TREATMENT OF CORONAVIRUS INFECTIONS

Abstract

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

Description

TECHNICAL FIELD

The present invention generally relates to treatment of infections and inflammatory diseases and the longer-term sequelae of these infectious diseases, and in particular to the use of dextran sulfate in treatment of such coronavirus infections and infectious diseases and inflammatory diseases that may be caused by coronavirus infections.

BACKGROUND

Coronaviruses (CoV) are a group of related enveloped viruses that cause diseases in mammals and birds. In humans, coronavirus infection may lead to respiratory tract infections that can be mild, such as some cases of the common cold, and others that can be lethal, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19).

Coronaviruses vary significantly in risk factor. Coronaviruses may cause colds with major symptoms, such as fever, and sore throat from swollen adenoids. Coronaviruses may also cause pneumonia, either direct viral pneumonia or a secondary bacterial pneumonia, and bronchitis, either direct viral bronchitis or a secondary bacterial bronchitis. Coronaviruses may also be associated with long-term organ disease and functional debilitation, sometimes called “long-COVID”.

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

The lungs are the organs typically most affected by SARS-CoV-2 because the virus accesses host cells via the enzyme angiotensin-converting enzyme 2 (ACE2), which within the lungs is most abundant in the type II alveolar cells of the lungs. The virus uses a special surface glycoprotein called a “spike” (peplomer) to engage ACE2 and enter the host cell. This binding between the spike protein and ACE2 is aided by heparan sulfate present on the host cell surface. As the alveolar disease progresses, respiratory failure might develop and death may follow.

The coronavirus may also affect gastrointestinal organs as ACE2 is abundantly expressed in the glandular cells of gastric, duodenal and rectal epithelium as well as endothelial cells and enterocytes of the small intestine.

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. Muscle pain, sputum production, diarrhea, and sore throat are less common. While the majority of cases result in mild symptoms, some progress to pneumonia and, in those most severely affected, COVID-19 may rapidly progress to acute respiratory distress syndrome (ARDS) causing respiratory failure, septic shock, hyperinflammation, oxidative stress, neural compromise, microthrombosis, fibrosis and/or multi-organ failure, in particular in long-COVID patients.

A lot of effort is put worldwide to develop a vaccine against SARS-CoV-2. Currently, there is a lack of effective treatments against COVID-19 and SARS-CoV-2 infections and the longer-term deleterious consequences for compromised tissues. There is therefore a general need for a treatment that is effective in treating coronavirus infections, including COVID-19.

SUMMARY

It is a general objective to provide a treatment of coronavirus infections or infectious diseases.

It is a particular objective to provide a treatment of SARS-CoV-2 infections and COVID-19.

These and other objectives are met by embodiments as disclosed herein.

An aspect of the invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in prevention, inhibition and/or treatment of a coronavirus infection or infectious disease.

Another aspect of the invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in preventing, inhibiting or treating an inflammatory disease selected from the group consisting of acute respiratory distress syndrome (ARDS) and systemic inflammatory response syndrome (SIRS).

A further aspect of the invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in preventing, inhibiting and/or treating of an infection or infectious disease caused by a pathogen capable of binding to cell surface heparan sulfate proteoglycans (HSPG).

Experimental data as presented herein indicates that dextran sulfate, or a pharmaceutically acceptable salt thereof, may be used in treating coronavirus infections and coronavirus infectious diseases and other infections and their longer term consequences caused by pathogens capable of binding to HSPG. Dextran sulfate, or the pharmaceutically acceptable salt thereof, is also capable of stimulating the release of reparative growth factors from tissue stores and suppressing pro-inflammatory cytokines in selected immune cells. This means that dextran sulfate, or the pharmaceutically acceptable salt thereof, prevents or at least significantly inhibits the increased levels of these cytokines in the circulating blood causing an inflammatory disease following coronavirus infection or in ARDS or SIRS. Dextran sulfate of the embodiment, also, has the potential to resolve inflammatory scarring caused by inflammatory conditions and promotes functional tissue regeneration. These effects are, together with, the effects of dextran sulfate in inducing metabolic normalization and muscle and liver improvement important for long COVID-19 patients.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:

FIG. 1 illustrates dextran sulfate competition for the protein-protein interaction between amyloid-β and PrP^c.

FIG. 2 illustrates 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).

FIG. 3 is a diagram illustrating LMW-DS-induced changes in brain glutamate levels.

FIGS. 4A-4D are diagrams illustrating LMW-DS-changed levels of adenine nucleotides (ATP, ADP, AMP) and ATP/ADP ratio as a measurement of mitochondrial phosphorylating capacity.

FIGS. 5A-5D are diagrams illustrating LMW-DS-changed levels of oxidative and reduced nicotinic coenzymes.

FIGS. 6A-6C are diagrams illustrating LMW-DS-changed levels of biomarkers representative of oxidative stress.

FIG. 7 is a diagram illustrating LMW-DS-changed levels of nitrate as a measurement of NO-mediated nitrosative stress.

FIGS. 8A-8C are diagrams illustrating LMW-DS-changed levels of N-acetylaspartate (NAA) and its substrates.

FIG. 9 illustrates concentrations of NAA measured in deproteinized brain homogenates of rats sacrificed at 2 days post severe traumatic brain injury (sTBI) without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after trauma induction. Controls are represented by sham-operated animals. Values are the mean of 12 animals. Standard deviations are represented by vertical bars. *significantly different from controls, p < 0.01. **significantly different from sTBI 2 days, p < 0.01.

FIG. 10 illustrates concentrations of ATP measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean of 12 animals. Standard deviations are represented by vertical bars. *significantly different from controls, p < 0.01. **significantly different from sTBI 2 days, p < 0.01.

FIG. 11 illustrates concentrations of ascorbic acid measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean of 12 animals. Standard deviations are represented by vertical bars. *significantly different from controls, p < 0.01. **significantly different from sTBI 2 days, p < 0.01.

FIG. 12 illustrates concentrations of glutathione (GSH) measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean of 12 animals. Standard deviations are represented by vertical bars. *significantly different from controls, p < 0.01. **significantly different from sTBI 2 days, p < 0.01.

FIG. 13 illustrates concentrations of NAA measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean of 12 animals. Standard deviations are represented by vertical bars. *significantly different from controls, p < 0.01. **significantly different from sTBI 2 days, p < 0.01.

FIG. 14 illustrates change in activated partial thromboplastin time (aPTT) in blood following LMW-DS administration.

FIG. 15 illustrates human trabecular meshwork cells were stimulated with transforming growth factor beta 2 (TGFβ2) (1.0 ng/ml) in the absence (saline) or presence of LMW-DS (4.0 µM) for 72 hours followed by immunofluorescence labelling of fibronectin (green) and nuclei staining with Hoecsht (cyan). (15A) Representative images are maximum intensity projections from confocal Z-stacks and (15B) the histogram shows quantification of fibronectin staining (mean ± SEM, n=6), P<0.05 (Wilcoxon test).

FIG. 16 illustrate (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), blocking AqH outflow and raising IOP. (16B) Line graph showing IOP measurements during IC TGFβ1 treatment for the first 14 days followed by IC TGFβ1 with daily subcutaneous saline vehicle control or LMW-DS treatment for a further 14 days. Normal IOP levels are indicated by the grey shaded area, ** P<0.01, **** P<0.0001 (2-way ANOVA). (16C + 16D) Representative images of ocular tissue sections including the angle of the anterior segment together with related histograms showing levels of immunoreactive laminin and fibronectin staining in the TM in the saline and LMW-DS treatment groups, ** P<0.01 *** P<0.001 (t-test). (16E) Representative images and histogram showing the RGC marker BRN3a in retinal sections from the saline and LMW-DS treatment groups, ** P<0.01 (Mann-Whitney test), GCL - ganglion cell layer. (16F) Representative optical coherence tomography images and related histogram showing the segmented RNFL (arrows) in the saline and LMW-DS treatment groups, **** P<0.0001 (t-test). Saline group n=5, LMW-DS group n=7. (16G) Schematic diagram showing the potential mechanism of LMW-DS in POAG.

FIG. 17 PBMC were cultured in the absence (media, unstimulated) or presence of stimulus: (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) plus vehicle (0.027% saline) or LMW-DS (ILB™ at either 60, 200 or 600 µg/ml) for 24 hours. Levels of IL-6 were quantified in the supernatant by ELISA. Data presented as mean ± SEM arising from six or 10-11 (LPS) donors. Data plotted as percentage stimulus + 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. *MannWhitney U test comparison between Vehicle + PHA-L and 600 µg/ml ILB™ + PHA-L p=0.048.

FIG. 18 Monocytes purified from PBMCs were cultured in the absence of stimulation (media) or stimulated with LPS (0.01 ng/ml) or peptidoglycan (30 ng/ml) in the absence (Vehicle) or presence of either (18A, 18D) LMW-DS (ILB™; 60 µg/ml, 200 µg/ml or 600 µg/ml), (18B, 18E) dexamethasone (3.0 µM) or (18C, 18F) heparin (2.0 µg/ml, 6.0 µg/ml or 20 µg/ml) for 24 hours. Levels of IL-6 were quantified in the cell culture supernatant by ELISA. Data presented as mean ± SEM, n=10. * indicates below the limit of detection (5 pg/ml). +P<0.05, +++P<0.001 Significant difference to stimulation (Mann Whitney U Test).

FIG. 19 PBMCs were cultured in the absence (media, unstimulated) or presence of stimulus: 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) plus vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200 or 600 µg/ml) for 24 hours. Levels of interferon gamma (IFNγ) were quantified in the supernatant by Luminex. Data presented as percentage stimulus + vehicle and mean ± SEM from 12 donors unless otherwise indicated. (-) 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 and (*) indicates data from 6 donors. Comparison to stimulation with vehicle: # P<0.05, ## P<0.01, ### P<0.001 and N.S indicates not-significant (Mann Whitney test, two tailed).

FIG. 20 PBMCs were cultured in the absence (media, unstimulated) or presence of stimulus: 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) plus vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200 or 600 µg/ml) for 24 hours. Levels of interleukin 8/chemokine (C-X-C motif) ligand 8 (IL-8/CXCL8) were quantified in the supernatant by Luminex. Data presented as percentage stimulus + vehicle and mean ± SEM from 12 donors unless otherwise indicated. (-) 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 and (*) indicates data from 6 donors. Comparison to stimulation with vehicle: # P<0.05, ## P<0.01, ### P<0.001 and N.S indicates not-significant (Mann Whitney test, two tailed).

FIG. 21 PBMCs were cultured in the absence (media, unstimulated) or presence of stimulus: 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) plus vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200 or 600 µg/ml) for 24 hours. Levels of tumor necrosis factor alpha (TNFα) were quantified in the supernatant by Luminex. Data presented as percentage stimulus + vehicle and mean ± SEM from 12 donors unless otherwise indicated. (-) 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 and (*) indicates data from 6 donors. Comparison to stimulation with vehicle: # P<0.05, ## P<0.01, ### P<0.001 and N.S indicates not-significant (Mann Whitney test, two tailed).

FIG. 22 PBMCs were cultured in the absence (media, unstimulated) or presence of stimulus: 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) plus vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200 or 600 µg/ml) for 24 hours. Levels of IL-1β were quantified in the supernatant by Luminex. Data presented as percentage stimulus + vehicle and mean ± SEM from 12 donors unless otherwise indicated. (-) 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 and (*) indicates data from 6 donors. Comparison to stimulation with vehicle: # P<0.05, ## P<0.01, ### P<0.001 and N.S indicates not-significant (Mann Whitney test, two tailed).

FIG. 23 PBMCs were cultured in the absence (media, unstimulated) or presence of stimulus: 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) plus vehicle (0.027% saline) or LMW-DS (ILB™ at 60, 200 or 600 µg/ml) for 24 hours. Levels of IL-10 were quantified in the supernatant by Luminex. Data presented as percentage stimulus + vehicle and mean ± SEM from 12 donors unless otherwise indicated. (-) 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 and (*) indicates data from 6 donors. Comparison to stimulation with vehicle: # P<0.05, ## P<0.01, ### P<0.001 and N.S indicates not-significant (Mann Whitney test, two tailed).

FIG. 24 (24A, 24B) Model of SARS-CoV-2 attachment to cell surface and (24C) inhibition of interaction between spike protein and ACE2 with LMW-DS.

FIG. 25 schematically illustrates beneficial effects and actions of LMW-DS in connection with COVID-19.

FIG. 26 serum NAA levels in patients after LMW-DS treatment.

FIG. 27 serum uric acid levels in patients after LMW-DS treatment.

FIG. 28 sum of oxypurines in serum in patients after LMW-DS treatment.

FIG. 29 serum nitrate levels in patients after LMW-DS treatment.

FIG. 30 serum nitrate + nitrite levels in patients after LMW-DS treatment.

FIG. 31 serum MDA levels in patients after LMW-DS treatment.

FIG. 32 serum ALA levels in patients after LMW-DS treatment.

FIG. 33 serum CITR levels in patients after LMW-DS treatment.

FIG. 34 serum ORN/CITR levels in patients after LMW-DS treatment.

FIG. 35 serum α-tocopherol levels in patients after LMW-DS treatment.

FIG. 36 serum γ-tocopherol levels in patients after LMW-DS treatment.

FIG. 37 impact of LMW-DS (ILB®) upon SARS-CoV-2 spike protein interacting with ACE-2 assessed using the RayBiotech® Life ELISA assay. Data represent the mean ± SEM (n=3).

FIG. 38 schematically illustrates serum lactate levels in ALS patients after dextran sulfate treatment.

FIG. 39 schematically illustrates ALSAQ-40 ADL scores in ALS patients after dextran sulfate treatment.

FIG. 40 schematically illustrates serum myoglobin levels in ALS patients after dextran sulfate treatment.

FIG. 41 schematically illustrates serum creatine kinase levels in ALS patients after dextran sulfate treatment.

FIG. 42 schematically illustrates serum hepatocyte growth factor (HGF) levels in ALS patients after dextran sulfate treatment.

FIG. 43 illustrates total bilirubin levels in the serum of ALS patients after dextran sulfate treatment.

DETAILED DESCRIPTION

The present invention generally relates to treatment of infections and inflammatory diseases and the longer-term sequelae of these infectious diseases, and in particular to the use of dextran sulfate in treatment of such coronavirus infections and infectious diseases and inflammatory diseases that may be caused by coronavirus infections.

