to 6.6E-04); and intercellular signaling. Nuclear communication and interaction (2.16E-13 to 6.65E-04); molecular transport (5.18E-12 to 4.95E-04); cell motility (1.86E-11 to 6.65E-04); cell death and survival (3.37E-11 to 6.41E-04); gene expression (1.27E-08 to 8.96E-05); protein synthesis (3.84E-07 to 8.69E-05); small molecule biochemistry (6 0.65E-07 to 5.18E-04; cell damage (7.12E-06 to 4.54E-04); protein degradation (1.62E-05 to 1.62E-05); amino acid metabolism (2.11E-05 to 4.25E-04); protein transport (3.4E-05 to 3.4E-05); intracellular signal transduction (8.69E-05 to 3E-04); post-translational modification (8.69E-05 to 2.15E-0 4); protein folding (2.15E-04 to 2.15E-04); cell cycle (2.69E-04 to 3.07E-04); DNA replication, recombination and repair (2.69E-04 to 4.77E-04); nucleic acid metabolism (2.69E-04 to 2.69E-04); lipid metabolism (3.12E-04 to 5.18E-04); and glucose metabolism (5.18E-04 to 5.18E-04).

[0273] LMW-DS induced differential expression of 609 genes in motor neurons as assessed by comparison of D0 control samples to D2 LMW-DS treated samples. The molecular functions affected by these genes are cell assembly and organization (3.91E-15 to 1.83E-03); cell function and maintenance (3.91E-15 to 1.83E-03); cell morphology (2.53E-13 to 1.43E-03); cell development (2.53E-13 to 1.81E-03); cell growth and proliferation (2.53E-13 to 1.83E-03); cell motility (4.95E-09 to 1.2E-03); cell-cell signaling and interaction (5.96E-09 to 1.47E-03); cell death and survival (2.25E-08 to 1.77E-03); molecular transport (7.08E-08 to 1.79E-03); DNA replication, recombination, and repair (3.03E-06 to 1.71E-03); and cell damage (9.23E-06 to 7. 0.65E-04); Amino Acid Metabolism (1.75E-05 to 1.64E-03); Cell Cycle (1.75E-05 to 1.77E-03); Small Molecule Biochemistry (1.75E-05 to 1.79E-03); Protein Synthesis (2.77E-05 to 1.5E-03); Protein Transport (2.77E-05 to 1.9E-04); Intracellular Signal Transduction (7.65E-05 to 1.73E-0 3); post-translational modification (3.01E-04 to 1.4E-03); gene expression (3.65E-04 to 1.15E-03); drug metabolism (6.49E-04 to 6.49E-04); glucose metabolism (6.95E-04 to 7.69E-04); vitamin and mineral metabolism (1.09E-03 to 1.09E-03); and nucleic acid metabolism (1.44E-03 to 1.73E-03).

[0274] LMW-DS induced differential expression of 247 genes in motor neurons as assessed by comparison of D0 control samples to D2 LMW-DS treated samples. The molecular functions affected by these genes were: cell morphology (6.01E-08 to 1.01E-02); cell development (7.46E-08 to 1.01E-02); cell growth and proliferation (7.46E-08 to 1.01E-02); cell death and survival (4.23E-07 to 1.01E-02); cell motility (2.69E-06 to 9.91E-03); cell assembly and organization (1.57E-05 to 1.01E-02); cell function and maintenance (1.57E-05 to 1.01E-02); cell cycle (1.01E-04 to 1.01E-02); cell signaling and interaction (1.01E-04 to 1.01E-02); and lipid metabolism (1.56E-04 to 1.01E-02). ~1.01E-02); small molecule biochemistry (1.56E-04~1.01E-02); gene expression (2.28E-04~3.38E-03); RNA damage and repair (2.28E-04~2.28E-04); RNA post-transcriptional modification (2.28E-04~2.28E-04); molecular transport (4.18E-04~8.32E-03); cellular damage (4.47E-04~2.2E-03); protein synthesis (2.66E-03~7.29E-03); protein transport (4.11E-03~8.32E-03); protein degradation (5.64E-03~7.29E-03); and DNA replication, recombination and repair (7.31E-03~1.01E-02). [Table 32]

[0275] Effects of LMW-DS on mitochondrial oxidative stress pathway Oxidative stress pathways occurring in mitochondria are important in various infectious diseases, as well as in aging and age-related degenerative diseases. Normal growth conditions induce a certain amount of oxidative stress in cells, which contributes to the aging process both in vivo and in vitro.

[0276] In Schwann cells cultured under normal conditions, complex I (NADH dehydrogenase) was inhibited, whereas complex IV (cytochrome c oxidase) was activated. When LMW-DS was added to the cultures, complex III (cytochrome bc1) was inhibited. Inhibition of complex III suppresses oxidative stress events involved in the pathogenesis of cancer and neurological diseases.

[0277] Complex III, sometimes called the coenzyme Q:cytochrome c oxidoreductase or cytochrome bc1 complex, is the third complex in the electron transport chain (EC 1.10.2.2) and plays a key role in the biochemical production of ATP (oxidative phosphorylation). Complex III is a multisubunit transmembrane protein encoded by both the mitochondrial (cytochrome b) and nuclear genomes (all other subunits). Complex III is present in the inner mitochondrial membrane of all animals, all aerobic eukaryotes, and most eubacteria. Mutations in complex III result in exercise intolerance and multisystemic 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.

[0278] In HUVECs, no significant modulation of the mitochondrial effects of oxidative stress was detected after treatment with LMW-DS.

[0279] Under normal culture conditions, motor neurons appear to undergo significant oxidative stress, which leads to the activation of several apoptotic mechanisms, including cytochrome C, AIF, and caspases 3, 8, and 9. Furthermore, motor neurons are characterized by the production of amyloid beta and fatty acid oxidation, which further exacerbate oxidative stress and FIAS1-mediated mitochondrial fragmentation in the cells. Furthermore, complex V is activated.

[0280] Addition of LMW-DS to cultures ameliorated its negative effects on amyloid-β production and mitochondrial fragmentation and dysfunction, and subsequent damage, by preventing and suppressing apoptosis through suppressing fatty acid oxidation. LMW-DS also inhibited pathways involving TRAK1 and PINK1, thereby contributing to improved mitochondrial function. LMW-DS also reduced H2O2 levels. An additional effect was the inhibition of HtrA2, which contributed to the suppression of apoptosis.

[0281] Under normal culture conditions, cortical neurons are exposed to considerable oxidative stress, leading to amyloid-β production and Lewy body formation accompanied by synuclein-α activation and increased levels of reactive oxygen species (ROS); apoptosis; mitochondrial fragmentation; and impaired mitochondrial function accompanied by C161. Addition of LMW-DS to the cultures prevented and reversed most of these adverse effects, including the accumulation of amyloid-β and Lewy body pathologies and mitochondrial dysfunction. Some apoptosis-inducing mechanisms remain active in the cultures, likely due to their potent activation.

[0282] Effect of LMW-DS on glutamate excitotoxicity Glutamate is an essential excitatory amino acid involved in long-term potentiation (LTP), i.e., learning and memory function. However, excess glutamate is also associated with excitotoxicity, leading to neuronal death. This latter phenomenon is postulated to be involved in neuronal death caused in chronic neurodegenerative conditions (including TBI). Genes involved in glutamate signaling were not expressed in HUVECs but are present in the Schwann and neuronal cell lines used in this study.

[0283] Glutamate production is suppressed by baseline conditions in motor neuron cultures. The suppression is unaffected by LMW-DS. Glutamate production is elevated in cortical neurons at baseline. Addition of LMW-DS did not alter glutamate production in these cells.

[0284] Addition of LMW-DS to the CM of Schwann cells induced the expression of protein complexes (CALM, Gβγ, GRM7, and PICK1). More importantly, LMW-DS increased the activity and / or levels of glutamate transporters, particularly SLC1A2 / 3, in Schwann cells, thereby removing 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.

[0285] SLC1A3, member 3 of the solute carrier family 1 (glial high-affinity glutamate transporter), is a protein encoded by the SLC1A3 gene in humans. SLC1A3 is also often referred to as glutamate aspartate transporter (GLAST) or excitatory amino acid transporter 1 (EAAT1). SLC1A3 is primarily expressed in the plasma membrane, which allows it to remove 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 glutamate to be transported into cells against its concentration gradient. SLC1A3 is expressed throughout the CNS and is highly expressed in astrocytes and Bergmann glia in the cerebellum. In the retina, SLC1A3 is expressed in Müller glial cells. SLC1A3 is also expressed in many other tissues, including cardiac myocytes.