Coronaviruses (CoV) constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. Six species of human coronaviruses are known, with one species subdivided into two different strains, making seven strains of human coronaviruses altogether. Four of these strains generally produce mild symptoms of the common cold; 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. Three strains produce symptoms that are potentially severe; all three of these are β-CoV strains; 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).

Coronavirus and coronavirus infection or infectious disease as used herein refer to any coronavirus and infection or infectious disease caused by such a coronavirus in a mammalian, preferably human, subject or patient. In an embodiment, the coronavirus is selected from the group consisting of MERS-CoV, SARS-CoV and SARS-CoV-2, and the coronavirus may cause a coronavirus infectious disease selected from the group consisting of MERS for MERS-CoV, SARS for SARS-CoV and COVID-19 for SARS-CoV-2. In a particular embodiment, the coronavirus is SARS-CoV-2 and the coronavirus infectious disease is COVID-19.

The present invention is related to the use of dextran sulfate in prophylaxis, inhibition and/or treatment of coronavirus infections and coronavirus infectious diseases and their consequences. As shown herein, dextran sulfate of the embodiments has a multimodal mechanism of action that is relevant to the initial infection, disease progression and adverse tissue responses of those infected with SARS-CoV-2 (FIG. 25). Dextran sulfate of the embodiments also has positive effects on patients suffering from long-term effects of COVID-19 symptoms, also referred to as long COVID and post-COVID conditions.

These multimodal mechanisms of action include disruption of the host-pathogen protein-protein interaction in the early infection phase, thereby inhibiting or suppressing binding of the coronavirus to the target molecule, such as ACE2 for SARS-CoV-2, thereby preventing or at least inhibiting the virus access to host cells.

As is schematically shown in FIGS. 24A and 24B, like other betacoronaviruses, attachment and cellular entry of SARS-CoV-2 is mediated by the spike glycoprotein (SPG). SPG interacts not only with its receptor, angiotensin converting enzyme 2 (ACE2), but also binds to glycosaminoglycans, such as heparan sulfate (HS), which is found on the surface of most mammalian cells in the form of heparan sulfate proteoglycan (HSPG). The binding of SPG to tethered HS on the cell surfaces increases the local concentration of virus particles at the cell surface and promotes binding of SPG to ACE2 (FIG. 24B). Soluble untethered dextran sulfate of the embodiments is capable of binding to SPG as HS prior to receptor presentation, and thereby inhibits the binding of SPG to HSPG and ACE2 on cell surfaces and prevents or at least significantly reduces local concentration, attachment and cellular entry of coronaviruses, such as SARS-CoV-2, in infected subjects (FIG. 24C). Accordingly, dextran sulfate of the embodiments can therefore be used not only to prevent or at least inhibit coronavirus infection but will also, once a subject has been infected by coronavirus, restrict spread and replication of coronavirus in the subject body by interfering with the interaction between the coronavirus and ACE2 and HSPG on cell surfaces.

Dextran sulfate of the embodiments may also reduce transmission of coronavirus, i.e., spread of coronavirus in a population. In more detail, dextran sulfate of the embodiments inhibits the coronavirus from accessing human cells and thereby inhibits viral replication and any subsequent viral shedding from infected cells. Such a reduced viral shedding load is benefitting not only for the subject by restricting spread in the subject body but also for the population since the infected subject is thereby less likely to spread the coronavirus to other subjects.

Dextran sulfate thereby has medical effects in stage I (early infection, FIG. 25) as shown in FIGS. 24A-24C, 37 by disrupting the interaction between ACE2 receptor and SPG by binding to the SPG on the virus particles.

Dextran sulfate of the embodiments has also effect in metabolic normalization and activation of tissue repair growth factors, such as hepatocyte growth factor (HGF) (FIGS. 2, 42). These actions of the dextran sulfate of the embodiments are of importance in suppressing the negative effects of the coronavirus in the infected subject during the pulmonary phase (stage II, FIG. 25) where the virus may cause pneumonia and negatively affect the respiratory tissue of the subject by effecting growth factor-mediated scar-free tissue repair. Dextran sulfate of the embodiments also has the ability to resolve and reverse established scarring, allowing healing of compromised tissues. Dextran sulfate of the embodiments is, in addition, capable of inducing metabolic normalization in cells, such as seen in improved mitochondrial function (FIGS. 4A-4D, 27-28; Tables 5-8, 15-18).

In more detail, dextran sulfate of the embodiment protects mitochondrial function and reduces oxidative stress as seen in, among others, improvement of the recovery of antioxidant status, protection of mitochondrial ATP energy supply by preserving ATP production and metabolism, normalization of mitochondrial phosphorylating capacity all as induced by dextran sulfate. As a consequence, dextran sulfate is capable of normalizing, protecting and preserving mitochondrial function in cells exposed to damage or disease, which is of importance in order to have optimally functional cells that can combat the infectious disease.

Dextran sulfate of the embodiment thereby has medical effects in stage II (pulmonary phase) of the infection by activating tissue repair growth factors and inducing metabolic normalization (FIG. 25).

Dextran sulfate of the embodiments also has effect in any following hyperinflammation phase that is 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. In fact, severely affected patients show a significant inflammatory response that may additionally cause DIC and provoke peripheral microthrombosis, including in the alveolar capillaries, which results in microclots in the affected blood vessels. Dextran sulfate of the embodiments has been shown to be anti-inflammatory and may therefore cause resolution or at least reduction of any cytokine storm and hyperinflammation in the subject and the deleterious consequences of this hyperinflammation (FIGS. 17A, 17D, 18A, 19-23). Dextran sulfate also has anti-coagulant effects, which would be useful in combating DIC and the microclots seen in severely affected subjects (FIG. 14).

The anti-inflammatory effects of dextran sulfate are selective in terms of acting on specific cells in the immune system and selectively reducing pro-inflammatory cytokines released by such cells. In more detail, experimental data as presented herein shows that dextran sulfate in particular targets monocytes and T lymphocytes and causing a concentration dependent and significant reduction in IL-6, IL-10, IL-1β, IL-8, TNFα and IFNγ by activated monocytes and IL-6, IL-10 TNFα and IFNγ by activated T lymphocytes (FIGS. 17-23). A significant advantage of the selective reduction of pro-inflammatory cytokines by dextran sulfate over, for instance, dexamethasone and other steroids is the dextran sulfate does not affect all cytokine production by the immune system and does not does not reduce cytokine production to very low levels. In infectious diseases, such as coronavirus infections, a controlled activation of the immune system is required in order to fight the infection. Dextran sulfate of the embodiments can achieve such a controlled activation by reducing, in clear contrast to shutting off, selected pro-inflammatory cytokines from selected cells of the immune system that reduces the risk of developing hyperinflammatory conditions, such as ARDS, SIRS and septic shock, while still allowing the immune system to fight the coronavirus infection.

An advantage of dextran sulfate over antibody-based inflammatory treatments is that dextran sulfate does not target a single pro-inflammatory cytokine as such antibodies do but rather reduces the levels of several key pro-inflammatory cytokines. Another advantage is the comparatively lower half-life of dextran sulfate in the body (C_max is 2 to 3 hours in humans) as compared to antibodies, thereby enabling a controlled anti-inflammatory effect during a well-defined period of time in synchrony with periods of risks of hyperinflammation following coronavirus infection.

Subjects recovering from COVID-19 may suffer from organ fibrosis caused by ARDS, SIRS and microthrombosis. Such organ fibroses include not only pulmonary fibrosis but may also include other organs, such as kidney fibrosis and cardiomyopathy. Dextran sulfate of the embodiments have anti-thrombotic effects, which is useful in such organ fibrosis (FIGS. 15-16). Additionally, dextran sulfate of the embodiments has been shown to have not only anti-fibrotic effects but also fibrolytic effects, i.e., capable of removing existing scars following organ fibrosis and facilitating scarless tissue remodelling.

Dextran sulfate of the embodiments therefore has medical effects in stage III (hyperinflammation phase) of the infection by resolving any cytokine storm and hyperinflammation and by having anti-fibrotic and anti-coagulant effects (FIG. 25).

There are emerging evidences that long COVID effects, such as chronic fatigue syndrome or myalgic encephalomyelitis, are caused by metabolic dysregulation. In more detail, mitochondrial dysfunction and oxidative/nitrosative stress is seen in long COVID patients, including altered ATP production and increased oxidative/nitrosative stress. Dextran sulfate of the embodiments is capable of counteracting such metabolic dysregulation and oxidative/nitrosative stress by normalizing metabolic and mitochondrial function (FIGS. 4A-4D, 27-28; Tables 5-8, 15-18).

Dextran sulfate of the embodiments was effective in restoring normal mitochondrial related energy metabolism, with positive effects on the concentration of triphosphate purine and pyrimidine nucleotides. In fact, dextran sulfate treatment could almost normalize ATP levels and NAA concentrations in compromised tissues as compared to healthy controls.

Dextran sulfate of the embodiments led to a significant reduction in oxidative/nitosative stress. In particular, the levels of ascorbic acid, as the main water-soluble brain antioxidant, and glutathione (GSH), as the major intracellular sulfhydryl group (SH) donor, were significantly improved. In addition, malondialdehyde (MDA) levels, as end product of polyunsaturated fatty acids of membrane phospholipids and therefore taken as a marker of reactive oxygen species (ROS) mediated lipid peroxidation, showed a significant reduction after dextran sulfate administration. In addition, the sum of nitrites/nitrates in compromised tissues was also significantly reduced (FIG. 30). The oxidative/nitrosative stress markers described above all indicated an improvement in the recovery of antioxidant/antinitrosative status after dextran sulfate treatment (FIGS. 29-31, 33-36).

Dextran sulfate of the embodiments also has medical effects in stage IV (recovery phase, FIG. 25) of the invention by having anti-thrombotic, anti-fibrotic and fibrolytic effects. Dextran sulfate of the embodiments additionally induces a metabolic normalization by improving mitochondrial function of the patient’s cells.

Dextran sulfate of the embodiments has further medical effects in stage IV (recovery phase, FIG. 25) by reducing muscle degeneration and improving muscle function (FIGS. 38 to 42).

There is emerging data showing that COVID-19, and in particular long term COVID-19, may cause liver damage. Experimental data as presented herein shows that dextran sulfate of the embodiments had positive effect on liver function and was capable of normalize perturbed liver function in human patients (FIG. 43), which is of benefit in stage IV (FIG. 25).

Dextran sulfate of the embodiments also have beneficial effect in the recovery phase (FIG. 25) by inducing a significant release of hepatocyte growth factor (HGF), which plays an important role in organ regeneration and wound healing. The elevated levels of plasma HGF induced by dextran sulfate of the embodiments (FIGS. 2, 42) are beneficial to COVID-19 patients by inducing tissue repair and regeneration and wound healing of organs and tissues negatively affected by the SARS-CoV-2 infection and the inflammatory responses induced by the SARS-CoV-2 infection.

An aspect of the invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in preventing, inhibiting and/or treating a coronavirus infection or infectious disease.

In an embodiment, the coronavirus infectious disease is selected from the group consisting of MERS, SARS and COVID-19. In a particular embodiment, the coronavirus infectious disease is COVID-19.

In an embodiment, the coronavirus infection is selected from the group consisting of a coronavirus infection caused by a coronavirus selected from the group consisting of MERS-CoV, SARS-CoV and SARS-CoV-2. In a particular embodiment, the coronavirus infection is caused by SARS-CoV-2.

The present invention also relates to use of dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention, inhibition and/or treatment of a coronavirus infection or infectious disease.

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

Treatment of a coronavirus infection or infectious disease as used herein does not necessarily mean curative treatment of the coronavirus infection or infectious disease but also encompass inhibition or reduction of the short- and long-term symptoms of the coronavirus infection or infectious disease. Hence, treatment also encompass delaying onset of the coronavirus infection or infectious disease, including delaying, preventing onset of symptoms or resolving established pathologies associated with the coronavirus infection or infectious disease.

Coronaviruses are not the only pathogens that use cell surface glycosaminoglycan heparan sulfate (HSPG) as a receptor. In clear contrast, HSPG serves as a receptor for several viruses and also 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, M.S.G Pavão (ed.)).

HSPG - pathogen interactions
Pathogen	Pathogen protein	HSPG	Function/interaction
Bacteria
Bacillus anthracis	AnIB, ANIO, InhA, Npr599	Syndecan-1	Shedding
Bacillus cereus	ClnA	Syndecan-1	Shedding
Borrelia burgdorferi	39 kDa protein	Unknown	Attachment
Bordetella pertussis	Filamentous hemagglutinin	Unknown	Attachment
Chlamydia pneumoniae	OmcB	Unknown	Attachment
Chlamydia trachomatis	Unknown	Unknown	Attachment, invasion
Haemophilus influenzae	High molecular weight protein (HMW)	Unknown	Attachment
Helicobacter pylori	Vacuolating cytotoxin (VacA)	Unknown	Toxin internalization
Listeria monocytogenes	ActA	Syndecan-1	Attachment, invasion
Mycobacterium tuberculosis	Hemagglutinin	Unknown	Attachment
Neisseria gonorrhoeae	Opa	Syndecan-1, -4	Attachment, invasion
Neisseria meningitidis	Opc	Unknown	Attachment, invasion
Neisseria meningitidis	GNA2132 (Neisserial Heparin Binding Antigen (NHBA))	Unknown	(presumptive) Attachment, resistance to serum killing
Orientia tsutsugamushi	Unknown	Syndecan-4	Attachment, invasion
Porphyromonas gingivalis	LPS, gingipains	Syndecan-1	Shedding
Pseudomonas aeruginosa	LasA	Syndecan-1	Shedding
Staphylococcus aureus	α-toxin, β-toxin	Syndecan-1	Shedding
Streptococcus agalactiae	Alpha C protein	Unknown	Attachment, invasion
Streptococcus pyogenes	M protein	Unknown	Attachment
Streptococcus pneumoniae	ZmpC	Syndecan-1	Shedding
Yersinia enterocolitica	LcrG	Unknown	Attachment, invasion
Adeno-associated virus type 2	Capsid protein VP3	Unknown	Attachment
Adenovirus	Ad3 Fiber knob	Unknown	Attachment
Coronavirus	Spike protein	Unknown	Attachment
Coxsackievirus	Capsid protein VP1	N- and 6-0-sulfated HSPGs	Attachment, endocytosis
Cytomegalovirus	gB	Unknown	Attachment
Dengue virus	E (envelope protein)	Unknown	Attachment, internalization
FMDV	VP3	Unknown	Attachment
HSV-1 and -2	gB, gC, gD	Syndecan-2	Attachment
Hepatitis B virus	Large viral envelope protein	Unknown	Attachment
Hepatitis C virus	Envelope glycoprotein E2	Unknown	Attachment
HHV-8 (KSHV)	gB, gpK8.1A	Unknown	Attachment
HIV-1	Tat	Perlecan	Tat internalization
Tat	Unknown	Lymphoid cell extravasation
gp120	Syndecan-3	Attachment
gp41	Agrin	Attachment
HPV	L1 carboxy terminal	Syndecan-1,-3, -4, glypican-1	Attachment
HTLV1	Surface glycoprotein gp46	Unknown	Attachment
Japanese encephalitis virus	Envelope (E) protein	Unknown	Attachment
Pseudorabies virus	Glycoprotein C	Unknown	Attachment
Respiratory syncytial virus	Fusion glycoprotein	Unknown	Attachment, infectivity
Rhinovirus	VP1	Unknown	Attachment
Sindbis virus	E2 envelope glycoprotein	Unknown	Attachment
Vaccinia virus	Viral envelope protein A27L	Unknown	Fusion
VCP (Vaccinia virus	Unknown	Anchoring of RCA (regulators of
	complement control protein)		complement activation) to cell membrane
West Nile virus	Envelope (E) protein	Unknown	Attachment
Yellow fever virus	Envelope (E) protein	Unknown	Attachment
Giardia lamblia	Alpha-1 giardin	Unknown	Attachment
Leishmania spp.	Unknown	Unknown	Attachment
Encephalitozoon spp.	Spore wall protein EnP1	Unknown	Attachment
Neospora caninum	Microneme protein NcMIC3	Unknown (CS)	Attachment
Plasmodium spp.	Circumsporozoite protein (CSP)	Multiple	CSP cleavage, productive invasion
Thrombospondin-related anonymous protein (TRAP)	Unknown	Invasion
BAEBL (EBA-140)	Multiple (HS)	Invasion
VAR2CSA	Chondroitin Sulfate A (CSA)	Attachment (to placenta)
Toxoplasma gondii	Surface antigen 3	Unknown	Attachment
Unknown	Unknown (N-sulfation required)	Replication in parasitophorous vesicle
Programmed cell death 5 (TgPDCD5)		Protein internalization and enhanced apoptotic activity
Trypanosoma cruzi	Cruzipain	HSPG	Enhanced enzymatic activity
Heparin-binding protein (HBP-Tc)	Unknown	Attachment
Prion	PrPC	Glypican-1	Lipid raft association, conversion to PrPSc