[0286] SLC1A2, solute transporter family 1 member 2, also known as excitatory amino acid transporter 2 (EAAT2) and glutamate transporter 1 (GLT-1), is a protein encoded by the SLC1A2 gene in humans. SLC1A2 is a member of the solute transporter family of proteins. This membrane-bound protein is the primary transporter for the removal of the excitatory neurotransmitter glutamate from the extracellular space of synapses in the CNS. Glutamate removal is necessary for proper synaptic activation and to prevent neuronal damage due to excessive activation of glutamate receptors. SLC1A2 is responsible for over 90% of glutamate reuptake in the brain. These findings indicate that LMW-DS may be useful in preventing glutamate excitotoxicity under conditions where its high extracellular levels are harmful, such as after TBI.

[0287] 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. 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 33). The aggregate effect of these molecules (17 molecules, see Table 33) on pathways regulating cell motility and adhesion in Schwann cells was such that cell adhesion could be inhibited and cell motility could be simultaneously activated, whereas in HUVECs (1 molecule, ADAM11), adhesion was not affected but angiogenesis could be activated. [Table 33] The effects of LMW-DS-induced differential gene expression in neurons were analyzed. In motor neurons, the same metallopeptidase-dependent pathway may be involved in the cell detachment observed in Schwann cells (see Table 34). [Table 34] However, none of the MMP-related genes were differentially expressed in cortical neurons. 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: 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, Ca2+, CA9, CADM1, CALR, calyculin A, caspase, CBL, CD209, CD36, CD44, CD46, CDH13, cerivastatin, chloramphenicol Chole, 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 receptors, EPHA8, EPHB1, eptifibatide, ethire dibenzodiaminetetraacetic acid, ETS1, F11R, F3, FBLN5, FBN1, Fc receptor, FCN2, FERMT2, FES, FGF2, FGFR1, fibrin, FN1, focal adhesion kinase, FSH, FUT3, FUT6, FUT7, FYN, HACD1, heparin, histone h3, histone h4, HRAS, HSPG2, HTN1, hyaluronic acid, hydrocortisone, hydrogen peroxide, ICAM1, ICAM2, IGF1R, IgG, Igg3, IL1, IL1B, IL6, ILK, integrin, integrin alpha 4 beta1, integrin alpha, IPO9, 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, Mn2+, NCK, NEDD9, NRG1, okadaic acid, OLR1, p38 MAPK, PDGFBB, 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.

[0288] 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.

[0289] Upstream regulatory pathways affected by LMW-DS As shown in Table 35, 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 35] In HUVECs, the number of growth factors whose effects were enhanced by LMW-DS was relatively small, but still highly significant (see Table 36). [Table 36] As shown in Table 37, in motor neurons, upstream regulator analysis revealed that LMW-DS affected the effects of several growth factors, either enhancing their activation or reducing their inhibition, present in the system. [Table 37] In normal culture conditions of cortical neurons, most pathway-dependent growth factors were significantly activated by normal medium. In most cases, this activation was unchanged by LMW-DS. However, LMW-DS activated molecules that are downstream effectors of GDF7, and the effects of this growth factor were enhanced by LMW-DS. GDF7 is a potent differentiation factor for neurons, and the additional activation of these growth factors in response to BDNF and NT3 activation provides a good explanation for the enhanced differentiation of these cells in culture.

[0290] Consideration Normal culture conditions for HUVECs mimic the environment following tissue hypoxia and reperfusion and include high nutrient content and growth factors, similarly supplemented with heparin. LMW-DS-treated cultures mimicked the effects of LMW-DS after 24 hours of hypoxia and reperfusion. A relevant real-life scenario is that of angiogenesis following an ischemic condition, such as a fibrotic state.

[0291] In Schwann cells, control cultures with high nutrient content and glucose reproduced Schwann cell activation. LMW-DS-treated cultures mimicked the effects of LMW-DS added 24 hours after glial activation. This reproduces a real-life scenario: glial activation after nervous system injury.

[0292] Normal culture conditions for neurons, both motor neurons and cortical neurons, containing high nutrient content and growth factors mimic the environment during normal neuronal differentiation. The only negative effect in these cultures is the oxidative stress to which the cells are subjected. A relevant real-life scenario is a degenerative state driven by oxidative stress in the presence of sufficient growth and differentiation factors. This corresponds to early stages of neurodegenerative diseases or conditions in which oxidative stress plays a central role.

[0293] The molecular effects observed in Schwann cells and HUVECs support a role for LMW-DS in protecting against apoptosis, inducing angiogenesis, increasing cell migration and migration, increasing cell viability and survival, and inducing cell differentiation across cell types. Analysis of key molecular pathways indicated that in neurons, LMW-DS should reduce the effects of oxidative stress on mitochondria and reduce neurodegeneration-related molecules such as amyloid-β and Lewy bodies.

[0294] Thus, the results from the HUVEC cell model indicate that LMW-DS can protect against cell injury and promote the development of new blood vessels in injured or affected tissues following stroke, etc. The results from Schwann cells indicate that LMW-DS can protect against cell loss in the diseased or injured nervous system.

[0295] 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 glutamate excitotoxicity in the diseased and injured nervous system.

[0296] Of particular importance, LMW-DS increased glial cell glutamate uptake, as demonstrated by Schwann cells. However, LMW-DS did not alter neuronal glutamate production. This is important because glutamate is necessary for LTP, i.e., learning and memory. Therefore, it is informative that LMW-DS did not alter neuronal glutamate production, since glutamate is necessary for normal neurotransmission in the processes described above. However, increased levels of glutamate released from damaged or dying cells are efficiently taken up by surrounding glial cells due to the effects of LMW-DS. Therefore, activation of glutamate transporters in glial cells by LMW-DS effectively removed glutamate released by damaged or dying neurons from the neuronal gap. This, in turn, prevented glutamate from exerting excitotoxic effects and further neuronal damage. Thus, LMW-DS induced the uptake of potentially harmful neurotoxic amounts of glutamate by glial cells.

[0297] Thus, the neuronal results confirm the potential therapeutic utility of LMW-DS in reducing secondary tissue damage due to oxidative stress, promoting repair, and reducing degeneration-associated protein accumulation.

[0298] 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 motility, increasing cell viability and survival, inducing cell differentiation, reducing the effects of oxidative stress, reducing glutamate excitotoxicity and reducing degeneration-associated protein products such as amyloid-β and Lewy bodies.

[0299] Cell adhesion was primarily affected in neurons and Schwann cells, where LMW-DS promoted cell detachment and migration. Cell adhesion was unaffected in HUVECs. The effect on cell adhesion was primarily due to the expression of metalloproteinase-type enzymes, although modulation of other adhesion molecules also contributed to this effect.

[0300] Scar formation as a pathological response is promoted by TGFβ. TGFβ induces a large interconnected network of 171 molecules that results in immune cell adhesion, cell activation, cell motility, cell aggregation, fibrosis, and the induction of TGFβ. Administration of LMW-DS completely abolished the TGFβ-induced effects on immune cell adhesion, cell activation, cell aggregation, fibrosis, and TGFβ autoactivation. These inactivating effects of LMW-DS on the molecular network promoted by TGFβ in Schwann cells were also observed even when TGFβ was activated, i.e., in the presence of excess TGFβ.

[0301] Thus, these studies confirm the potential therapeutic utility of LMW-DS in inhibiting signaling by the pro-inflammatory, fibrogenic cytokine TGFβ, which in turn inhibits tissue fibrosis, which otherwise contributes to the severity of the disease in COVID-19 subjects.

[0302] Example 8 This example demonstrates that LMW-DS resolves inflammatory scarring, stimulates matrix remodeling, and enables functional tissue regeneration in rodent and human fibrotic disease models.