Hence, dextran sulfate of the embodiments can also be used to prevent, inhibit and/or treat a pathogen infection or infectious disease caused by a pathogen capable of binding to cell surface heparan sulfate proteoglycans (HSPG), and in particular capable of binding to HSPG to facilitate initial cellular attachment and/or subsequent cellular entry.

Another aspect of the invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in preventing, inhibiting and/or treating of an infection or infectious disease caused by a pathogen capable of binding to cell surface heparan sulfate proteoglycans (HSPG).

In a particular embodiment, the pathogen is capable of binding to HSPG to facilitate initial cellular attachment and/or subsequent cellular entry.

In another particular embodiment, the pathogen is capable of binding to the heparan sulfate part of HSPG.

In an embodiment, the pathogen is a bacterium, virus, prion or parasite selected from Table 1. In a particular embodiment, the pathogen is other than human immunodeficiency virus (HIV), such as other than HIV-1 and HIV-2.

In an embodiment, 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 meningitides, Neisseria meningitides, Orientia tsutsugamushi, Porphyromonas gingivalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumoniae and Yersinia enterocolitica.

B. anthracis causes anthrax; B. cereus causes nausea, vomiting and diarrhea; B. burgdorferi causes Lyme disease; B. pertussis causes pertussis; C. pneumoniae causes pneumonia; C. trachomatis causes chlamydia; H. influenzae causes bacteremia, pneumonia, epiglottitis, acute bacterial meningitis, cellulitis, osteomyelitis, and infectious arthritis; H. pylori causes gastritis and ulcers, but has also been associated with a wide range of other diseases, e.g., idiopathic thrombocytopenic purpura, iron deficiency anemia, atherosclerosis, 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, the antiphospholipid syndrome, plasma cell dyscrasias, reactive arthritis, central serous chorioretinitis, open angle glaucoma, blepharitis, diabetes mellitus, the metabolic syndrome, various types of allergies, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, hepatic fibrosis, and liver cancer; L. monocytogenes causes listeriosis, M. tuberculosis causes tuberculosis; N. gonorrhoaea causes gonorrhea; N. meningitides causes meningitis and other forms of meningococcal disease such as meningococcemia, a life-threatening sepsis; O. tsutsugamushi causes scrub typhus; P. gingivalis causes periodontal diseases, as well as in the upper gastrointestinal tract, the respiratory tract and the colon, and has also been linked to Alzheimer’s disease and rheumatoid arthritis; P. aeruginosa causes pneumonia and various sepsis conditions; S. aureus causes skin infections including abscesses, respiratory infections, such as sinusitis, and food poisoning; S. agalactiae causes neonatal infections including neonatal infection sepsis, pneumonia, and meningitis; S. pyogenes causes skin infections, neonatal infections but may also cause rheumatic fever, acute post-infectious glomerulonephritis and PANDAS; S. pneumoniae causes pneumonia; and Y. enterocolitica causes yersiniosis.

In an embodiment, 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 viruses 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.

In another embodiment, 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.

Coxsackieviruses cause aseptic meningitis; cytomegaloviruses cause mononucleosis, and pneumonia; DENV causes dengue fever; FMDV causes foot-and-mouth disease; HSV causes cold sores, genital herpes and contagious; Hepatitis B virus causes hepatitis B; Hepatitis C virus causes hepatitis C, hepatocellular carcinoma and lymphomas; HHV-8 causes Kapsi’s sarcoma, primary effusion lymphoma, HHV-8-associated multicentric Castleman’s disease and KSHV inflammatory cytokine syndrome; HPV causes precancerous lesions, cervical cancer, HPV-positive oropharyngeal cancers, genital warts and laryngeal papillomatosis; HTLV1 causes adult T-cell lymphoma (ATL), HTLV-I-associated myelopathy, uveitis, and Strongyloides stercoralis hyper-infection; JEV causes Japanese encephalitis; Pseudorabies virus causes Aujeszky’s disease; RSV causes infections of the respiratory tract, including bronchiolitis and pneumonia; Rhinoviruses cause common cold; SINV causes sindbis fever; WNV causes West Nile fever; and Yellow fever virus causes yellow fever.

In an embodiment, 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.

Giardia lamblia causes giardiasis, Leishmania spp. 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 surra in horses, and a brucellosis-like disease in cattle.

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

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 HSPG. The method comprises administering an effective amount of dextran sulfate, or a pharmaceutically acceptable salt thereof, to a subject suffering from the infection or infectious disease caused by the pathogen capable of binding to cell surface HSPG or having a risk of suffering from the infection or infectious disease caused by the pathogen capable of binding to cell surface HSPG.

Treatment of an infection or infectious disease caused by a pathogen capable of binding to cell surface HSPG as used herein does not necessarily mean curative treatment of the infection or infectious disease but also encompass inhibition or reduction of the symptoms or resolving established pathologies associated with the infection or infectious disease. Hence, treatment also encompass delaying onset of the infection or infectious disease, including delaying onset of symptoms associated with the infection or infectious disease.

ARDS is a type of respiratory failure characterized by rapid onset of widespread inflammation in the lungs. ARDS may be caused by sepsis, pancreatitis, trauma, pneumonia, and aspiration. The underlying mechanism involves diffuse injury to cells which form the barrier of the microscopic air sacs of the lungs, surfactant dysfunction, activation of the immune system, and dysfunction of the body’s regulation of blood clotting. In effect, ARDS impairs the lungs’ ability to exchange oxygen and carbon dioxide. Adult diagnosis is based on a PaO₂/FiO₂ ratio (ratio of partial pressure arterial oxygen and fraction of inspired oxygen) of less than 300 mm Hg despite a positive end-expiratory pressure (PEEP) of more than 5 cm H₂O.

SIRS is an inflammatory state affecting the whole body. It is the body’s response to an infectious or noninfectious insult. SIRS is frequently complicated by failure of one or more organs or organ systems, and may cause acute kidney injury, shock and multiple organ dysfunction syndrome.

SIRS is a serious condition related to systemic inflammation, organ dysfunction, 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 satisfy criteria for SIRS and have a suspected or proven infection. Manifestations of SIRS include body temperature less than 36° C. or greater than 38° C., heart rate greater than 90 beats per minute, tachypnea (high respiratory rate), with greater than 20 breaths per minute; or, an arterial partial pressure of carbon dioxide less than 4.3 kPa (32 mmHg), white blood cell count less than 4000 cells/mm³ (4 × 10⁹ cells/L) or greater than 12,000 cells/mm³ (12 × 10⁹ cells/L); or the presence of greater than 10% immature neutrophils (band forms). When two or more of these criteria are met with or without evidence of infection, patients may be diagnosed with SIRS. Patients with SIRS and acute organ dysfunction may be termed severe SIRS.

Dextran sulfate of the embodiments has been shown to be anti-inflammatory and may therefore cause resolution or at least reduction of any cytokine storm and inflammation associated with ARDS and SIRS in the subject (FIGS. 17A, 17D, 18A, 19-23).

The anti-inflammatory effects of dextran sulfate are selective in terms of acting on specific cells in the immune system and selectively reducing pro-inflammatory cytokines released by such cells. In more detail, experimental data as presented herein shows that dextran sulfate in particular targets monocytes and T lymphocytes and causing a concentration dependent and significant reduction in IL-6, IL-10, IL-1B, IL-8, TNFα and IFNγ by activated monocytes and IL-6, IL-10, TNFα and IFNγ by activated T lymphocytes (FIGS. 17-23). A significant advantage of the reduction of pro-inflammatory cytokines by dextran sulfate over, for instance, dexamethasone and other steroids is the dextran sulfate does not totally shut down the cytokines and does not affect all cytokine production by the immune system. In infectious diseases, such as coronavirus infections, a controlled activation of the immune system is required in order to fight the infection. Dextran sulfate of the embodiments can achieve such a controlled activation by reducing in clear contrast to shutting off selected pro-inflammatory cytokines from selected cells of the immune system that reduces the risk of developing ARDS and SIRS.

A further aspect of the invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in preventing, inhibiting or treating an inflammatory disease selected from the group consisting of ARDS and SIRS.

In an embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is for preventing, inhibiting or treating ARDS.

In another embodiment, dextran sulfate, or the pharmaceutically acceptable salt thereof, is for preventing, inhibiting or treating SIRS.

In a particular embodiment, the inflammatory disease is caused by an infection, such as a coronavirus infection or an infection by another HSPG binding pathogen.

The present invention also relates to use of dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for preventing, inhibiting and/or treating an inflammatory disease selected from the group consisting of ARDS and SIRS.

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

Treatment of an inflammatory disease selected from the group consisting of ARDS and SIRS as used herein does not necessarily mean curative treatment of the inflammatory disease but also encompass inhibition or reduction of the short- and long-term symptoms of the inflammatory disease. Hence, treatment also encompass delaying onset of the infection or infectious disease, including delaying onset of symptoms associated with the inflammatory disease and also resolving long-term pathologies such as fibroproliferative, myopathies and chronic fatigue conditions that are established following such disease.

The present invention also relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in preventing, inhibiting or treating long-term effects COVID-19 symptom caused by a SARS-CoV-2 infection.

Long-term effects COVID-19 symptoms include organ damages in particular damages to the heart, liver and lungs of the subjects. Imaging tests taken months after recovery from COVID-19 have shown lasting damage to the heart muscle, even in people who experienced only mild COVID-19 symptoms. Furthermore, COVID-19 can cause long-standing damage to the tiny air sacs (alveoli) in the lungs. The resulting scar tissue can lead to long-term breathing problems. A high portion of hospitalized COVID-19 patients have abnormal liver function. Common long-term symptoms are mainly fatigue, shortness of breath, cough, palpitations and impaired sense of smell. Other symptoms include chest pain, muscle and joint pain, weight loss, and gastrointestinal disorders. The reported clinical measures for long-term effects COVID-19 patients were impaired lung function and lung damage, cardiovascular effects such as myocardial inflammation, cerebral changes and impaired sense of smell and taste (anosmia and hyposmia).

The present invention also relates to use of dextran sulfate, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for preventing, inhibiting and/or treating long-term effects COVID-19 symptom caused by a SARS-CoV-2 infection.

The present invention further relates to a method for preventing, inhibiting and/or treating long-term effects COVID-19 symptom caused by a SARS-CoV-2 infection. The method comprises administering an effective amount of dextran sulfate, or a pharmaceutically acceptable salt thereof, to a subject suffering from long-term effects COVID-19 symptom caused by a SARS-CoV-2 infection or having a risk of suffering from the long-term effects COVID-19 symptom caused by a SARS-CoV-2 infection.

In the following, reference to (average) molecular weight and sulfur content of dextran sulfate applies also to any pharmaceutically acceptable salt of dextran sulfate. Hence, the pharmaceutically acceptable salt of dextran sulfate preferably has the average molecular weight and sulfur content as discussed in the following embodiments.

Dextran sulfate outside of the preferred ranges of the embodiments are believed to have inferior effect and/or causing negative side effects to the cells or subject.

For instance, dextran sulfate of a molecular weight exceeding 10,000 Da (10 kDa) generally has a lower effect vs. side effect profile as compared to dextran sulfate having a lower average molecular weight. This means that the maximum dose of dextran sulfate that can be safely administered to a subject is lower for larger dextran sulfate molecules (>10,000 Da) as compared to dextran sulfate molecules having an average molecular weight within the preferred ranges. As a consequence, such larger dextran sulfate molecules are less appropriate in clinical uses when the dextran sulfate is to be administered to subjects in vivo.

Dextran sulfate is a sulfated polysaccharide and in particular a sulfated glucan, i.e., polysaccharide made of many glucose molecules. Average molecular weight as defined herein indicates that individual sulfated polysaccharides may have a molecular weight different from this average molecular weight but that the average molecular weight represents the mean molecular weight of the sulfated polysaccharides. This further implies that there will be a natural distribution of molecular weights around this average molecular weight for a dextran sulfate sample.

Average molecular weight, or more correctly weight average molecular weight (M_w), of dextran sulfate is typically determined using indirect methods such as gel exclusion/penetration chromatography, light scattering or viscosity. Determination of average molecular weight using such indirect methods will depend on a number of factors, including choice of column and eluent, flow rate, calibration procedures, etc.

Weight average molecular weight (M_w):

$\frac{{\displaystyle \sum M_i^2{N}_i}}{{\displaystyle \sum M_i{N}_i}},$

typical for methods sensitive to molecular size rather than numerical value, e.g., light scattering and size exclusion chromatography (SEC) methods. If a normal distribution is assumed, then a same weight on each side of M_w, i.e., the total weight of dextran sulfate molecules in the sample having a molecular weight below M_w is equal to the total weight of dextran sulfate molecules in the sample having a molecular weight above M_w. The parameter N_i indicates the number of dextran sulfate molecules having a molecular weight of M_i in a sample or batch.

In an embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_w equal to or below 10,000 Da. In a particular embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_w within an interval of from 2,000 Da to 10,000 Da.

In another embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_w within an interval of from 2,500 Da to 10,000 Da, preferably within an interval of from 3,000 Da to 10,000 Da. In a particular embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_w within an interval of from 3,500 Da to 9,500 Da, such as within an interval of from 3,500 Da to 8,000 Da.

In another particular embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_w within an interval of from 4,500 Da to 7,500 Da, such as within an interval of from 4,500 Da and 6,500 Da or within an interval of from 4,500 Da and 5,500 Da.

Thus, in some embodiments, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_w equal to or below 10,000 Da, equal to or below 9,500 Da, equal to or below 9,000 Da, equal to or below 8,500 Da, equal to or below 8,000 Da, equal to or below 7,500 Da, equal to or below 7,000 Da, equal to or below 6,500 Da, equal to or below 6,000 Da, or equal to or below 5,500 Da.

In some embodiments, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_w equal to or above 1,000 Da, equal to or above 1,500 Da, equal to or above 2,000 Da, equal to or above 2,500 Da, equal to or above 3,000 Da, equal to or above 3,500 Da, equal to or above 4,000 Da. or equal to or above 4,500 Da. Any of these embodiments may be combined with any of the above presented embodiments defining upper limits of the M_w, such combined with the upper limit of equal to or below 10,000 Da.

In a particular embodiment, the M_w of dextran sulfate, or the pharmaceutically acceptable salt thereof, as presented above is average M_w, and preferably determined by gel exclusion/penetration chromatography, size exclusion chromatography, light scattering or viscosity-based methods.

Number average molecular weight

$\left({\text{M}}_{\text{n}}\right):\frac{{\displaystyle \sum M_i{N}_i}}{{\displaystyle \sum N_i}},$

typically derived by end group assays, e.g., nuclear magnetic resonance (NMR) spectroscopy or chromatography. If a normal distribution is assumed, then a same number of dextran sulfate molecules can be found on each side of M_n, i.e., the number of dextran sulfate molecules in the sample having a molecular weight below M_n is equal to the number of dextran sulfate molecules in the sample having a molecular weight above M_n.

In an embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_n as measured by NMR spectroscopy within an interval of from 1,850 to 3,500 Da.

In a particular embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_n as measured by NMR spectroscopy within an interval of from 1,850 Da to 2,500 Da, preferably within an interval of from 1,850 Da to 2,300 Da, such as within an interval of from 1,850 Da to 2,000 Da.

Thus, in some embodiments, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_n equal to or below 3,500 Da, equal to or below 3,250 Da, equal to or below 3,000 Da, equal to or below 2,750 Da, equal to or below 2,500 Da, equal to or below 2,250 Da, or equal to or below 2,000 Da. In addition, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_n equal to or above 1,850 Da.

In an embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average sulfate number per glucose unit within an interval of from 2.5 to 3.0.

In a particular embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average sulfate number per glucose unit within an interval of from 2.5 to 2.8, preferably within an interval of from 2.6 to 2.7.

In an embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average number of glucose units within an interval of from 4.0 to 6.0.

In a particular embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average number of glucose units within an interval of from 4.5 to 5.5, preferably within an interval of from 5.0 to 5.2.

In an embodiment, the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a M_n as measured by NMR spectroscopy within an interval of from 1,850 to 3,500 Da, an average sulfate number per glucose unit within an interval of from 2.5 to 3.0, and an average sulfation of C2 position in the glucose units of the dextran sulfate is at least 90%.

In an embodiment, the dextran sulfate has an average number of glucose units of about 5.1, an average sulfate number per glucose unit within an interval of from 2.6 to 2.7 and a M_n within an interval of from 1,850 Da and 2,000 Da.

In an embodiment, the pharmaceutically acceptable salt of dextran sulfate is a sodium salt of dextran sulfate. In a particular embodiment, the sodium salt of dextran sulfate has an average number of glucose units of about 5.1, an average sulfate number per glucose unit within an interval of from 2.6 to 2.7 and a M_n including the Na+ counter ion within an interval of from 2,100 Da to 2,300 Da.

In an embodiment, the dextran sulfate has an average number of glucose units of 5.1, an average sulfate number per glucose unit of 2.7, an average M_n without Na+ as measured by NMR spectroscopy of about 1,900-1,950 Da and an average M_n with Na+ as measured by NMR spectroscopy of about 2,200-2,250 Da.

The dextran sulfate according to the embodiments can be provided as a pharmaceutically acceptable salt of dextran sulfate, such as a sodium or potassium salt.

A currently preferred dextran sulfate according to the embodiments is disclosed in WO 2016/076780.

The subject is preferably a mammalian subject, more preferably a primate and in particular a human subject. The dextran sulfate, or the pharmaceutically acceptable salt thereof, can, however, be used also in veterinary applications. Non-limiting example of animal subjects include primate, cat, dog, pig, horse, mouse, rat.

The dextran sulfate, or the pharmaceutically acceptable salt thereof, is preferably administered by injection to the subject and in particular by intravenous (i.v.) injection, subcutaneous (s.c.) injection or (i.p.) intraperitoneal injection, preferably i.v. or s.c. injection. Other parenteral administration routes that can be used include intramuscular and intraarticular injection. Injection of the dextran sulfate, or the pharmaceutically acceptable derivative thereof, could alternatively, or in addition, take place directly in, for instance, a tissue or organ or other site in the subject body, at which the target effects are to take place.

The dextran sulfate, or the pharmaceutically acceptable salt thereof, of the embodiments is preferably formulated as an aqueous injection solution with a selected solvent or excipient. The solvent is advantageously an aqueous solvent and in particular a buffer solution. A non-limiting example of such a buffer solution is a citric acid buffer, such as citric acid monohydrate (CAM) buffer, or a phosphate buffer. For instance, dextran sulfate of the embodiments can be dissolved in saline, such as 0.9% NaCl saline, and then optionally buffered with 75 mM CAM and adjusting the pH to about 5.9 using sodium hydroxide. Also, non-buffered solutions are possible, including aqueous injection solutions, such as saline, i.e., NaCl (aq). Furthermore, other buffer systems than CAM could be used if a buffered solution are desired.

The embodiments are not limited to injections and other administration routes can alternatively be used including orally, nasally, bucally, rectally, dermally, tracheally, bronchially, or topically. The active compound, dextran sulfate, is then formulated with a suitable excipient or carrier that is selected based on the particular administration route.

Suitable dose ranges for the dextran sulfate, or the pharmaceutically acceptable salt thereof, may vary according to the application, such as in vitro versus in vivo, the size and weight of the subject, the condition for which the subject is treated, and other considerations. In particular for human subjects, a possible dosage range could be from 1 µg/kg to 100 mg/kg of body weight, preferably from 10 µg/kg to 50 mg/kg of body weight.

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

The dextran sulfate, or the pharmaceutically acceptable derivative thereof, can be administered at a single administration occasion, such as in the form of a single bolus injection. This bolus dose can be injected quite quickly to the subject but is advantageously infused over time so that the dextran sulfate solution is infused over a few minutes of time to the patient, such as during 5 to 10 minutes.

Alternatively, the dextran sulfate, or the pharmaceutically acceptable salt thereof, can be administered at multiple, i.e., at least two, occasions during a treatment period.

The dextran sulfate, or the pharmaceutically acceptable salt thereof, can be administered together with other active agents, either sequentially, simultaneously or in the form of a composition comprising the dextran sulfate, or the pharmaceutically acceptable salt thereof, and at least one other active agent. The at least one active agent can be selected among any agent useful in any of the above mentioned diseases, disorders or conditions. The at least one active agent could also be in the form of cells in cell therapy, such as stem cells including, but not limited to, embryonic stem cells (ESCs) and mesenchymal stromal cells (MSCs).

EXAMPLES

In the following Examples, a sodium salt of dextran sulfate, denoted low molecular weight dextran sulfate (LMW-DS) herein, was used (ILB®, Tikomed AB, Viken, Sweden, WO 2016/076780).

Example 1

Attachment to host tissues is a critical step for coronavirus invasion and dissemination. Hence, disrupting host-pathogen protein-protein interactions may be an effective way of inhibiting coronavirus invasion.

The current study investigated the ability of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016/076780) to inhibit the protein-protein interaction between oligomeric β-amyloid and the pathogen protein PrP^c in an attempt to reveal its potential to inhibit protein-protein interaction.

Material and Methods

Chemicals and Antibodies

Streptavidin HRP was from BioLegend; β-amyloid-(1-42)-biotin was from Innovagen; normal human cellular prion protein (PrP^c) was from Merck; TMB was from eBioscience; 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was from Sigma; anti-amyloid β antibody clone 6E10 was from BioLegend; anti-mouse HRP was from Cell Signaling; dextran sulphate sodium salt (DSSS) with an average M.W. > 500,000 Da was from Sigma; dextran (M.W. 450,000 - 650,000 Da) was from Sigma; Maxisorp plates were from Sigma.

Preparation of Amyloid-β Oligomers

Oligomerization of β-amyloid was optimized based on previous methods (Stine et al., Methods Mol. Biol. 2011, 670: 13-32; Aimi et al., J Neurochem. 2015, 134: 611-617). Briefly amyloid-β was dissolved in HFIP to a final concentration of 1.0 mM, subject to protected sonication and the HFIP carefully evaporated. Arising peptide films were stored at -20° C. in a sealed container. Prior to use, the peptide films were slowly dissolved in DMSO to a final concentration of 5.0 mM and subject to protected 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 37° C. (β-amyloid-biotin) for 16 hours. To prepare monomers, the DMSO solution was diluted in ice-cold 18 MOhm water to a final concentration of 100 µM and used immediately.

Identification of Amyloid-βmonomers and Oligomers

Preparations optimized to generate monomers or oligomers of amyloid-β were solubilized in nonreducing gel sample buffer containing 5% SDS. Proteins were run on a 15% Bis-Tris gel using nonreducing MES running buffer. Gels were transferred to PVDF, blocked in 10% non-fat milk, before incubation with anti-amyloid-β antibody overnight at 4° C. and developed with anti-mouse HRP followed by ECL and exposed to film.

ELISA Method to Quantify the Protein-protein Interaction Between Oligomeric Amyloid-β and PrP^c

PrP^c was diluted to 10× the coating amount (in 100 µl; final amount of 500 ng PrP^c per well) in carbonate coating buffer and applied to Maxisorp plates. Plates were then sealed and left overnight at 4° C. Coated plates were carefully washed in PBS-TWEEN 20® and blocked with 2% BSA in PBS. Plates were washed and 100 µl of oligomeric amyloid-β-biotin peptide preparation (final concentration 200 nM) carefully mixed with test compound before adding to each well. Plates were incubated for 60 minutes at room temperature, washed and treated with streptavidin-HRP and after further washes the color was developed using TMB (reaction stopped with 2 N H₂SO₄). Absorbance was read at 450 nm within 30 minutes.

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

Curve Fitting

Quantitative pharmacological analysis was performed by iterative curve fitting to a floating four parameter logistic equation.

Results

DSSS competed for the protein-protein interaction between oligomeric amyloid-β and PrP^c in a concentration dependent manner as did LMW-DS (FIG. 1; Table 2). Quantitative pharmacological analysis indicated that LMW-DS displayed comparable overall affinity to DSSS yet apparent differences in the side-by-side levels of competable binding and Hill coefficients suggest a differential interaction between the two compounds (FIG. 1; Table 2). In contrast to DSSS and LMW-DS, dextran failed to compete appreciably for the protein-protein interaction between oligomeric amyloid-β and PrP^c.

Quantitative pharmacological analysis of ability to compete for protein-protein interaction between amyloid-β and PrPc
Compound	Competable binding (%)	IC50 (µg/mL)	Hill coefficient
DSSS	101±2	0.62±0.07	1.51±0.06
LMW-DS	85±4	0.42±0.16	1.00±0.21

Discussion

High-molecular weight dextran sulfate (DSSS) has previously been reported to compete with the protein-protein interaction between oligomeric amyloid-β and PrP^c with effective concentrations in the low µg/ml range (Aimi et al., J Neurochem. 2015, 134: 611-617). In the present study, optimization of the methodology resulted in the generation of an apparent greater proportion of oligomeric amyloid-β relative to the study of Aimi et al. The optimization of the protein-protein interaction ELISA resulted in a greater degree of specific protein-protein interaction; the greater dynamic range of competition facilitated quantitative pharmacological analysis of the interaction by competing compounds. The present study therefore represents an improvement over the study reported by Aimi et al.

DSSS and LMW-DS displayed comparable affinity to compete for the protein-protein interaction between oligomeric amyloid-β and PrP^c, yielding IC₅₀ values of 0.62±0.07 and 0.42±0.16 µg/mL, respectively. Hill analysis of the nature of the competition indicated that LMW-DS displayed shallower competition curves in comparison to the relatively high Hill coefficients associated with DSSS, which provides evidence for a differential pharmacological action between DSSS and LMW-DS.

LMW-DS thereby competed for the protein-protein interaction between oligomeric amyloid-β and PrP^c and can thereby be used to prevent or at least inhibit protein-protein interactions. This effect as seen with LMW-DS has potentials in coronavirus infections and coronavirus infectious diseases involving protein-protein interaction. Hence, LMW-DS has potential in disrupting the interaction of the SARS-CoV-2 trimeric spike protein with ACE2, which is further verified in Example 13.

Example 2

This Example investigated the potential of LMW-DS in enhancing the release and activation of a tissue repair growth factor in the blood of ALS patients.

Materials & Methods

This was a single-center, open single-arm study where the safety, tolerability and efficacy of subcutaneous (s.c.) administered LMW-DS was evaluated in patients with ALS. There were 10 planned visits to the clinic: 1 screening visit divided in 2 parts (Visit 1a and Visit 1b), 5 investigational medicinal product (IMP) administration visits (Visit 2 to Visit 6) and 3 follow-up visits (Visit 7 to Visit 9). Each individual patient’s study participation was planned to be approximately 4 months, including the screening and follow-up visits.