[0303] result POAG is an example of a chronic intraocular fibrotic disease. In the eye, TGFβ is known to increase ECM deposition in the TM, most notably fibronectin. Fibronectin is found within the sheath material connecting Schlemm's canal to the innermost region of the TM and helps regulate tissue contractility and consequent AqH drainage and IOP. Higher levels of TGFβ and fibronectin have been detected in patients with glaucoma. Here, we showed that LMW-DS significantly reduced fibronectin levels in cultured human trabecular meshwork cells treated with TGFβ2 (1136 ± 155 vs. 1579 ± 210; P < 0.05; Figures 15A and 15B). Following this, we evaluated the effects of subcutaneous (SC) injection of LMW-DS in a rodent model of anterior segment fibrosis, an experimental model of glaucoma. In this model, fibrosis was induced by biweekly intracameral (IC) injections of TGFβ1 (Figure 16A). By establishing TM fibrosis, twice-weekly IC TGFβ1 injections significantly elevated IOP to 13 mmHg by day 14, at which point daily SC injections of LMW-DS or saline were initiated. In control rats receiving SC injections of IC TGFβ1 plus saline, IOP continued to rise, reaching 17.6 ± 2.7 mmHg by day 28 (Figure 16B). In contrast, IC TGFβ1 with SC LMW-DS injections reduced IOP to 11.4 ± 0.2 mmHg by day 28 (P < 0.0001; Figure 16B), a value within the normal range (10–12 mmHg). Immunohistochemical analysis of fibrosis in the TM showed a significant decrease in the ECM molecules laminin (25.6 ± 3.5%, P < 0.001; Figure 16C ) and fibronectin (40.2 ± 4.0%, P < 0.01; Figure 16D ) compared with SC saline-treated control rats (63.7 ± 6.8% and 63.6 ± 2.9%, respectively).This was accompanied by a significantly increased number of surviving retinal ganglion cells (RGCs) as measured by optical coherence tomography (OCT) (12.6 ± 0.4 RGCs / mm in SC LMW-DS-injected rats compared with 9.0 ± 0.3 RGCs / mm in control SC saline-injected rats; P < 0.01; Figure 16E) and preserved retinal nerve fiber layer (RNFL) thickness (59.9 ± 1.2 μm in SC LMW-DS-injected rats compared with 44.0 ± 1.6 μm in control SC saline-injected rats; P < 0.01; Figure 16F).

[0304] Consideration Because inflammation precedes fibrosis in inflammatory fibroproliferative conditions, it is important to understand how LMW-DS regulates inflammatory and tissue remodeling pathways in human cells. Relevant protein and gene expression data from cultured human cells suggest that, through reprogramming of immune activation, inflammation resolution, and tissue remodeling (Example 7), LMW-DS regulates important innate and adaptive responses that lead to improved healing and functional remodeling of diseased and injured human tissues.

[0305] In Example 6, the mechanistic profile of LMW-DS at its protein level was investigated using the BioMAP® Diversity PLUS assay, which models inflammatory and wound healing responses in various human tissue types, consisting of 12 different validated human primary cell culture systems. The data demonstrated that LMW-DS has a distinct phenotypic profile with multimodal actions, exerting effects on different cell types and after stimulation with different inflammatory mediators. In particular, LMW-DS reduced the levels of three chemokines: CXCL9, CXCL10, and CXCL11, all of which are structurally related, signal through CXC receptor 3 (CXCR3), and are induced by interferon and TNFα. CXCR3 and its related chemokines play important roles in immune cell recruitment and function and have been implicated in numerous inflammatory and autoimmune diseases. In addition, they have been used as prognostic and predictive biomarkers for disease. The CXCR3 pathway plays an important role in the pathogenesis of glaucoma, and its expression level correlates with the progression of POAG. In models of ocular hypertension, antagonism of CXCR3 reduces IOP, increases AqH outflow, reduces inflammation, and reduces apoptosis of TM and retinal cells.

[0306] Both the gene expression data (Example 7) and results from the glaucoma model support the unique multimodal effects of LMW-DS through its actions on the TGFβ signaling pathway and other ECM remodeling signals. In POAG, TGFβ is involved in the progressive accumulation of ECM proteins in the TM, preventing the normal outflow of AqH, leading to elevated IOP. The rodent glaucoma model used in this example models TGFβ-induced anterior segment fibrosis and provides a robust, clinically relevant means to evaluate antifibrotic therapies. Here, we show that daily SC injections of LMW-DS ablated inflammatory signaling, reversed existing TM fibrosis, and reduced IOP, thereby rescuing RGCs from progressive death and supporting the potential of LMW-DS to modulate the progression of fibroproliferative disorders by modulating the TGFβ pathway. Furthermore, these results achieved with weekly SC injections of LMW-DS are comparable to those evident in the same in vivo model with twice-weekly IC injections of decorin, which demonstrate similar antifibrotic efficacy in experimental models of ocular, spinal cord, and brain fibrosis. Interestingly, as shown by gene expression analysis (Example 7), LMW-DS may function in part to modulate inflammation and promote tissue remodeling by increasing decorin expression.

[0307] In conclusion, this is the first study to demonstrate that subcutaneous injection of LMW-DS can resolve inflammation and nascent / pre-existing fibrosis in both rodent and human models of inflammation and fibrosis, thereby supporting functional tissue regeneration. Consequently, in the evaluated ocular disease models, inflammatory fibrosis was eliminated, IOP was rapidly normalized, and damaged retinal cells were protected from progressive death. We also provided clues about the multimodal mechanism of action of LMW-DS. Collectively, our study provides proof of concept that LMW-DS has potential as a novel disease-modifying therapy for the treatment of POAG and other acute and chronic fibroproliferative conditions. The antifibrotic and fibrolytic effects of LMW-DS have the advantage of preventing or at least inhibiting scar formation after the recovery phase (stage IV) and hyperinflammatory phase (stage III) in COVID-19 subjects (Figure 25). LMW-DS may thereby not only reduce the formation of harmful tissue scarring in, for example, the lungs, liver and heart, but may also resolve such scarring.

[0308] material and method LMW-DS was obtained at a stock concentration of 20 mg / ml from Tikomed AB, Viken, Sweden (WO 2016 / 076780) and kept in a temperature-monitored refrigerator. Immediately before use, an aliquot of LMW-DS was diluted to the appropriate concentration in sterile saline and administered by subcutaneous injection.

[0309] Culturing TM cell populations Human trabecular meshwork cells (TMC-6590; ScienCell Research Laboratories, Carlsbad, CA) were cultured in complete medium with TMC (6591; ScienCell Research Laboratories) and maintained at 37°C in 5% CO until they reached 80–90% confluence. Cells were seeded at a density of 5,000–10,000 cells per well in a Greiner-Bio Cell 96-well black cell culture microplate (655090, Sigma) and allowed to adhere overnight. The next day, cells were treated with TGFβ2 (1.0 ng / ml; Preprotech) and / or LMW-DS (4.0 μM; Tikomed AB) under serum-free conditions for 72 h.

[0310] Immunostaining and confocal imaging of TM cells After 72 hours of treatment, cells were fixed with 4% PFA (28908, Thermo Fisher Scientific) in PBS for 10 minutes, washed with PBS, and then with PBS containing saponin-based permeabilization buffer (00-8333-56, Thermo Fisher Scientific). Cells were then immunostained with fibronectin Alexa Fluor® 488 (53-9869-82, Thermo Fisher Scientific) and nuclear stain Hoechst 33342 (H21492, Thermo Fisher Scientific) in permeabilization buffer for 1 hour at room temperature in the dark. Finally, cells were washed twice in PBS containing saponin-based permeabilization buffer and once with PBS before image analysis by confocal microscopy. Images were acquired on a Yokogawa CQ1 spinning disk microscope using a 40x objective and appropriate excitation / emission settings for Hoechst and Alexa Fluor® 488. Z-series were acquired, and maximum intensity z-projections are displayed. Images were analyzed using Yokogawa image analysis software.

[0311] Experimental design for ocular hypertension model Prior to induction of elevated IOP, rats were randomly assigned to two groups (saline vehicle and LMW-DS treatment). Induction of elevated IOP was performed as previously described (Hill LJ, et al. Decorin reduces intraocular pressure and retinal ganglion cell loss in rodents through fibrolysis of the scarred trabecular meshwork. Investigative Ophthalmology & Visual Science. 2015;56(6):3743-57). On day 0, a self-sealing tunnel was created through the cornea using a 15° disposable blade. This access point was used for all subsequent twice-weekly IC injections of 3.5 μl of 5 ng / ml TGFβ1 (Peprotech, London, UK) performed using a sterile glass micropipette (Harvard Apparatus, Kent, UK) over the 28-day experiment. IOP was also measured twice weekly (immediately prior to IC injection) throughout the 28-day experiment. On day 14, non-responders to TGFβ (i.e., their IOP did not increase above baseline at any time) were excluded from further analysis, leaving eligible subjects: n = 7 rats (14 eyes) in the LMW-DS treatment group and n = 5 rats (10 eyes) in the saline vehicle treatment group. On day 14, rats received subcutaneous injections of 15 mg / kg LMW-DS (Tikomed AB) or an equivalent volume of saline, which continued for 14 days. OCT measurements of RFNL as a surrogate for RGC density were performed on day 28, and ocular tissues from both groups were processed for immunohistochemistry to assess the level of TM fibrosis and RGC survival.

[0312] Animals and surgery Rodent studies were carried out at the Biomedical Services Unit at the University of Birmingham (UK) under UK Home Office Licence No. 70 / 8611, in accordance with Home Office guidelines set out in the Animal Act 1986 (UK), in compliance with the ARRIVE guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

[0313] Adult (8-10 week old) male Sprague-Dawley rats (Charles River, Kent, UK) were housed under a 12-h light / dark cycle with food and water available ad libitum. Surgeries were performed at the Biomedical Services Unit at the University of Birmingham (UK) in accordance with the Home Office guidelines set out in the Animal Act 1986 (UK) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Intraocular injections, optical coherence tomography, and intraocular pressure measurements were all performed while the rats were under inhalation anesthesia using 2%-5% isofluorane / 98%-95% O2 (National Vet Supplies, UK). The health of all rats was closely monitored at all times.

[0314] IOP measurement IOP measurements were obtained using an iCare Tonolab (ICare, Helsinki, Finland) calibrated for rats. IOP was recorded twice weekly between 9 AM and 11 AM throughout the 28-day experiment to avoid confounding diurnal IOP variability. Immediately after induction of anesthesia, six rebound measurements were taken with a tonometer from the center of the cornea and averaged to obtain a single reading (mmHg). Each graph represents the mean ± SEM of three readings (six rebounds) taken from each rat to ensure accurate measurements.

[0315] Optical coherence tomography Retinal nerve fiber layer (RNFL) measurements were performed on day 28 using a Spectralis HRA3 confocal scanning laser ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). Images were acquired from rats under inhalation anesthesia (as described in the "Animals and Surgery" section above). OCT images were acquired from the retina around the optic nerve head, and built-in software was used to segment the images and quantify RNFL thickness.

[0316] Tissue preparation for immunohistochemistry Rats were euthanized on day 28 by intraperitoneal injection of sodium pentobarbital (National Veterinary Supplies) and, while under terminal anesthesia, perfused intracardially with 4% paraformaldehyde (PFA; TAAB, Aldermaston, UK) in PBS. Eyes were removed and immersion-fixed in 4% PFA in PBS for 2 hours at room temperature, followed by cryoprotection in increasing concentrations of sucrose (10%, 20%, and 30%) at 4°C for 24 hours each. Eyes were then embedded in optimal cutting temperature (OCT) embedding medium (Thermo Shandon, Runcorn, UK) in peel-away mold containers (Agar Scientific, Essex, UK) and rapidly frozen on crushed dry ice before storage at -80°C. Eyes were sectioned (15 μm) in the parasagittal plane through the optic nerve head (to account for RGC changes) using a Bright cryostat microtome (Bright, Huntingdon, UK) at −22°C, mounted on positively charged glass slides (Superfrost plus, Fisher Scientific, USA), dried at 37°C for 2 hours, and stored at −20°C.

[0317] Immunohistochemistry Unless otherwise specified, all reagents were purchased from Sigma (Poole, UK). Frozen tissue sections were allowed to equilibrate to room temperature for 30 minutes, then hydrated in PBS for 3 x 5 minutes, followed by permeabilization in 0.1% Triton X-100 for 20 minutes. After an additional 3 x 5 minute washes in PBS, the eye sections were isolated with a hydrophobic PAP pen (Vector Laboratories, Peterborough, UK). Nonspecific antibody binding sites were blocked with 0.5% BSA, 0.3% Tween 20®, and 15% normal goat serum in PBS. Sections were then placed in primary antibodies (laminin L9393, fibronectin F3648, Sigma, Brn3a SC-31984, Santa Cruz) and left overnight at 4°C, then washed 3x5 min in PBS and incubated with secondary antibodies (goat anti-rabbit Alexa Fluor 594 or goat anti-mouse Alexa Fluor 488) for 1 h at room temperature. Sections were washed 3x5 min in PBS before mounting with DAPI-containing Vectashield (Vector Laboratories). Control tissue sections containing secondary antibodies but no primary antibodies were negatively stained (not shown).

[0318] Microscopy and analysis Fluorescently stained sections were analyzed using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss Ltd, Oberkochen, Germany) by using randomization numbers with an operator masked to treatment group, as previously described (Hill, LJ et al. Decorin reduces intraocular pressure and retinal ganglion cell loss in rodents through fibrolysis of the scarred trabecular meshwork. Investigative Ophthalmol. Vis. Sci. 56, 3743-3757 (2015)). Briefly, images for each antibody were captured with the same exposure (250 ms), and pixel intensity was assessed relative to ECM levels. The percentage of immunofluorescent pixels above threshold within the region of interest was measured using ImageJ software.

[0319] Example 9 This example investigated the ability of LMW-DS to affect the in vitro release of IL-6 from stimulated human PBMCs. Human PBMCs can be stimulated in vitro with a variety of agents that directly and indirectly activate different cell subsets. Monitoring cytokine release allows for investigating the potential effects of drugs and predicting their action in patients. IL-6 is a typical pro-inflammatory cytokine that has been shown to be associated with numerous pathologies, including neurodegenerative conditions such as ALS, inflammatory diseases such as arthritis, and the serious consequences of viral infections, e.g., COVID-19.

[0320] material and method Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Paque PLUS (GE Healthcare; 11778538) density gradient centrifugation. 2 × 10 PBMCs were collected. 5Cells per well were cultured for 24 hours at 37°C under 5% CO2 in the presence or absence (vehicle) of three concentrations of LMW-DS (Tikomed AB, Viken, Sweden, WO 2016 / 076780)—60 μg / ml, 200 μg / ml, and 600 μg / ml—with or without stimulation (unstimulated PBS vehicle control) or stimulation (LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, or Cytostim). After centrifugation, cell culture supernatants were removed and stored at -20°C until analysis of IL-6 by ELISA. IL-6 levels in the supernatants were quantified by ELISA (R&D Systems; DY206) according to the manufacturer's instructions.

[0321] result Previously collected internal data guided the selection of stimulatory concentrations, using submaximal concentrations of LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, and Cytostim. In PBMC mixtures, all stimuli increased IL-6 release into the cell culture supernatant. Due to the encouraging results generated from cells from the first six donors regarding the effect of LMW-DS on LPS stimulation, we decided to extend the LPS testing to six additional donors. Furthermore, because the original selection concentrations of CpG + IL-15 and Cytostim gave relatively small increases in IL-6 release compared to unstimulated PBMCs, higher concentrations of CpG + IL-15 and Cytostim were investigated in cells from the additional six donors. These higher concentrations induced greater increases in IL-6 release compared to unstimulated cells.

[0322] LPS LPS (lipopolysaccharide) is a Toll-like receptor (TLR) 4 agonist. In human PBMC mixtures, the primary cell type directly activated by LPS is monocytes, which express TLR4. Monocytes are part of the innate immune system, and these myeloid cells can also be used to model responses to other myeloid cells, such as macrophages and microglia. In this study, LPS caused a substantial increase in IL-6 release into the cell culture supernatant (Figure 17A), and LMW-DS produced a concentration-dependent and statistically significant decrease in IL-6 (Figure 17A). This indicates that LMW-DS has the potential to reduce the pro-inflammatory consequences of IL-6 after TLR4 activation in monocytes.

[0323] peptidoglycan Peptidoglycan is a TLR2 agonist and is expressed primarily by monocytes and B lymphocytes in PBMC mixtures, the latter being a component of the acquired immune system well known for its important role in the generation of specific antibodies to antigens. Although peptidoglycan resulted in a substantial increase in IL-6 release into cell culture supernatants, experiments with cells from six donors showed little evidence that LMW-DS caused an overall reduction in IL-6 release, even at the highest concentration tested.

[0324] pokeweed mitogen Phytolacca mitogen is a lectin derived from pokeweed (Phytolacca americana). It induces T-lymphocyte-dependent activation of B lymphocytes. In this study, pokeweed mitogen significantly increased IL-6 release by PBMC mixtures, but this release was largely unaffected by LMW-DS (Figure 17C).

[0325] PHA-L PHA-L (phytohemagglutinin-L) is an L-type subunit lectin derived from kidney beans (kintoki beans) that crosslinks T lymphocyte surface receptors upon their activation. PHA-L induced a relatively small increase in IL-6 release from PBMC mixtures, which was significantly inhibited by LMW-DS in a concentration-dependent manner (Fig. 17D).