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

Results

LMW-DS induced a significant increase in plasma HGF levels in ALS patients with a maximum peak at about 2 hr after LMW-DS administration (FIG. 2, Table 3), which had reduced but were still significantly elevated at 6 hours following LMW-DS administration (Table 3).

LMW-DS exponentially increases plasma HGF in ALS patients
Sample Time	Plasma HGF (pg/mL) Day 7	Plasma HGF (pg/mL) Day 36
15 min before LMW-DS administration	750 ± 317	637 ± 126
Max Peak 2 h after LMW-DS administration	32 438 ± 5 348*	41 691 ± 8 005*
Last sample 6 h after LMW-DS administration	10 687 ± 5 795*	6 423 ± 3 370*
*P<0.01 vs before LMW-DS administration

Discussion

HGF, sometimes referred to as scatter factor (SF), is a paracrine cellular growth, motility and morphogenic factor. It has been shown to have a major role in embryonic organ development but also in adult organ regeneration and wound healing.

The elevated levels of plasma HGF induced by LMW-DS would be beneficial in COVID-19 patients by inducing tissue repair and regeneration and wound healing of organs and tissues negatively affected by the SARS-CoV-2 infection and the inflammatory responses induced by the SARS-CoV-2 infection, including ARDS, SIRS and organ fibrosis.

Example 3

The effects of daily sub-cutaneous injections of LMW-DS on glutamate excitotoxicity and mitochondrial function after severe traumatic brain injury (sTBI) in rats were evaluated by high-performance liquid chromatography (HPLC) analysis of frozen brain samples. The results suggest that LMW-DS interferes with mitochondrial function to improve energy metabolism and also decreases glutamate excitotoxicity.

Materials and Methods

Induction of sTBI and Drug Administration Protocol

The experimental protocol used in this study was approved by the Ethical Committee of the Catholic University of Rome, according to international standards and guidelines for animal care. Male Wistar rats of 300-350 g body weight (b.w.) were fed with standard laboratory diet and water ad libitum in a controlled environment.

They were divided into three groups:

• 1) n = 6 animals subjected to sTBI, with drug administration after 30 minutes and sacrifice at 2 days post-TBI (Acute phase 1)
• 2) n= 6 animals subjected to sTBI, with drug administration after 30 minutes and sacrifice at 7 days post-TBI (Acute phase 2).
• 3) n= 6 animals subjected to sTBI, with drug administration after 3 days and sacrifice at 7 days post-TBI (Chronic phase).

As the anesthetic mixture, animals received 35 mg/kg b.w. ketamine and 0.25 mg/kg b.w. midazolam by i.p. injection. sTBI was induced by dropping a 450 g weight from 2 m height on to the rat head that had been protected by a metal disk previously fixed on the skull, according to the “weight drop” impact acceleration model (Marmarou et al., J Neurosurg. 1994; 80: 291-300). Rats that suffered from skull fracture, seizures, nasal bleeding, or did not survive the impacts, were excluded from the study. At the end of each period of treatment, rats were anesthetized again and then immediately sacrificed.

The drug treatment was a subcutaneous injection of 0.5 ml of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016/076780; 15 mg/kg) and administered according to the aforementioned schematic protocol.

Cerebral Tissue Processing

An in vivo craniectomy was performed in all animals during anesthesia, after carefully removing the rat’s skull, the brain was exposed and removed with a surgical spatula and quickly dropped in liquid nitrogen. After the wet weight (w.w.) determination, tissue preparation was affected as previously disclosed (Tavazzi et al., Neurosurgery. 2005; 56: 582-589; Vagnozzi et al., Neurosurgery. 2007; 61: 379-388; Tavazzi et al., Neurosurgery. 2007; 61: 390-395; Amorini et al., J Cell Mol Med. 2017; 21: 530-542.). Briefly, whole brain homogenization was performed with 7 ml of ice-cold, nitrogen-saturated, precipitating solution composed by CH₃CN + 10 mM KH₂PO₄, pH 7.40, (3:1; v:v), and using an Ultra-Turrax set at 24,000 rpm/min (Janke & Kunkel, Staufen, Germany). After centrifugation at 20,690 x g, for 10 min at 4° C., the clear supernatants were saved, pellets were supplemented with 3 ml of the precipitating solution and homogenized again as described above. A second centrifugation was performed (20,690 x g, for 10 min at 4° C.), pellets were saved, supernatants combined with those previously obtained, extracted by vigorous agitation with a double volume of HPLC-grade CHCl₃ and centrifuged as above. The upper aqueous phases containing water-soluble low-molecular weight compounds were collected, subjected to chloroform washings for two more times (this procedure allowed the removal of all the organic solvent and of any lipid soluble compound from the buffered tissue extracts), adjusted in volumes with 10 mM KH₂PO₄, pH 7.40, to have ultimately aqueous 10% tissue homogenates and saved at -80° C. until assayed.

HPLC Analyses of Purine-pyrimidine Metabolites

Aliquots of each deproteinized tissue samples were filtered through a 0.45 µm HV Millipore filter and loaded (200 µl) onto a Hypersil C-18, 250 × 4.6 mm, 5 µm particle size column, provided with its own guard column (Thermo Fisher Scientific, Rodano, Milan, Italy) and connected to an HPLC apparatus consisting of a Surveyor System (Thermo Fisher Scientific, Rodano, Milan, Italy) with a highly sensitive diode array detector (equipped with a 5 cm light path flow cell) and set up between 200 and 300 nm wavelength. Data acquisition and analysis were performed by a PC using the ChromQuest® software package provided by the HPLC manufacturer.

Metabolites belonging to the purine-pyrimidine profiles (listed below) and related to tissue energy state, mitochondrial function and relative to oxidative-nitrosative stresses were separated, in a single chromatographic run, according to slight modifications of existing ion-pairing HPLC methods (Lazzarino et al., Anal Biochem. 2003; 322: 51-59; Tavazzi et al., Clin Biochem. 2005; 38: 997-1008). Assignment and calculation of the compounds of interest in chromatographic runs of tissue extracts were carried out at the proper wavelengths (206, 234 and 260 nm) by comparing retention times, absorption spectra and areas of peaks with those of peaks of chromatographic runs of freshly-prepared ultra-pure standard mixtures with known concentrations.

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), Thyimidine, Orotic Acid, Nitrate (—NO₃^-), Uridine monophosphate (UMP), Nicotinamide adenine dinucleotide, oxidized (NAD+), Adenosine (ADO), Inosine monophosphate (IMP), Guanosine monophosphate (GMP), Uridine diphosphate-glucose (UDP-Glc), UDP-galactose (UDP-Gal), oxidized glutathione (GSSG), UDP-N-acetyl-glucosamine (UDP-GlcNac), UDP-N-acetylgalactosamine (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 (NADH), Adenosine triphosphate (ATP), Nicotinamide adenine dinucleotide phosphate, reduced (NADPH), Malonyl-CoA, Coenzyme A (CoA-SH), Acetyl-CoA, N-acetylaspartate (NAA).

HPLC Analyses of Free Amino Acids and Amino Group Containing Compounds

The simultaneous determination of primary free amino acids (FAA) and amino group containing compounds (AGCC) (listed below) was performed using the precolumn derivatization of the sample with a mixture of Ortho-phthalaldehyde (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, the derivatization mixture composed by 25 mmol/l OPA, 1% MPA, 237.5 mmol/l sodium borate, pH 9.8 was prepared daily and placed in the autosampler. The automated precolumn derivatization of the samples (15 µl) with OPA-MPA was carried out at 24° C. and 25 µl of the derivatized mixture were loaded onto the HPLC column (Hypersil C-18, 250 × 4.6 mm, 5 µm particle size, thermostated at 21° C.) for the subsequent chromatographic separation. In the case of glutamate, deproteinized brain extracts were diluted 20 times with HPLC-grade H₂O prior to the derivatization procedure and subsequent injection. Separation of OPA-AA and OPA-AGCC was carried out at a flow rate of 1.2 ml/min using two mobile phases (mobile phase A = 24 mmol/l CH₃COONa + 24 mmol/l Na₂HPO₄ + 1% tetrahydrofurane + 0.1% trifluoroacetic acid, pH 6.5; mobile phase B = 40% CH₃OH + 30% CH₃CN + 30% H₂O), 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).

Assignment and calculation of the OPA-AA and OPA-AGCC in chromatographic runs of whole brain extracts were carried out at 338 nm wavelengths by comparing retention times and areas of peaks with those of peaks of chromatographic runs of freshly-prepared ultra-pure standard mixtures with known concentrations.

List of FAA and ACGC compounds: aspartate (ASP), glutamate (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).

Statistical Analysis

Normal data distribution was tested using the Kolmogorov-Smirnov test. Differences across groups were estimated by the two-way ANOVA for repeated measures. Fisher’s protected least square was used as the post hoc test. Only two-tailed p-values of less than 0.05 were considered statistically significant

Results

The most evident result among the cerebral values of the 24 standard and non-standard amino acids and primary amino-group containing compounds was that LMW-DS treatment had a remarkable inhibition of the increase in glutamate (GLU) induced by sTBI (FIG. 3), thus certainly causing a decrease of excitotocity consequent to excess of this compound.

This effect was, however, visible only if the drug was administered early post-injury (30 min following sTBI), with no efficacy on this excitotoxicity marker when LMW-DS was injected at 3 days after sTBI. It is also worth underlining that LMW-DS had significant beneficial effects on compounds involved in the so-called methyl cycle (Met, L-Cystat, SAH), see Table 4.

concentrations of cerebral compounds
	ASP	GLU	ASN	SER	GLN	HIS
Control	2.67±0.45	8.95±1.76	0.11±0.02	0.56±0.14	3.70±0.72	0.045±0.01
TBI 2 days	3.86±0.80	11.8±1.15	0.12±0.02	0.85±0.17	4.81±0.78	0.060±0.01
TBI 5 days	3.85±0.91	12.77±1.17	0.09±0.03	0.69±0.19	3.57±0.62	0.046±0.008
Acute phase 1	2.40±0.56d,i	9.81±1.66i	0.12±0.02i	0.88±0.25a	4.78±1.09a	0.068±0.015b
Acute phase 2	2.94±0.98f,j	9.93±1.56e,i	0.13±0.03i	0.71±0.28b	3.66±0.41	0.055±0.019
Chronic phase	4.46±0.70a,f	13.58±1.28a	0.18±0.02a	0.93±0.27a,e	3.98±0.34	0.047±0.021
	GLY	THR	CITR	ARG	ALA	TAU
Control	0.65±0.10	0.58±0.15	0.018±0.002	0.16±0.034	0.30±0.067	3.60±0.89
TBI 2 days	1.54±0.16	0.78±0.17	0.017±0.006	0.098±0.029	0.66±0.17	4.93±0.79
TBI 5 days	0.84±0.13	0.60±0.12	0.017±0.007	0.13±0.52	0.35±0.047	4.00±0.97
Acute phase 1	0.83±0.25a,c	0.92±0.29a	0.018±0.004	0.13±0.02b,d	0.50±0.12a	4.86±00.85b
Acute phase 2	0.71±0.16f,i	0.66±0.23	0.018±0.008	0.16±0.03	0.52±0.24a,e	3.80±1.19
Chronic phase	1.05±0.13a,f	0.75±0.24a,e	0.020±0.006	0.14±0.02	0.57±0.28a,e	4.49±0.43a
	GABA	TYR	SAH	L-Cystat	VAL	MET
Control	1.15±0.40	0.120±0.022	0.26±0.010	0.147±0.080	0.049±0.005	0.015±0.002
TBI 2 days	1.74±0.35	0.160±0.023	0.077±0.009	0.337±0.011	0.057±0.005	0.011±0.001
TBIl 5 days	1.50±0.30	0.123±0.013	0.043±0.013	0.202±0.061	0.042±0.014	0.010±0.001
Acute phase 1	1.43±0.25a	0.15±0.03	0.033±0.008b,c,j	0.185±0.031b,c,i	0.042±0.011	0.016±0.005d,j
Acute phase 2	1.60±0.24a	0.172±0.046b,f	0.026±0.010f,i	0.173±0.038b,f,i	0.057±0.017	0.022±0.006b,e,i
Chronic phase	1.85±0.65a	0.21±0.05f	0.050±0.013a	0.26±0.05a,f	0.040±0.016b	0.009±0.004b
Control	0.013±0.002	0.023±0.001	0.030±0.010	0.015±0.002	0.012±0.003	0.206±0.042
TBI 2 days	0.023±0.004	0.046±0.011	0.043±0.005	0.014±0.007	0.013±0.015	0.202±0.023
TBI 5 days	0.012±0.003	0.033±0.006	0.038±0.010	0.014±0.005	0.009±0.002	0.19±0.092
Acute phase 1	0.030±0.007b,dg,i	0.031±0.011b,d	0.038±0.007	0.021±0.005a,c	0.014±0.007	0.236±0.057b,d,h
Acute phase 2	0.015±0.006	0.028±0.010	0.048±0.017a	0.018±0.004	0.011±0.005	0.32±0.04a,e,i
Chronic phase	0.012±0.007	0.033±0.011b	0.041±0.016b	0.024±0.032b,f	0.017±0.009a,e	0.179±0.036
a p<0.01 (comparison with control), b p<0.05 (comparison with control), c p<0.01 (comparison with TBI 2 days), d p<0.05 (comparison with TBI 2 days), e p<0.01 (comparison with TBI 5 days), f p<0.05 (comparison with TBI 5 days), g p<0.01 (comparison with Acute phase 2), h p<0.05 (comparison with Acute phase 2), i p<0.01 (comparison with Chronic phase), j p<0.05 (comparison with Chronic phase) Table 3 lists the compounds in µmol/g (w.w.)