[0326] CpG+IL-15 CpG-ODN is a short single-stranded DNA molecule that activates TLR9, which is expressed primarily by monocytes and B cells in PBMC mixtures. IL-15 synergizes with CpG in stimulating B lymphocytes. Unlike pokeweed mitogen, CpG + IL-15 directly activates B lymphocytes, i.e., is T lymphocyte-independent. In a first-round experiment with PBMCs from six donors, the selected concentrations of CpG-ODN + IL-15 caused only a slight increase in IL-6 release into the cell culture supernatant. While this relatively low level of IL-6 release was overall unaffected by LMW-DS (Figure 17E), in additional experiments performed with additional donors to increase the number of LPS stimulations, these same additional donors were subjected to higher concentrations of CpG+IL-15 to elicit a more potent IL-6 release beyond that evident from unstimulated cells; this was achieved, yet there was no overall effect of LMW-DS (Figure 17F).

[0327] Cytostim Cytostim is an antibody-based T lymphocyte activator. It binds to the T cell receptor (TCR) and crosslinks it with major histocompatibility complex (MHC) molecules on antigen-presenting cells. Thus, Cytostim stimulates both CD4 and CD8 T lymphocytes. In this study, initial rounds of experiments with PBMCs from six donors used concentrations of Cytostim that resulted in only a relatively small increase in IL-6 release into the cell culture supernatant, which was generally unaffected by LMW-DS (Figure 17G). PBMCs from these same additional donors were examined with higher concentrations of Cytostim to produce greater IL-6 release compared to that from unstimulated cells, yet there was no overall effect of LMW-DS (Figure 17H). Although all stimuli investigated in this study were able to increase IL-6 release from cells in the PBMC mixture, the targeted effect of LMW-DS (versus LPS and PHA-L) on IL-6 release suggests a refined mode of action rather than a general ability to reduce IL-6 release.

[0328] Example 10 Monocytes are circulating innate immune cells that are a key component of the immune system. In addition, because they are readily available, they can be used to model other less accessible myeloid cells, such as macrophages and microglia.

[0329] Similar to various myeloid cells, monocytes can be activated via Toll-like receptor (TLR) agonists, such as TLR2 and TLR4 receptor agonists peptidoglycan and lipopolysaccharide (LPS), respectively. Activation can be monitored by the expression of activation markers (via flow cytometry) and / or by the secretion of cytokines, such as the pro-inflammatory cytokine interleukin-6 (IL-6). In Example 9, LMW-DS reduced IL-6 secretion in human peripheral blood mononuclear cell (PBMC) preparations in response to LPS stimulation. However, the diverse cell types in PBMC mixtures hinder interpretation of the exact cell type mediating this response. This study investigated the ability of LMW-DS to modulate LPS-stimulated IL-6 release from purified human monocytes to identify the precise intracellular target of LMW-DS. IL-6 is a typical pro-inflammatory cytokine that has been associated with numerous pathologies, including neurodegenerative conditions such as ALS, inflammatory diseases such as arthritis, and viral / bacterial infections, the serious consequences of which can result from COVID-19.

[0330] material and method Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Paque PLUS (GE Healthcare; 11778538) density gradient centrifugation. Monocytes were purified using the EasySep™ Human Monocyte Enrichment Kit (StemCell), which purifies monocytes "intactly" to preserve their phenotype.

[0331] Monocytes were cultured for 24 hours at 37°C under 5% CO2 in the presence or absence (vehicle) of three concentrations of LMW-DS (Tikomed AB, Viken, Sweden, WO 2016 / 076780)—maximum concentrations of 600 μg / ml, 200 μg / ml, and 60 μg / ml—with or without heparin (2.0, 6.0, or 20 μg / ml; equivalent to 0.406, 1.218, and 4.06 units / ml; Sigma-Aldrich) or dexamethasone (3.0 μM; Sigma-Aldrich)—either unstimulated (PBS vehicle control) or stimulated (LPS or peptidoglycan). After centrifugation, cell culture supernatants were removed and stored at -20°C until analysis of IL-6 by ELISA. The levels of IL-6 in the supernatants were quantified by ELISA (R&D Systems) according to the manufacturer's instructions.

[0332] result Previously collected internal data guided the selection of stimulatory concentrations and the use of submaximal concentrations of LPS and peptidoglycan, which also correspond to the same concentrations of LPS and peptidoglycan used in Example 9 when a human PBMC mixture was used as the IL-6 source. The TLR2 agonist peptidoglycan and the TLR4 agonist LPS resulted in the release of IL-6 into cell culture supernatants from human purified monocytes (FIG. 18). Average results from monocytes from 10 healthy donors showed a statistically significant, concentration-dependent inhibition of LPS-stimulated IL-6 release into the cell culture supernatant by LMW-DS (Fig. 18A), and heparin produced a statistically significant, concentration-dependent enhancement of LPS-stimulated IL-6 release into the cell culture supernatant (Fig. 18C). As expected, the glucocorticoid steroid dexamethasone statistically significantly inhibited LPS-stimulated IL-6 release into the cell culture supernatant (Fig. 18B).

[0333] Average results from monocytes from 10 healthy donors showed a statistically significant, concentration-dependent increase in peptidoglycan-stimulated IL-6 release into the cell culture supernatant by LMW-DS (Figure 18D). This enhancement was mirrored to some extent by heparin, although the trend did not reach statistical significance (Figure 18F). As expected, the presence of dexamethasone statistically significantly inhibited peptidoglycan-stimulated IL-6 release into the cell culture supernatant (Figure 18E).

[0334] Monocytes are part of the innate immune system, and these myeloid cells can also be used to model responses to other myeloid cells, such as macrophages and microglia. In this study, the clear and substantial effect of LMW-DS in suppressing the LPS-induced increase in IL-6 release from purified monocytes provides strong evidence that these cells are targets of LMW-DS.

[0335] Example 11 Activation of the immune response in diseased, injured, or infected tissues is reflected by changes in the phenotypic balance of peripheral blood mononuclear cells (PBMCs). Thus, evaluation of the impact of test agents on components of the adaptive and innate immune systems may reveal mechanistic cellular pathways to better understand clinical changes associated with the test therapy and, potentially, the identification of cellular and / or molecular biomarkers predictive of therapeutic efficacy for different disease states.

[0336] Human PBMCs can be directly and indirectly stimulated in vitro with a variety of agents that activate different cell subsets and mimic immune responses associated with compromised tissue. Monitoring cytokine release from PBMCs allows for the investigation of potential drug effects and the prediction of drug action in specific patients.

[0337] material and method Example 9 investigated the ability of LMW-DS to modulate IL-6 secretion from human PBMCs using various stimuli. This study performed a more extensive analysis of the cell culture supernatants resulting from Example 9. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Paque PLUS density gradient centrifugation. PBMCs were collected at 2 x 10 5 Cells per well were cultured at 37°C in 5% CO2 in the presence or absence (vehicle) of three concentrations of LMW-DS (Tikomed AB, Viken, Sweden, WO 2016 / 076780) at a maximum concentration of 600 μg / ml, 200 μg / ml, and 60 μg / ml, either unstimulated (unstimulated PBS vehicle control) or stimulated (LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, or Cytostim). Therefore, treatment was as follows for each PBMC donor: i. Vehicle ii. Stimulation iii. Stimulation + LMW-DS (60μg / ml) iv. Stimulation + LMW-DS (200μg / ml) v. Stimulation + LMW-DS (600μg / ml) Each was repeated three times, resulting in a total number of samples: 1. LPS: 165 samples (from 11 donors) 2. Peptidoglycan from Bacillus subtilis; 90 samples (from 6 donors) 3. PHA-L; 90 samples (from 6 donors) 4. 0.2 μM CpG + IL-15; 90 samples (from 6 donors) 5. 1.0 μM CpG + IL-15; 90 samples (from 6 donors) 6. Phytolacca mitogen; 90 samples (from 6 donors) 7. 10 μl of Cytostim; 90 samples (from 6 donors) 8. 30 μl of Cytostim; 90 samples (from 6 donors) Total number of supernatant samples from all stimulations (all cell types) = 795

[0338] After treatment, cell culture supernatants were removed, centrifuged, and stored at -20°C until thawed for multiplex analysis of various cytokines using a 5-plex human magnetic Luminex assay (R&D Systems; catalog number LXSAHM-05). Luminex analysis was performed exactly according to the manufacturer's protocol.

[0339] result Previously collected internal data guided the selection of stimulatory concentrations to use submaximal concentrations of LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, and Cytostim; submaximal concentrations of stimuli tend to allow both increased and decreased modulation (if present) to be identified. In PBMC mixtures, all stimuli increased cytokine release into the cell culture supernatant, although some stimuli were more effective than others (Figures 19-23).

[0340] LPS LPS (lipopolysaccharide) is a Toll-like receptor (TLR) 4 agonist. In human PBMC mixtures, the primary cell type directly activated by LPS is monocytes, which express TLR4. Monocytes are part of the innate immune system, and these myeloid cells can also be used to model responses to other myeloid cells, such as macrophages and microglia. In this study, when assessing the effects of LPS, there was a slight increase in the secretion of IFNγ, TNFα, IL-1β, and IL-10, but a significant increase in IL-8 secretion (Figures 19-23). ​​LMW-DS caused a concentration-dependent, but slight decrease in IFNγ and IL-10 (Figures 19 and 23), and the presence of the highest concentration of LMW-DS resulted in a slight decrease in stimulated secretion of IL-1β, IL-8, and TNFα (Figures 20-22).

[0341] peptidoglycan Peptidoglycan is a TLR2 agonist expressed primarily by monocytes and B lymphocytes in PBMC mixtures, the latter being a component of the acquired immune system well known for its important role in the generation of specific antibodies to antigens. Peptidoglycan causes a substantial increase in the release of IL-1β, IL-8, and TNFα into cell culture supernatants. However, examination of results from all donors revealed little evidence that LMW-DS, even at the highest concentration tested, resulted in an overall reduction in cytokine release, although there was a corresponding increase in IL-1β and TNFα secretion (Figures 19-23).

[0342] PHA-L PHA-L (phytohemagglutinin-L) is an L-type subunit lectin derived from kidney beans (kintoki beans) that crosslinks T lymphocyte surface receptors upon their activation. In PBMC mixtures, PHA-L induced increases in IFNγ, IL-8, IL-10, and TNFα, with an overall small increase in IL-1β (Figures 19-23). ​​LMW-DS produced a small increase in the stimulated release of IFNγ, IL-8, and TNFα, but not IL-1β (Figures 19-22). In contrast, LMW-DS produced a large, concentration-dependent decrease in IL-10 secretion (Figure 23).

[0343] pokeweed mitogen Phytolacca mitogen is a lectin derived from Phytolacca americana. It induces T-lymphocyte-dependent activation of B lymphocytes. In this study, Phytolacca mitogen significantly increased secretion of IFNγ, IL-1β, IL-8, IL-10, and TNFα by PBMC mixtures (Figures 19-23), but this release was largely unaffected by LMW-DS, except for a concentration-dependent reduction in IL-10 secretion (Figure 23).

[0344] CpG+IL-15 CpG-ODN is a short, single-stranded DNA molecule that activates TLR9, which is expressed primarily by monocytes and B cells in PBMC mixtures. IL-15 synergizes with CpG in stimulating B lymphocytes. Unlike pokeweed mitogen, CpG + IL-15 directly activates B lymphocytes, i.e., in a T lymphocyte-independent manner. In this study, there was only strong evidence for this stimulation to increase IL-8 secretion. Overall, in these experiments, LMW-DS had little effect on this response (Figures 19-23).

[0345] Cytostim Cytostim is an antibody-based T lymphocyte activator. It binds to the T cell receptor (TCR) and crosslinks it with major histocompatibility complex (MHC) molecules on antigen-presenting cells. Thus, Cytostim stimulates both CD4 and CD8 T lymphocytes. Overall, Cytostim led to increased secretion of IFNγ, IL-1β, IL-8, IL-10, and TNFα (Figures 19-23), with only a slight decrease associated with the presence of LMW-DS, except for IL-10 secretion (Figure 23), which showed a significant concentration-dependent decrease in the presence of LMW-DS. Overall, the data support the role of LMW-DS in benefiting patients with post-infectious inflammatory responses, such as sepsis. Post-infectious sepsis is considered a dysregulated immune response that results in organ damage. Sepsis is responsible for the majority of morbidity, mortality, and healthcare expenditures. The urgent need for novel therapeutics has been highlighted once again during the current pandemic, with the largest number of deaths among critically ill COVID-19 patients.

[0346] During sepsis in response to infection, excessive production of inflammatory cytokines (cytokine storm) can lead to septic shock. Several cytokines regulated by LMW-DS are elevated and thought to be involved in the pathogenesis of sepsis. For example, IFNγ, IL-1β, IL-6, IL-8, and TNFα continue to increase in non-survivors. TNFα and IL-1β play a key role in sepsis, acting on cells such as macrophages, where they amplify the inflammatory cascade, increasing the release of other pro-inflammatory cytokines and reactive oxygen and nitrogen species, and also acting on endothelial cells, where they mediate inflammation-induced activation of coagulation. An additional role of TNFα includes promoting neutrophil extravasation through its effect on endothelial cells. Blockade of TNFα with monoclonal antibodies has also been demonstrated to improve survival in patients with severe sepsis. IL-6 can enhance the activation of T cells, B cells, and the coagulation system, and IL-6 levels correlate with the clinical severity of sepsis. Knockout of IL-6 reduces lung injury in a mouse model of acute lung injury. IL-8 acts to potently attract and activate neutrophils, and levels correlate with the severity of sepsis. Despite the promise of targeting cytokines described above, their role in sepsis remains a "double-edged sword," as, in addition to their pathological role in sepsis, cytokines may play host-protective and immunomodulatory roles in host defense.

[0347] One challenge in inflammatory diseases such as sepsis is how to target components of the response without producing long-term immunosuppression. While the benefits of neutralizing TNFα in sepsis have been demonstrated, monoclonal antibodies with long half-lives, such as infliximab, adalimumab, and certolizumab, have half-lives of approximately 14 days, creating challenges in the timing and route of administration. Drugs that target multiple cytokines, such as LMW-DS, with a relatively short duration of action at specific stages of the disease, could therefore potentially provide additional clinical benefit to patients with inflammatory diseases such as sepsis. In the case of COVID-19-associated sepsis, an additional benefit of LMW-DS may arise from its direct effect on the virus; thus, incubation of SARS-CoV-2 with exogenous LMW-DS reduces binding to human epithelial cell lines and reduces viral replication in ex vivo human lung tissue explants.

[0348] Example 12 The purpose of this example was to evaluate the effect of repeated LMW-DS administration on serum metabolite concentrations in serum samples from a cohort of ALS patients in Sweden who participated in a study titled "A Single-Center, Single-Arm, Open-Label Study Evaluating the Safety, Tolerability, and Efficacy of Subcutaneously Administered ILB® in Patients with Amyotrophic Lateral Sclerosis." Using a longitudinal study, the study aimed to measure changes in selected serum metabolites in each ALS patient before and after weekly LMW-DS treatment. Changes in measured metabolites indicate the patient's biochemical response to LMW-DS, potentially underlying disease-modifying and potentially mediated mechanisms of action of the drug in this patient population.

[0349] material and method After the initial screening visit, patients received a single injection of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780) in saline at 1.0 mg / kg body weight administered five times a week into the subcutaneous fat of the lower abdomen.

[0350] Peripheral venous blood samples were collected from patients using standard tourniquet procedures from the antecubital vein into single VACUETTE® polypropylene tubes containing a serum separator and clot activator (Greiner-Bio One GmbH, Kremsmunster, Austria) at least 15 minutes after complete withdrawal. After 30 minutes at room temperature, the blood samples were centrifuged at 1,890 × g for 10 minutes, and the resulting serum samples were stored at −20°C until analysis.

[0351] After thawing, a 500 μl aliquot of each serum sample was supplemented with 1 ml of HPLC-grade acetonitrile, vortexed for 60 seconds, and centrifuged at maximum speed in a benchtop centrifuge to precipitate proteins. The supernatant was washed with a large volume of HPLC-grade chloroform to remove organic solvents, centrifuged, and the upper aqueous phase was transferred to different tubes, clearly labeled to distinguish between samples, and stored at -80°C until analysis to determine the various water-soluble compounds.

[0352] A second, approximately 300 μl aliquot of each serum sample was light-protected and then processed to extract fat-soluble antioxidants as described in detail elsewhere (Lazzarino et al., Single-step preparation of selected biological fluids for the high-performance liquid chromatographic analysis of fat-soluble vitamins and antioxidants. J Chromatogr A. 2017;1527:43-52). Briefly, samples were supplemented with 1 ml of HPLC-grade acetonitrile, vortexed vigorously for 60 seconds, and incubated at 37°C for 1 hour in a water bath under agitation to allow complete extraction of lipid-soluble compounds. Samples were then centrifuged at 20,690 x g for 15 minutes at 4°C to precipitate proteins, and the clear supernatant was stored at -80°C until HPLC analysis of fat-soluble vitamins and antioxidants.