As is seen in Table 5, LMW-DS positively affected various compounds related to energy metabolism and mitochondrial functions. Particularly interesting are the concentrations of adenine nucleotides and ATP/ADP ratio as measurement of mitochondrial phosphorylating capacity (FIGS. 4A-4D).

concentrations of energy metabolites
	cytosine	creatinine	uracil	β-pseudouridine	cytidine
Control	12.89±1.77	18.77±2.09	10.65±1.11	6.32±1.11	12.54±1.84
TBI 2 days	23.58±5.62	28.61±3.33	17.32±1.54	8.45±0.98	11.33±1.23
TBI 5 days	21.56±2.88	76.03±8.19	24.31±2.60	18.66±1.29	26.12±2.37
Acute phase 1	17.69±2.50b,d	24.55±3.20b,g,i	14.56±5.44	6.65±1.30g,i	15.40±3.04
Acute phase 2	15.70±4.10f	37.27±5.82a,e,j	19.40±7.52a,e	13.26±3.16a,e,j	16.18±4.21e
Chronic phase	15.58±2.50b,f	51.25±10.17a,f	16.57±2.99a f	18.62±2.80a	14.71±2.83e
	hypoxanthine	guanine	xanthine	CDP choline	ascorbic acid
Control	7.21±1.22	3.12±0.78	8.09±1.48	7.50±1.01	4954.36±212.43
TBI 2 days	11.36±1.52	5.42±0.87	13.15±2.88	9.83±1.71	3186.09±287.87
TBI 5 days	16.83±2.13	4.56±1.29	14.14±2.11	8.12±1.55	2234.51±198.62
Acute phase 1	14.47±2.87a	4.80±1.24b	9.46±2.34d	10.93±3.22b,h	3733.10±277.88a,d
Acute phase 2	12.90±2.58a,j	4.73±1.07	10.41±2.11f	6.91±1.86	3512.58±224.62a,e
Chronic phase	17.97±4.49a	5.31±1.04b	9.35±0.83f	8.37±2.19	3375.03±856.41 a,e
	uridine	adenine	NO2	GSH	inosine
Control	56.17±3.88	23.14±2.16	151.21±16.79	3810.29±200.65	94.33±17.48
TBI 2 days	112.09±15.65	54.85±8.88	233.14±25.48	2109.89±156.71	126.36±14.06
TBI 5 days	94.8±10.75	76.55±6.33	256.28±28.07	1902.56±183.42	137.73±24.82
Acute phase 1	76.35±12.85a,c	44.82±6.31 a,d,g	216.03±41.74a	2649.50±397.31a,d	92.55±31.20c
Acute phase 2	63.02±9.66b,e	58.16±6.36a,f	226.40±30.95b	2821.50±242.82a,e	85.52±20.36e
Chronic phase	63.28±3.37f	52.94±8.59a,f	217.67±55.04a	2608.67±358.07a,e	105.81±25.57f
	uric acid	guanosine	CMP	MDA	thymidine
Control	2.75±0.35	18.96±2.90	12.16±1.61	1.13±0.25	0.54±0.16
TBI 2 days	30.84±5.13	17.52±2.44	30.83±4.81	28.37±3.37	0.67±0.19
TBI 5 days	23.63±3.40	21.32±3.04	27.20±3.76	7.69±2.18	0.97±0.32
Acute phase 1	23.62±3.77a,d,h	20.71±5.66	30.12±9.97a,h	12.47±2.09a,c,g	0.69±0.11
Acute phase 2	19.17±2.15a,h,i	17.90±3.24j	15.68±2.12f,j	4.82±1.73a,e,i	0.49±0.20f
Chronic phase	27.77±3.60a	28.87±7.60a,f	20.51±3.73a,f	11.62±3.90a,e	0.71±0.11
Control	5.67±0.85	178.66±37.75	96.21±10.51	506.88±59.15	50.73±8.29
TBI 2 days	10.09±1.54	265.31±47.68	116.06±13.55	322.37±30.87	66.19±11.06
TBI 5 days	14.27±1.67	325.19±60.08	128.70±28.28	261.67±49.97	78.91±20.42
Acute phase 1	8.80±2.45b,h,j	210.64±91.95d	107.80±21.62	404.63±51.10a,c,i	71.67±15.87
Acute phase 2	13.34±3.65a	198.56±25.93e,i	138.73±32.01b	401.18±34.53a,e,i	82.11±16.51a
Chronic phase	12.05±1.50a	241.27±18.84e	103.11±29.79	301.13±29.90a	89.97±12.98a
Control	54.09±12.15	98.93±10.42	47.23±3.14	120.18±10.99	189.21±20.19
TBI 2 days	50.82±10.45	181.94±27.20	45.17±6.67	131.19±18.49	179.51±29.17
TBI 5 days	124.46±18.97	158.35±40.43	41.43±5.14	112.26±17.36	196.65±33.48
Acute phase 1	67.71±10.63g,i	177.00±32.39a,g	32.14±4.59g	119.45±12.50	185.21±48.10
Acute phase 2	102.63±22.09a	91.47±12.35e,i	44.44±7.59j	145.14±27.76	219.54±53.36
Chronic phase	99.29±13.82a	148.56±31.21a	35.79±3.45b	122.29±12.15	231.08±44.34b,f
Control	93.71±14.16	35.09±3.07	30.31±5.12	34.89±8.18	14.08±1.14
TBI 2 days	93.71±14.16	20.17±3.33	73.32±12.88	39.16±6.87	18.31±2.15
TBI 5 days	129.54±21.21	10.56±2.89	98.32±10.99	59.88±12.54	19.03±6.45
Acute phase 1	95.85±19.73h,i	19.17±4.01a	53.61±17.91a,c,j	38.71±6.86	25.53±6.83a,c
Acute phase 2	130.65±28.41a	19.90±3.12a,e	57.70±23.01a,e,j	49.25±10.33a	24.29±6.76a
Chronic phase	129.42±15.88b	21.84±2.80a,e	90.01±21.24a	43.85±5.06b	23.55±6.45a
Control	26.06±7.32	61.78±17.09	27.52±2.58	48.88±5.61	38.90±4.64
TBI 2 days	55.47±6.70	149.02±19.09	16.36±4.41	133.31±30.02	21.57±3.19
TBI 5 days	43.71±8.81	113.11±28.34	12.50±2.97	221.80±36.72	18.79±3.69
Acute phase 1	61.83±10.23a,g	158.72±24.57a	17.95±3.28a	137.87±43.18a	18.98±6.58a,g
Acute phase 2	40.38±8.50a,i	126.70±31.35a,j	21.27±4.19b,e,j	141.96±23.56a,e,j	32.63±3.99e,i
Chronic phase	57.40±5.88a,f	173.05±28.68a,e	16.44±2.66a,f	173.94±8.45a	25.23±2.93a,f
Control	233.19±21.33	138.95±28.89	567.33±54.79	14.50±2.75	2441.66±257.71
TBI 2 days	264.71±26.31	107.77±12.83	208.13±28.36	8.54±1.73	1350.25±140.87
TBI 5 days	328.26±31.30	90.50±18.69	191.81±37.56	6.77±1.58	1195.81±137.82
Acute phase 1	279.34±29.59b	123.46±15.42d	255.29±45.21a,g	15.49±2.05c,j	1464.25±99.09a,h
Acute phase 2	264.07±28.29b,e,j	146.71±32.68e	336.65±35.18a,e,j	13.12±4.19e	1632.23±90.07a,e,j
Chronic phase	315.53±46.53a	136.80±33.25f	290.92±34.68a,f	11.78±3.32e	1381.03±212.64a
Control	7.95±1.38	15.83±1.31	28.91±3.19	38.97±5.79	9141.22±366.64
TBI 2 days	8.14±1.69	10.46±2.56	19.64±2.37	21.76±4.49	5570.00±912.08
TBI 5 days	9.24±2.07	11.89±1.96	21.77±1.44	18.94±3.75	4300.00±480.84
Acute phase 1	6.22±1.73	12.33±1.82b	21.61±3.42a,h	21.56±6.22a,g,i	6147.91±989.12a
Acute phase 2	7.05±2.21	11.29±2.27b	30.57±6.02f	36.86±4.11e	7262.84±749.73a,e
Chronic phase	7.34±2.65f	10.00±1.95b	27.58±6.24f	35.68±6.55e	6375.36±974.12a,e
a p<0.01 (comparison with control), b p<0.05 (comparison with control), c p<0.01 (comparison with TBI 2 days), d p<0.05 (comparison with TBI 2 days), e p<0.01 (comparison with TBI 5 days), f p<0.05 (comparison with TBI 5 days), g p<0.01 (comparison with Acute phase 2), h p<0.05 (comparison with Acute phase 2), i p<0.01 (comparison with Chronic phase), j p<0.05 (comparison with Chronic phase) Table 4 lists the compounds in nmol/g (w.w.)

Remarkable changes of oxidative and reduced nicotinic coenzymes were also observed (FIGS. 5A-5D).

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

In addition, MDA, as end product of polyunsaturated fatty acids of membrane phospholipids and therefore taken as a marker of ROS-mediated lipid peroxidation, was also measured. MDA levels showed a significant reduction after administration of LMW-DS. The oxidative stress markers described above all indicated an improvement in the recovery of antioxidant status after treatment with LMW-DS (FIGS. 6A-6C).

Indices of representative of NO-mediated nitrosative stress (nitrite and nitrate) were also analyzed. LMW-DS administration significantly decreased the nitrate concentrations in both the acute and chronic phases of sTBI (FIG. 7).

NAA is a brain specific metabolite and a valuable 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, the molecule must move between cellular compartments to reach oligodendrocytes where it is degraded into acetate and aspartate by aspartoacylase (ASPA). An upregulation of the catabolic enzyme ASPA and an NAA decrease in order to supply the availability of the substrates aspartate and acetyl-CoA are an indication of the status of metabolic impairment. In this study NAA and its substrates were measured after sTBI and showed significant improvements in levels after LMW-DS administration (FIGS. 8A-8C).

These effects on energy metabolites were particularly evident when animals received the LMW-DS administration early post-injury (30 mins). It is important to note that the overall beneficial effects of LMW-DS were observed either when the animals were sacrificed 2 days after sTBI or when sacrifice occurred 7 days post sTBI. In these groups of animals, the general amelioration of metabolism connected to AGCC and energy metabolites was more evident, suggesting a long-lasting positive effect of the LMW-DS administration on brain metabolism.

Discussion

The data presented herein suggests that early administration of LMW-DS reduced levels of glutamate excitotoxicity and ameliorated adverse changes in metabolic homeostasis by protecting mitochondrial function.

In more detail, LMW-DS protects mitochondrial function and reduces oxidative stress as seen in, among others, improvement of the recovery of antioxidant status, protection of mitochondrial ATP energy supply by preserving ATP production and metabolism, normalization of mitochondrial phosphorylating capacity all as induced by LMW-DS. As a consequence, LMW-DS is capable of protecting and preserving mitochondrial function in cells exposed to a damage or disease, which is of importance in order to have functional cells that can combat the infectious disease. The metabolic normalization as induced by LMW-DS is of benefit for COVID-19 patients both during the pulmonary phase (stage II) and the recovery phase (stage IV) (FIG. 25).

Example 4

The aim of this study was to evaluate the potential neuroprotective effects of LMW-DS on biochemical, molecular and histo-anatomical damages produced by the experimental model of closed-head diffuse severe TBI (sTBI) in the rat. In the present study, results were obtained through HPLC analyses of low molecular weight metabolites representative of energy metabolism, oxidative/nitrosative stress, antioxidants and free amino acids in cerebral tissue extracts of treated animals.

Materials and Methods

Induction of sTBI and Drug Administration Protocol

Male Wistar rats (n=160) of 300-350 g body weight were used in this study. They were fed with standard laboratory diet and water ad libitum in a controlled environment.

As the accepted anesthetic mixture, animals received 35 mg/kg b.w. ketamine and 0.25 mg/kg body weight midazolam by intramuscular injection. Diffuse sTBI was induced according to the “weight drop” impact acceleration model set up by Marmarou et al. J. Neurosurg. 1994, 80: 291-300. This model causes diffuse axonal injury and it is able to reproduce the physical and mechanical characteristics of the diffuse TBI in humans.

Severe TBI was induced by dropping a 450 g weight from 2 meters height onto the rat head protected by a helmet (metal disk previously fixed on the skull using dental cement) in order to uniformly distribute the mechanical force to the brain. Rats were placed prone on a bed of specific polyurethane foam inserted in a special container; this foam dissipates the major part of the potential energy (deriving from the mechanical forces) and prevents any rebound of the animal after the impact that could produce spinal damages.

Rats suffering from skull fracture, seizures, nasal bleeding, or did not survive the impact, were excluded from the study. After 2 or 7 days from TBI induction, rats were anesthetized again and then immediately sacrificed. These time points are coincident with the worst biochemical derangement (2 days) or, in the case of a mildly injured brain, with a full metabolic recovery (7 days).

The drug treatment consisted in a subcutaneous injection of 0.5 ml of LMW-DS (ILB®, Tikomed, Viken, Sweden, WO 2016/076780) and administered at 3 different concentrations (1, 5 and 15 mg/kg body weight), according to the schematic protocol described below. Sham-operated animals underwent the same procedure of anesthesia but TBI and were used as the control group.

Experimental Design

Rats used in this study were divided into 4 groups in order to carry out a study on the efficacy of three different concentrations of LMW-DS at two different times post TBI. As subsequently specified, in each group there were animals subjected to a specific treatment for metabolic analyses and other animals intended to histo-morphological studies, according to the procedures described below.

Group-1

Controls (n = 12) dedicated to the biochemical evaluation. Four additional animals were used for the histo-morphological studies. Total rats in this group: n = 16

Group-2

Rats subjected to sTBI with no pharmacological treatment were divided into the following subgroups:

• 1. 12 animals subjected to sTBI and sacrificed after 2 days post-TBI
• 2. 12 animals subjected to sTBI and sacrificed after 7 days post-TBI

Four additional rats to each subgroup were used for the histo-morphological studies. Total rats in this group: n = 32.

Group-3

Rats subjected to sTBI and receiving a single administration of LMW-DS after 30 minutes post-TBI, with sacrifice at 2 days post-TBI. Animals were divided in the following subgroups:

• 1. 12 animals subjected to sTBI and treated with 1 mg/kg b.w. LMW-DS
• 2. 12 animals subjected to sTBI and treated with 5 mg/kg b.w. LMW-DS
• 3. 12 animals subjected to sTBI and treated with 15 mg/kg b.w. LMW-DS

Four additional rats to each subgroup were used for the histo-morphological studies. Total rats in this group: n = 48.

Group-4

Rats subjected to sTBI and receiving a single administration of LMW-DS after 30 minutes post-TBI, with sacrifice at 7 days post-TBI. Animals were divided in the following subgroups:

• 1. 12 animals subjected to sTBI and treated with 1 mg/kg b.w. LMW-DS
• 2. 12 animals subjected to sTBI and treated with 5 mg/kg b.w. LMW-DS
• 3. 12 animals subjected to sTBI and treated with 15 mg/kg b.w. LMW-DS

Four additional rats to each subgroup were used for the histo-morphological studies. Total rats in this group: n = 48.