[0353] A method described elsewhere (Tavazzi et al., Simultaneous high performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino and acids, and dicarboxylic acids for the chemical diagnosis of inborn errors of metabolism. Clin Biochem. 2005;38:997-1008; Romitelli et al., Comparison of nitrite / nitrate concentration in human plasma and serum samples measured by the enzymatic batch Griess assay, ion-pairing HPLC and ion-trap GC-MS:The importance of a correct removal of proteins in the Griess assay.J Chromatogr B Analyt Technol Biomed Life Sci.2007;851:257-267;Amorini et al.,Metabolic profile of amniotic fluid as a biochemical tool to screen for inborn errors of metabolism and fetal anomalies.Mol Cell According to the method described in Biochem. 2012;359:205-216, the following water-soluble compounds were separated and quantified by HPLC in deproteinized serum samples: hypoxanthine, xanthine, uric acid, malondialdehyde (MDA), nitrite, nitrate, N-acetylaspartic acid (NAA), citrulline (CITR), alanine (ALA), and ornithine (ORN).

[0354] The following fat-soluble vitamins and antioxidants in deproteinized serum samples were separated and quantified by HPLC according to a previously described method (Lazzarino et al., Cerebrospinal fluid ATP metabolites in multiple sclerosis. Multiple Scler J. 2010;16:549-554). In addition to the fat-soluble antioxidants and vitamins mentioned above, the amount of total bilirubin in serum samples was also measured.

[0355] statistics Comparisons of pre- and post-treatment subgroups were performed using paired-samples two-tailed Student's t-tests. Comparisons of each subgroup with control healthy subjects were performed using two-tailed nonparametric Mann-Whitney U tests for unpaired observations. Differences of P<0.05 were considered statistically significant.

[0356] result Statistical analysis showed that the two pre- and post-treatment subgroups of patients differed significantly in serum concentrations of NAA (Figure 26), uric acid (Figure 27), MDA (Figure 31), nitrate (NO3) (Figure 29), nitrite + nitrate (NO3 + NO2) (Figure 30), total oxypurines (Figure 28), ALA (Figure 32), CITR (Figure 33), ORN / CITR ratio (Figure 34), vitamin E (α-tocopherol and γ-tocopherol) (Figures 35 and 36), and total bilirubin (Figure 43).

[0357] Data for the above compounds are presented in the box plots of Figures 26-36 (reporting minimum, maximum, median, 25th and 75th percentiles), where values ​​for control healthy subjects (ages 25-65 years) were compared with both groups of patients (before and after treatment), including those from a previously collected dataset derived from an Italian cohort of patients. In all figures, differences relative to control values ​​are indicated with a single asterisk (*), and differences between the two patient subgroups are indicated with two asterisks (**).

[0358] ALS patients had higher serum NAA levels than those measured in healthy control subjects, likely due to a reduction in viable neurons (Figure 26). Significantly lower NAA values ​​were measured even after LMW-DS treatment, thus suggesting a protective effect of the drug on cell survival.

[0359] ALS patients have higher serum concentrations of uric acid and the sum of oxypurines (hypoxanthine + xanthine + uric acid) than measured in controls (Figure 28), likely the result of an imbalance in energy metabolism leading to activation of the adenine nucleotide degradation pathway and resulting increase in circulating purine compounds. LMW-DS treatment reduces both of these parameters, thus suggesting restoration of mitochondrial function, including an increase in cellular energy status.

[0360] ALS patients have higher serum concentrations of nitrate (Figure 29), nitrite + nitrate (Figure 30), and MDA (Figure 31) than measured in controls, strongly suggesting persistent oxidative / nitrosative stress leading to increased circulating concentrations of ROS-mediated lipid peroxidation (MDA) and stable end products of nitric oxide metabolism (nitrate and nitrite + nitrate). LMW-DS treatment reduced the levels of these parameters, thus indicating direct scavenging activity of the compound, favorable effects on genes such as BDNF, modulation of scavenging enzyme levels, and / or favorable effects on mitochondrial function ultimately leading to lower levels of ROS production.

[0361] ALS patients had higher serum concentrations of AA than those measured in controls (Figure 32), likely due to a higher rate of muscle protein breakdown. LMW-DS treatment normalized circulating ALA levels (equal to those of controls and significantly lower than those of the pretreatment subgroup), suggesting a favorable effect of the drug on muscle metabolism and function.

[0362] ALS patients had higher CITR serum concentrations (Figure 33) and lower ORN / CITR ratios (Figure 34) than those measured in controls, thereby confirming the presence of persistent nitrosative stress, which leads to excessive nitric oxide production and subsequent increases in reactive nitrogen species (RNS). LMW-DS treatment improved both parameters, likely by reducing the expression of inducible nitric oxide synthase, an enzyme involved in inducing nitrosative stress.

[0363] ALS patients have serum concentrations of both α- and γ-tocopherol (the two major forms of vitamin E) that are lower than those measured in controls (Figures 35 and 36), thereby suggesting a significant reduction in these major fat-soluble antioxidants, which play an important role as inhibitors of ROS-mediated peroxidation of fatty acids in membrane phospholipids. LMW-DS treatment improves both parameters.

[0364] ALS patients have higher serum concentrations of total bilirubin than those measured in controls, likely reflecting disturbed liver function. LMW-DS treatment reduces this parameter, again tending to normalize levels of this serum metabolite, indicating improved liver function.

[0365] LMW-DS can improve liver function, thereby suppressing the deterioration of liver function, which has been observed in some COVID-19 cases. Therefore, the effect of this LMW-DS treatment is particularly beneficial in convalescent (stage IV) and long-term COVID-19 patients (Figure 25).

[0366] Example 13 This example investigated the ability of LMW-DS to affect the interaction between SARS-CoV-2 spike protein and ACE2, as assessed using the RayBio® COVID-19 Spike-ACE2 Binding Assay Kit.

[0367] COVID-19 results from infection with the virus SARS-CoV-2. One mechanism by which the virus enters human cells is currently understood to be through direct interaction between the SARS-CoV-2 spike protein and angiotensin I-converting enzyme 2 (ACE2); the latter is expressed on the plasma membrane of a variety of human cells. This interaction can be assessed using the RayBio® COVID-19 Spike-ACE2 Binding Assay Kit, a rapid ELISA-based colorimetric assay.

[0368] material and method The RayBio® COVID-19 Spike-ACE2 Binding Assay Kit (catalog no. CoV-SACE2) was used according to the manufacturer's instructions (version 1.6) to assess the ability of LMW-DS to disrupt the interaction between the SARS-CoV-2 spike protein and ACE2. LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780) was added at concentrations ranging from 3.0 μg / ml to 600 μg / ml in saline.

[0369] result There was clear evidence that LMW-DS could reduce the interaction between the SARS-CoV-2 spike protein and ACE2 (Figure 37). More specifically, LMW-DS caused a concentration-dependent disruption of the SARS-CoV-2 spike protein-ACE2 interaction. Inhibition of the interaction between the SARS-CoV-2 spike protein and ACE2, as occurred with LMW-DS, would be predicted to benefit patients by reducing the ability of the SARS-CoV-2 virus to enter human cells.

[0370] Example 14 Using a longitudinal study, this study aimed to measure changes in selected serum metabolites in ALS patients before dextran sulfate administration and at various times after the start of treatment. The changes in measured metabolites indicate the patient's biochemical response to dextran sulfate, which is the underlying potential disease-modifying and mechanism of action of this ALS drug.

[0371] material and method Dextran sulfate (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780) was administered at 2 mg / kg by subcutaneous injection daily and once weekly for 10 weeks to eight human ALS patients.

[0372] Peripheral venous blood samples were collected from patients using standard tourniquet procedures from the antecubital vein into single VACUETTE® polypropylene tubes containing a serum separator and clot activator (Greiner-Bio One GmbH, Kremsmunster, Austria) before (week 0) and after (weeks 5 and 10) dextran sulfate administration, with at least 15 minutes of complete withdrawal. After 30 minutes at room temperature (20–25°C), the blood samples were centrifuged at 1,890 × g for 10 minutes to obtain serum aliquots.

[0373] A 500 μl serum aliquot was supplemented with 1 ml of HPLC-grade acetonitrile, vortexed for 60 seconds, and centrifuged at maximum speed in a benchtop centrifuge to precipitate proteins. The supernatant was washed with a large volume of HPLC-grade chloroform to remove the organic solvent, centrifuged, and the upper aqueous phase was transferred to different tubes, clearly labeled to distinguish between samples, and stored at -80°C until analysis to determine the various water-soluble compounds.

[0374] Patients were assessed pretreatment with the ALSAQ-40, then weekly for 10 treatment weeks, and at follow-up visits.