Group-5

Rats (n = 12) subjected to sTBI and receiving repeated administrations of the maximal dose of LMW-DS (15 mg/kg b.w.) after 30 minutes, 3 days and 5 days post-TBI, with sacrifice at 7 days post-TBI. Four additional rats were used for the histo-morphological studies. Total rats in this group: n=16

Cerebral Tissue Processing for Biochemical and Gene Expression Analyses

To minimize metabolite loss, an in vivo craniectomy was performed in all animals during anesthesia. The rat skull was carefully removed, the brain was exposed, sharply cut along the sagittal fissure and the two hemispheres were separated. The hemispheres dedicated to biochemical analyses were freeze-clamped by aluminum tongues pre-cooled in liquid nitrogen and then immersed in liquid nitrogen. The freeze-clamping procedure was introduced to accelerate freezing of the tissue, thus minimizing potential metabolite loss.

The remaining hemispheres, dedicated to molecular biology analyses, were placed in 5-10 volumes of RNAlater® Solution (Invitrogen Life Technologies), a RNA stabilization solution that stabilize and protect RNA from degradation. Brain samples were stored at 4° C. overnight to allow the solution completely penetrate tissue.

Tissue homogenization for metabolite analyses was effected as described below. After the wet weight (w.w.) determination, the frozen hemispheres were placed into 7 ml of ice-cold, nitrogen-saturated, precipitating solution (1:10 w/v) composed by CH₃CN + 10 mM KH₂PO₄, pH 7.40, (3:1; v:v), and the homogenization was performed using an Ultra-Turrax homogenizer set at 24,000 rpm/min (Janke & Kunkel, Staufen, Germany). After centrifugation at 20,690 × g, for 10 min at 4° C., the clear supernatants were saved, pellets were supplemented with an aliquot of 10 mM KH₂PO₄ and homogenized again as described above and saved overnight at -20° C. in order to obtain a complete recovery of aqueous phase from tissue. A second centrifugation was performed (20,690 × g, for 10 min at 4° C.) and supernatants combined with those previously obtained were extracted by vigorous agitation with a double volume of HPLC-grade CHCl₃ and centrifuged as above. The upper aqueous phases (containing water-soluble low-molecular weight compounds) were collected, subjected to chloroform washings for two more times (this procedure allowed the removal of all the organic solvent and of any lipid soluble compound from the buffered tissue extracts), adjusted in volumes with 10 mM KH₂PO₄, pH 7.40, to have ultimately aqueous 10% tissue homogenates and saved at -80° C. until assayed.

HPLC Analysis of Energy Metabolites, Antioxidants and Oxidative/nitrosative Stress Biomarkers

Aliquots of each deproteinized tissue sample were filtered through a 0.45 µm HV Millipore filter and loaded (200 µl) onto a Hypersil C-18, 250 × 4.6 mm, 5 µm particle size column, provided with its own guard column (Thermo Fisher Scientific, Rodano, Milan, Italy) and connected to an HPLC apparatus consisting of a Surveyor System (Thermo Fisher Scientific, Rodano, Milan, Italy) with a highly sensitive diode array detector (equipped with a 5 cm light path flow cell) and set up between 200 and 300 nm wavelength. Data acquisition and analysis were performed by a PC using the ChromQuest® software package provided by the HPLC manufacturer.

Metabolites (listed below) related to tissue energy state, mitochondrial function antioxidants and representative of oxidative/nitrosative stress were separated, in a single chromatographic run, according to slight modifications of existing ion-pairing HPLC methods formerly (Lazzarino et al., Anal Biochem. 2003, 322: 51-59; Tavazzi et al., Clin Biochem. 2005, 38: 997-1008). Assignment and calculations of the compounds of interest in chromatographic runs of tissue extracts were carried out at the proper wavelengths (206, 234 and 260 nm) by comparing retention times, absorption spectra and areas of peaks with those of peaks of chromatographic runs of freshly-prepared ultra-pure standard mixtures with known concentrations.

List of compounds: Cytosine, Creatinine, Uracil, Beta-Pseudouridine, Cytidine, Hypoxanthine, Guanine, Xanthine, CDP-Choline, Ascorbic Acid, Uridine, Nitrite (NO₂), reduced glutathione (GSH), Inosine, Uric Acid, Guanosine, CMP, Malondialdehyde (MDA), Nitrate (NO₃), UMP, NAD+, ADO, IMP, GMP, UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetyl-glucosamine (UDP-GlcNac), UDP-N-acetyl-galactosamine (UDP-GalNac), AMP, GDP-glucose, UDP, GDP, NADP+, ADP-Ribose, CTP, ADP, UTP, GTP, NADH, ATP, NADPH, Malonyl-CoA, Coenzyme A (CoA-SH), Acetyl-CoA, N-acetylaspartate (NAA).

HPLC Analysis of Free Amino Acids and Amino Group Containing Compounds

The simultaneous determination of primary free amino acids (FAA) and amino group containing compounds (AGCC) (listed below) was performed using the precolumn derivatization of the sample with a mixture of OPA and MPA, as described in (Amorini et al., J Cell Mol Med. 2017, 21(3): 530-542; Amorino et al., Mol Cell Biochem. 2012, 359: 205-216). Briefly, the derivatization mixture composed by 25 mmol/l OPA, 1% MPA, 237.5 mmol/l sodium borate, pH 9.8 was prepared daily and placed in the autosampler. The automated precolumn derivatization of the samples (15 µl) with OPA-MPA was carried out at 24° C. and 25 µl of the derivatized mixture were loaded onto the HPLC column (Hypersil C-18, 250 × 4.6 mm, 5 µm particle size, thermostated at 21° C.) for the subsequent chromatographic separation. In order to quantify correctly Glutamate, deproteinized brain extracts were diluted 20 times with HPLC-grade H₂O prior to the derivatization procedure and subsequent injection. Separation of OPA-AA and OPA-AGCC was carried out at a flow rate of 1.2 ml/min using two mobile phases (mobile phase A = 24 mmol/l CH₃COONa + 24 mmol/l Na₂HPO₄ + 1% tetrahydrofurane + 0.1% trifluoroacetic acid, pH 6.5; mobile phase B = 40% CH₃OH + 30 CH₃CN + 30% H₂O), using an appropriate step gradient.

Assignment and calculation of the OPA-AA and OPA-AGCC in chromatographic runs of whole brain extracts were carried out at 338 nm wavelengths by comparing retention times and areas of peaks with those of peaks of chromatographic runs of freshly-prepared ultra-pure standard mixtures with known concentrations.

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

Brain Tissue Processing for Histo-morphological Analyses

After adequate anesthesia rats were transcardially perfused as described in (Di Pietro et al., Sci Rep. 2017, 7(1): 9189). Briefly, a thoracotomy was performed and a heparin solution was administered into the portal vein to avoid blood coagulation during all the operation. Afterwards, a right atrial incision was carried out and the perfusion needle was advanced into the ascending aorta. Perfusion was performed with 100 ml of Phosphate Buffer Solution (PBS) at pH 7.4 in order to wash out blood before further perfusion with 100 ml 4% paraformaldehyde (PFA) in PBS solution at pH 7.4. After rapid removal from the skull, each brain was post fixed by immersion in 4% PFA in PBS solution for 2 hours at 4° C. Cryoprotection was obtained by immersing the whole brain in PBS enriched with increasing sucrose solutions (10%, 20%, and 30%) for 24 hours at 4° C., then implanted in optimal cutting temperature embedding medium (OCT) (Thermo Shandon, Runcorn, UK) in peel-away mould containers (Agar Scientific, Essex, UK). Brains immersed in OCT were rapidly frozen in crushed dry ice before storage at -80° C.

Statistical Analysis

Differences across groups were estimated by the Student’s t-test. Only two-tailed p-values of less than 0.05 were considered statistically significant.

Results

SUMMARY OF BIOCHEMICAL DATA RECORDED AT 2 DAYS POST sTBI

Effects of Increasing Doses of LMW-DS on Brain Energy Metabolism Measured

Table 6 summarizes values referring to phosphorylated high-energy purine and pyrimidine compounds. It is particularly evident the depletion of triphosphate nucleotides (ATP, GTP, UTP and CTP) caused by sTBI, that was accompanied by an increase in ADP and in the N-acetylated derivatives of UDP-glucose (UDP-GlcNac) and UDP-galactose (UDP- GalNac).

At this time post injury, treatment with LMW-DS was only partly effective in improving cell energy metabolism: Significantly higher values of high energy phosphates (ATP, GTP, and CTP) were recorded with all the three dosages of the drug tested. No effects were seen on the concentrations of UTP and ADP. It is worth recalling that 48 hours post TBI in rats represents a critical time point for brain metabolism, coincident with maximal alterations of mitochondrial functions including changes in the mitochondrial quality control. In this experimental model of TBI, this time point could be considered a sort of “turning point” at which recovery or no recovery of cerebral metabolism is defined.

Concentrations of energy metabolites (phosphorylated purines and pyrimidines) measured in deproteinized brain homogenates of rats sacrificed at 2 days post-sTBI, without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only)	LMW-DS 1	LMW-DS 5	LMW-DS 15
CMP	13.52 ± 3.44	34.85 ± 7.11	29.39 ± 6.00	28.94 ± 5.91	25.61 ± 5.23
UMP	82.30 ± 9.82	151.45 ± 20.92	148.04 ± 20.45	97.98 ± 13.53	147.53 ± 20.38
IMP	51.57 ± 4.610	55.06 ± 10.36	45.97 ± 8.65	33.19 ± 6.25	68.95 ± 12.98
GMP	82.81 ± 7.821	186.08 ± 23.36	205.06 ± 25.74	167.44 ± 8.47	178.88 ± 22.46
UDP-Glc	51.00 ± 10.89	48.87 ± 7.24	45.14 ± 6.68	28.60 ± 4.23	43.41 ± 6.43
UDP-Gal	131.00 ± 13.26	127.11 ± 10.61	118.50 ± 9.89	116.42 ± 9.72	116.34 ± 9.71
UDP-GlcNac	88.77 ± 19.55	102.34 ± 9.32	96.62 ± 8.80	140.58 ± 12.80	108.74 ± 9.90
UDP- GalNac	38.82 ± 9.83	22.10 ± 3.26	21.24 ± 3.13	20.75 ± 3.06	22.37 ± 3.30
GDP Glucose	85.35 ± 12.76	89.05 ± 39.68	65.66 ± 41.61	83.81 ± 37.35	84.24 ± 37.54
AMP	43.59 ± 9.90	65.13 ± 41.27	62.04 ± 7.46	67.03 ± 11.85	66.26 ± 10.74
UDP	23.94 ± 6.75	64.40 ± 6.60	83.06 ± 8.52	70.93 ± 7.27	80.00 ± 8.20
GDP	57.40 ± 14.06	167.28 ± 23.11	189.85 ± 26.23	183.27 ± 25.32	194.61 ± 26.88
ADP-Ribose	12.69 ± 1.43	13.85 ± 2.78	25.69 ± 5.16	21.65 ± 4.35	23.06 ± 4.63
CTP	41.85 ± 10.32	28.32 ± 5.73	33.01 ± 7.63	37.72 ± 7.63	37.53 ± 7.59
ADP	222.67 ± 30.99	297.53 ± 25.59	333.90 ± 28.72	364.92 ± 31.39	346.37 ± 29.79
UTP	152.64 ± 17.39	100.79 ± 15.83	104.07 ± 16.34	142.82 ± 22.43	108.21 ± 16.99
GTP	569.00 ± 45.32	202.19 ± 21.33	169.98 ± 17.93	180.01 ± 18.99	179.07 ± 18.89
ATP	2390.14 ± 213.98	1330.60 ± 77.96	1696.96 ± 99.43	1683.87 ± 98.66	1556.54 ± 91.20

In Tables 6 - 25, bold indicates significantly different from controls (p < 0.05); bold underlined indicates significantly different from TBI (p < 0.05); and bold italic indicates significantly different from both controls and TBI (p < 0.05).

Effects of Increasing Doses of LMW-DS on Nicotinic Coenzymes

Values of oxidized (NAD+ and NADP+) and reduced (NADH and NADPH) nicotinic coenzymes are summarized in Table 7. Table 7 also reports the calculated, adimensional values of the NAD+/NADH ratio which is suitable to evaluate how much metabolism is dependent on glycolysis or on mitochondrial oxidative phosphorylation.

As previously observed herein, sTBI caused decrease of NAD+, NADP+ and of the NAD+/NADH ratio. At this time point, treatment with LMW-DS was effective only at the maximal dose tested (15 mg/kg b.w.) that produced significant protection of the nicotinic coenzyme pool and avoid the metabolic switch towards glycolysis, thereby indirectly suggesting overall better mitochondrial functions.

Concentrations of nicotinic coenzymes measured in deproteinized brain homogenates of rats sacrificed at 2 days post-sTBI, without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w. The NAD+/NADH ratio is adimensional.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15
NAD+	485.74 ± 37.06	379.70 ± 64.64	325.87 ± 55.47	376.85 ± 64.15	475.32 ± 80.91
NADH	13.57 ± 1.94	12.45 ± 1.82	9.42 ± 1.38	10.37 ± 1.19	10.83 ± 1.58
NADP+	23.17 ± 4.58	17.68 ± 4.04	11.79 ± 2.70	11.86 ± 2.71	17.75 ± 4.06
NADPH	8.51 ± 0.71	7.94 ± 0.66	13.07 ± 1.09	37.48 ± 3.11	8.93 ± 0.74
NAD+/NADH	36.47 ± 5.46	34.99 ± 6.05	33.91 ± 9.32	36.61 ± 6.09	44.40 ± 7.67

Effects of Increasing Doses of LMW-DS on CoA-SH Derivatives

Table 8 reports data referring to free CoA-SH and CoA-SH derivatives. Particularly Acetyl-CoA is a crucial compound for mitochondrial metabolism allowing correct functioning of the tricarboxylic acid cycle (TCA cycle), thus ensuring continuous electron supply for the electron transfer chain (ETC). TCA is the major cell cycle for the generation of reduced coenzymes (NADH and FADH₂) which, by transferring their electrons to mitochondrial complexes I and II, respectively, are the fuel for ETC and oxidative metabolism. All compounds, particularly Acetyl-CoA, are significantly affected by sTBI. A partial rescue of this compound was observed when 5 or 15 mg/kg b.w. LWM-DS was administered to animals 30 minutes post injury.