[0375] result Lactic acid is produced by muscles and accumulates in the blood during exercise in normal and ALS patients. Elevated levels of serum lactate indicate improved muscle function / use. Figure 38 shows serum lactate levels in ALS patients before (week 0) and after dextran sulfate administration. The data show that circulating lactate levels, primarily derived from muscle cell metabolism, significantly increased with increasing time of dextran sulfate administration (*significant difference compared to week 0, p<0.01). After 5 weeks of dextran sulfate treatment, serum lactate levels increased by 29.8% from 1.78±0.59 to 2.31±1.02 μmol / L (p<0.01, Wilcoxon signed-rank test), whereas after 10 weeks of dextran sulfate treatment, serum lactate levels increased by 70% to 3.02±1.59 μmol / L. Thus, dextran sulfate administration enhanced muscle activity in ALS patients.

[0376] The ALSAQ-40 subscores, termed "Activities of Daily Living / Independence" (ADL) and "Physical Mobility" (PM), reflect patients' views of their level of physical activity and independence. A decrease in score reflects improved physical activity. After 10 weeks of dextran sulfate treatment, the ADL subscore significantly decreased by 18.6%, from 58.9±21.4 to 44.4±24.7 (p<0.05) (see FIG. 39), while the PM subscore decreased by 16%, from 27.2±22.2 to 22.7±20.2. These results indicated an improvement in patients' physical activity during treatment.

[0377] The improvement in muscle function induced by dextran sulfate administration is beneficial for COVID-19 patients who may otherwise suffer from muscle pain and worsening muscle function. Thus, muscle improvement achieved by dextran sulfate is beneficial during the recovery phase (Stage IV) (Figure 25).

[0378] Example 15 material and method The safety, tolerability, and potential efficacy of subcutaneously administered dextran sulfate were evaluated in patients with intermediate-rate ALS in a single-center, single-arm, open-label phase 2a clinical trial. The clinical trial was conducted at Sahlgrenska University Hospital, Gothenburg, Sweden, and was overseen and approved by the Ethics Committee of the University of Gothenburg and the Swedish Medical Products Agency. Dextran sulfate (Tikomed AB, Viken, Sweden, WO 2016 / 076780) was administered at 1 mg / kg by subcutaneous injection daily and once a week for 5 weeks to 13 human patients with ALS. Blood samples were collected at defined test intervals into vacutainer tubes via a venous catheter. Laboratory analysis of plasma was performed immediately after collection by the Clinical Chemistry Laboratory of Sahlgrenska University Hospital. The ALSFRS-R was assessed pre-treatment and then weekly for the 5 treatment weeks and at follow-up visits.

[0379] result Myoglobin is a protein typically found in cardiac and skeletal muscles. When injury / disease causes muscle damage, elevated myoglobin levels are found in the bloodstream. A decrease in serum myoglobin levels indicates a decrease in muscle degeneration. Serum myoglobin data from the patients showed a statistically significant 30% decrease in myoglobin levels from 133.92±126.28 to 103.69±72.16 μg / L (compared to Day 1, p=0.021) after 4 weeks of dextran sulfate treatment, indicating a drug-related reduction in the patient's muscle tissue degeneration and muscle twitch rate during treatment (see FIG. 40). The appearance of the muscle enzyme creatine kinase in the blood is generally considered a biomarker of muscle damage and is particularly useful in diagnosing conditions involving muscle spasms, including ALS. Elevated creatine kinase levels are a common feature of ALS patients. Decreased levels of serum creatine kinase indicate remission of disease-associated myopathy. After 4 weeks of dextran sulfate treatment, serum creatine data from patients showed a statistically significant (p<0.05, Wilcoxon signed-rank test) 13.3% decrease from 7.15±5.74 to 6.2±5.08 μkat / L, indicating a drug-related reduction in muscle spasms in treated patients (see FIG. 41). Hepatocyte growth factor (HGF) is a naturally occurring growth factor that has been shown to act as a potent neuroprotective and myogenic agent and to be beneficial against degenerative disease progression in multiple animal models, including ALS models. Interestingly, Hauerslev S et al. (2014, Plos One 9:e100594) demonstrated an 18% increase in muscle mass after two weeks of recombinant HGF treatment in a mouse model of muscle spasm. The observation that HGF treatment can induce such a rapid regenerative response in skeletal muscle in this animal model of muscle spasm is related to the rapid muscle response observed in ALS patients in response to dextran sulfate.

[0380] Pharmacokinetic data from ALS patients showed a statistically significant (p<0.001) increase in circulating HGF to pharmacologically relevant levels after dextran sulfate injection, from 820±581 to a peak of 37863±14235 μg / L at 2.5 hours (see FIG. 42). This indicates a direct myogenic, as well as an indirect neurotrophic, HGF-mediated effect on muscle contraction after dextran sulfate administration.

[0381] Biochemical evidence of reduced muscle degeneration supports clinical observations of improved muscle function. For example, in the ALSFRS-R, function mediated by the cervical, trunk, lumbosacral, and respiratory muscles is assessed by three items each, and scores in these categories show close agreement with objective measures of muscle strength. Notably, two patients with severe bulbar paresis experienced near-complete resolution of this condition during the 5-week treatment period.

[0382] The improvement in muscle function induced by dextran sulfate administration is beneficial for COVID-19 patients who may otherwise suffer from muscle pain and worsening muscle function. Thus, the muscle improvement achieved by dextran sulfate is beneficial during the recovery phase (Stage IV) (Figure 25). In addition, increased levels of HGF activate tissue repair and wound healing in COVID-19 patients, which is relevant during the pneumonia phase (Stage II) (Figure 25).

[0383] The above-described embodiments should be understood as a few illustrative examples of the present invention. It will be apparent to 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 feasible.

Claims

1. Use of dextran sulfate having an average molecular weight of 10,000 Da or less, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention, suppression, or treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or coronavirus disease 2019 (COVID-19).

2. Use of dextran sulfate having an average molecular weight of 10,000 Da or less, or a pharmaceutically acceptable salt thereof, in the manufacture of a drug for preventing, suppressing, or treating an inflammatory disease selected from the group consisting of acute respiratory distress syndrome (ARDS) and systemic inflammatory response syndrome (SIRS).

3. The use described in claim 1 or 2, wherein the drug comprises dextran sulfate or a pharmaceutically acceptable salt thereof and is formulated for systemic administration to the subject.

4. The use described in claim 3, wherein the drug comprises dextran sulfate or a pharmaceutically acceptable salt thereof and is formulated for intravenous or subcutaneous administration to the subject.

5. The use described in claim 3, wherein the drug comprises dextran sulfate or a pharmaceutically acceptable salt thereof and is formulated for subcutaneous administration to the subject.

6. The use according to any one of claims 1 to 5, wherein the average molecular weight is in the range of 2,000 to 10,000 Da.

7. The use according to claim 6, wherein the average molecular weight is in the range of 4,500 to 7,500 Da.

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

9. The dextran sulfate or a pharmaceutically acceptable salt thereof has a number average molecular weight (M) in the range of 1,850 to 3,500 Da as measured by nuclear magnetic resonance (NMR) spectroscopy. ｎ 9. The use according to any one of claims 1 to 8, wherein 10. The use of claim 9, wherein the dextran sulfate or pharmaceutically acceptable salt thereof has an M n in the range of 1,850 to 2,500 Da as measured by NMR spectroscopy.

11. The use of claim 9, wherein the dextran sulfate or pharmaceutically acceptable salt thereof has an M n in the range of 1,850 to 2,300 Da as measured by NMR spectroscopy.

12. The dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 1,850 to 2,000 Da as measured by NMR spectroscopy. ｎ The use according to claim 11, wherein

13. The use according to any one of claims 1 to 12, wherein the dextran sulfate or pharmaceutically acceptable salt thereof has an average number of sulfates per number average glucose unit in the range of 2.5 to 3.

0.

14. The use of claim 13, wherein the dextran sulfate or pharmaceutically acceptable salt thereof has an average number of sulfates per number average glucose unit in the range of 2.5 to 2.

8.

15. The use of dextran sulfate or a pharmaceutically acceptable salt thereof according to claim 14, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of sulfates per number average glucose unit in the range of 2.6 to 2.

7.

16. The use according to any one of claims 1 to 15, wherein the dextran sulfate or pharmaceutically acceptable salt thereof has an average of 5.1 glucose units and an average number of sulfates per glucose unit of 2.6 to 2.

7.

17. The use of any one of claims 1 to 16, wherein the drug comprises dextran sulfate or a pharmaceutically acceptable salt thereof and is formulated as an injectable aqueous solution.

18. The use according to any one of claims 1 to 17, wherein the pharmaceutically acceptable salt thereof is the sodium salt of dextran sulfate.

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