Concentrations of free CoA-SH and CoA-SH derivatives (Acetyl-CoA and Malonyl-CoA) measured in deproteinized brain homogenates of rats sacrificed at 2 days post-TBI without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15
Malonyl-CoA	15.02 ± 2.38	11.82 ± 2.50	19.06 ± 4.04	35.58 ± 7.54	28.73 ± 6.09
CoA-SH	26.31 ± 3.86	21.00 ± 2.32	9.42 ± 1.04	7.46 ± 0.82	9.35 ± 1.03
Acetyl-CoA	36.97 ± 5.43	28.32 ± 3.29	27.74 ± 3.23	34.85 ± 4.05	32.38 ± 3.77

Effects of Increasing Doses of LMW-DS on Antioxidants and Oxidative/nitrosative Stress Biomarkers

Table 9 shows the concentrations of the main water-soluble brain antioxidants (ascorbic acid and GSH) and of biomarkers of oxidative (MDA) and nitrosative stress (—NO₂ and —NO₃^-). Malondialdehyde (MDA) originates from decomposition of unsaturated fatty acids of membrane phospholipids as a consequence of ROS-mediated lipid peroxidation. Nitrites (—NO₂—) and nitrates (—NO₃—) are stable end products of nitric oxide (NO) metabolism which, under pathological conditions, is generated in excess by an inducible form of nitric oxide synthase (iNOS) and gives raise to reactive nitrogen species (RNS) through the reaction with ROS:

At two days post impact, 25 to 45% decrease in both water-soluble antioxidants occurred in rats experiencing sTBI. Consequent increase in signatures of oxidative/nitrosative stress was also recorded. Administration of LWM-DS significantly ameliorated the concentrations of both ascorbic acid and reduced glutathione (GSH) with evident decrease of cerebral tissue nitrites and nitrates. These effects were more remarkable when 15 mg kg/b.w. where used.

Concentrations of antioxidants and oxidative/nitrosative stress biomarkers measured in deproteinized brain homogenates of rats sacrificed at 2 days post-TBI without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15
ASCORBIC ACID	3315.38 ± 351.59	2577.87 ± 148.36	2567.35 ± 147.76	2626.68 ± 151.17	2783.04 ± 160.17
GSH	3521.63 ± 275.04	1972.14 ± 287.59	2337.06 ± 340.81	2067.79 ± 301.54	2418.94 ± 352.75
MDA	0.85 ± 0.26	27.30 ± 4.45	44.00 ± 7.17	32.73 ± 5.33	18.28 ± 2.98
NO2	142.93 ± 28.19	232.31 ± 27.99	158.36 ± 19.08	218.12 ± 26.28	72.29 ± 8.71
NO3	169.51 ± 20.79	266.82 ± 58.06	99.16 ± 21.58	148.41 ± 32.30	56.50 ± 12.30

Effects of Increasing Doses of LMW-DS on De-phosphorylated Purines and Pyrimidines

The majority of the compounds reported in Table 10 originates from the degradation pathways of purine and pyrimidine nucleotides and are indirectly connected to cell energy metabolism. Rats receiving sTBI had higher cerebral concentrations of all these compounds, but CDP-choline, most of which were positively affected by the drug administration.

Concentrations of de-phosphorylated purines and pyrimidines measured in deproteinized brain homogenates of rats sacrificed at 2 days post-TBI without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15
CYTOSINE	14.14 ± 3.38	20.19 ± 2.47	13.47 ± 1.65	13.65 ± 1.67	13.57 ± 1.66
CREATININE	17.12 ± 2.49	31.08 ± 5.79	17.66 ± 3.29	10.22 ± 1.90	11.77 ± 2.19
URACIL	10.91 ± 2.27	15.64 ± 3.06	17.18 ± 3.36	17.83 ± 3.48	15.55 ± 3.04
β-PSEUDOURIDINE	6.89 ± 1.27	8.51 ± 1.71	11.64 ± 2.3	10.41 ± 2.09	7.84 ± 1.57
CYTIDINE	12.76 ± 2.59	10.07 ± 1.82	13.79 ± 2.49	7.47 ± 1.35	11.46 ± 2.07
HYPOXANTHINE	7.57 ± 0.93	15.22 ± 2.49	4.02 ± 0.66	4.18 ± 0.68	6.82 ± 1.12
GUANINE	3.34 ± 0.88	5.11 ± 1.28	1.62 ± 0.41	1.68 ± 0.42	1.61 ± 0.40
XANTHINE	7.61 ± 1.39	15.82 ± 1.64	13.79 ± 1.43	6.71 ± 0.70	13.87 ± 1.44
CDP choline	7.97 ± 1.370	8.25 ± 1.23	8.23 ± 1.22	5.16 ± 0.77	7.07 ± 1.05
URIDINE	64.08 ± 14.14	131.59 ± 23.17	79.93 ± 14.07	117.21 ± 20.64	87.55 ± 15.41
INOSINE	89.43 ± 15.04	134.31 ± 17.51	113.85 ± 14.84	114.89 ± 14.98	142.91 ± 18.63
URIC ACID	3.36 ± 0.64	37.73 ± 7.74	52.42 ± 10.75	11.22 ± 2.30	26.00 ± 5.33
GUANOSINE	21.10 ± 5.56	19.69 ± 3.27	16.46 ± 2.73	30.97 ± 5.15	24.35 ± 4.05
ADENOSINE	46.71 ± 7.39	68.07 ± 16.30	68.91 ± 16.50	92.58 ± 22.16	53.25 ± 12.75

Effects of Increasing Doses of LMW-DS on N-acetylaspartate (NAA)

NAA is the most abundant N-acetylated amino acid of the mammalian brain, with concentrations almost equaling those of the neurotransmitter glutamate in humans. Notwithstanding the biological role of NAA has not yet been fully elucidated. It has previously been shown, in both preclinical and clinical studies, that TBI decreases NAA concentrations and that its time course changes following head injury mirrors those of ATP. Particularly, sTBI causes an irreversible modification in NAA homeostasis, NAA is a good surrogate marker of brain energy metabolism and decrease and recovery of NAA levels are much slower than symptom clearance in post-concussed athletes. Hence, NAA has a particular relevance in studies on TBI.

Decrease by 40% in whole brain NAA was observed in sTBI rats (FIG. 9) at two days post impact. LMW-DS produced beneficial effects on NAA concentrations when administered at 5 or 15 mg/kg b.w. Although significantly lower than controls, NAA in rats administered with either one of the two drug dosages was significantly higher than values found in sTBI rats, with highest NAA levels found in rats receiving the highest dose of LMW-DS.

Effects of Increasing Doses of LMW-DS on Free Amino Acids Involved in Neurotransmission

Compounds listed in Table 11 are amino acids directly (GLU, GABA) of indirectly (GLN, ASP, ASN, GLY, SER, THR, ALA) involved in neurotransmission. Particularly, GLU is the main excitatory amino acid, counteracted in its action by GABA. Excitotoxicity of GLU is modulated by SER, GLY, THR and ALA and it is linked to the function of the GLU-GLN cycle involving neurons and astrocytes. As shown in a previous study, we here found that most of these amino acids increased in sTBI rats at two days post injury. Treating animals with a single administration of LMW-DS was partly effective when the drug was subcutaneously infused at 5 or 15 mg/kg b.w. In most cases, values of the different compounds were significantly better than those found in the group of untreated sTBI animals but not than those of controls.

Concentrations of free amino acids with neurotransmitter functions measured in deproteinized brain homogenates of rats sacrificed at 2 days post-TBI without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15
ASP	2.88 ± 0.88	4.55 ± 0.63	4.17 ± 0.99	4.15 ± 0.95	3.05 ± 0.42
GLU	9.92 ± 0.83	12.88 ± 0.60	12.52 ± 0.91	11.93 ± 0.55	11.79 ± 0.55
ASN	0.10 ± 0.03	0.14 ± 0.02	0.13 ± 0.02	0.17 ± 0.03	0.17 ± 0.03
SER	0.64 ± 0.17	0.82 ± 0.07	0.91 ± 0.07	0.91 ± 0.07	0.76 ± 0.06
GLN	3.89 ± 0.87	4.34 ± 0.42	4.37 ± 0.59	4.55 ± 0.44	4.21 ± 0.51
GLY	0.78 ± 0.13	1.38 ± 0.27	1.35 ± 0.26	1.43 ± 0.28	1.18 ± 0.23
THR	0.69 ± 0.18	0.76 ± 0.16	0.70 ± 0.15	0.77 ± 0.17	0.61 ± 0.13
ALA	0.41 ± 0.11	0.58 ± 0.06	0.76 ± 0.08	0.79 ± 0.08	0.68 ± 0.07
GABA	1.36 ± 0.22	1.93 ± 0.17	1.87 ± 0.17	1.99 ± 0.18	1.58 ± 0.14

Effects of Increasing Doses of LMW-DS on Free Amino Acids Involved in the Methyl Cycle

Free amino acids reported in Table 12 are involved either in the so called methyl cycle, regulating the homeostasis of compounds acting as methyl donors in cell metabolism, or in the formation of cysteine, the sole amino acid having a free -SH group. Severe head trauma caused significant changes in the main actors of this important metabolic pathway. Restoration of methionine was accomplished by LWM-DS at any dose tested. Drug treatment was partly effective in normalizing the other amino acids. Comments to changes in L-Cystathionine (L-Cystat) will be given in the corresponding Table at 7 days post impact.

Concentrations of free amino acids involved in the methyl cycle and homeostasis of -SH groups measured in deproteinized brain homogenates of rats sacrificed at 2 days post-TBI without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5 (	LMW-DS 15
SAH	0.03 ± 0.01	0.07 ± 0.01	0.07 ± 0.02	0.07 ± 0.02	0.06 ± 0.02
L-Cystat	0.15 ± 0.03	0.31 ± 0.06	0.25 ± 0.05	0.31 ± 0.06	0.41 ± 0.08
MET	0.03 ± 0.01	0.02 ± 0.01	0.04 ± 0.01	0.05 ± 0.01	0.03 ± 0.01

Effects of Increasing Doses of LMW-DS on Free Amino Acids Involved in the Generation of Nitric oxide (NO)

Table 13 illustrates concentrations of the free amino acids directly involved in the generation of NO, in the reaction catalyzed by nitric oxide synthases (NOS), a family of enzymes existing in three isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS). The last isoform (iNOS) is the one involved in nitrosative stress. Nitric oxide is generated through a complex reaction in which arginine (ARG) donates a nitrogen atom undergoing a partial oxidation and forming citrulline (CITR) and NO. Animals at 2 days post sTBI showed concomitant decrease in ARG and increase in CITR, in line with data showing increase in the stable NO end products nitrites and nitrates (Table 8). Administration of LMW-DS was particularly effective when the 15 mg/kg b.w. dose was used.

Concentrations of free amino acids involved in nitric oxide formation measured in deproteinized brain homogenates of rats sacrificed at 2 days post-TBI without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15
CITR	0.03 ± 0.01	0.03 ±0.01	0.01 ± 0.01	0.02 ± 0.01	0.01 ± 0.01
ARG	0.17 ± 0.03	0.11 ± 0.03	0.13 ± 0.03	0.13 ± 0.03	0.16 ± 0.04
ORN	0.02 ± 0.01	0.02 ± 0.01	0.02 ± 0.01	0.01 ± 0.01	0.02 ± 0.01

Effects of Increasing Doses of LMW-DS on Long-chain Free Amino Acids

The free amino acids reported in Table 14 represents a source of carbon skeleton useful to generate α-ketoacids that cells use to replenish the TCA cycle. Among these compounds, only isoleucine (ILE) was significantly affected by sTBI and restored in rats receiving drug treatment.

Concentrations of long chain free amino acids measured in deproteinized brain homogenates of rats sacrificed at 2 days post-TBI without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15
VAL	0.07 ± 0.02	0.06 ± 0.03	0.07 ± 0.03	0.08 ± 0.03	0.06 ± 0.03
ILE	0.03 ± 0.01	0.05 ± 0.01	0.10 ± 0.02	0.10 ± 0.02	0.06 ± 0.01
LEU	0.04 ± 0.01	0.04 ± 0.01	0.09 ± 0.02	0.10 ± 0.02	0.04 ± 0.01
LYS	0.23 ± 0.03	0.28 ± 0.10	0.29 ± 0.11	0.37 ± 0.14	0.32 ± 0.12

Effects of Increasing Doses of LMW-DS on Free Amino Acids Acting as Osmolytes and Aromatic Free amino acids

Results summarized in Table 15 clearly show that sTBI caused the increase in the concentrations of all these free amino acids. Particularly, the increase in taurine (TAU) may suggest the attempt to counteract cell edema by increasing the levels of one of the most important brain osmolyte. Differently, increase in aromatic free amino acids may suggest reduced biosynthesis of the neurotransmitters serotonin (formed from tryptophan) and dopamine (generated from the biotransformation of phenylalanine first and tyrosine then). No remarkable effects of LMW-DS administration were observed at this time point after impact.

Concentrations of free amino acids acting as osmolytes and aromatic free amino acids measured in deproteinized brain homogenates of rats sacrificed at 2 days post-TBI without and with a single administration of increasing doses of LWM-DS (1, 5 and 15 mg/kg b.w.), performed 30 minutes after brain trauma induction. Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15
TAU	3.82 ± 0.61	4.84 ± 0.46	4.98 ± 0.47	5.15 ± 0.49	4.59 ± 0.43
HYS	0.05 ± 0.01	0.06 ± 0.01	0.08 ± 0.01	0.11 ± 0.02	0.10 ± 0.01
TYR	0.13 ± 0.03	0.17 ± 0.03	0.18 ± 0.03	0.20 ± 0.03	0.17 ± 0.03
TRP	0.01 ± 0.01	0.02 ± 0.01	0.02 ± 0.01	0.03 ± 0.01	0.04 ± 0.01
PHE	0.03 ± 0.01	0.05 ± 0.01	0.07 ± 0.03	0.07 ± 0.03	0.06 ± 0.01

SUMMARY OF BIOCHEMICAL DATA RECORDED AT 7 DAYS POST sTBI

Effects of Increasing Doses of LMW-DS on Brain Energy Metabolism Measured

Table 16 summarizes values referring to phosphorylated high-energy purine and pyrimidine compounds. It is particularly evident the no amelioration of the depletion of triphosphate nucleotides (ATP, GTP, UTP and CTP) was observed at 7 days post sTBI. Concomitant increase in AMP and ADP was accompanied by significant changes in the concentrations of UDP derivatives (UDP-Glc, UDP-Gal, UDP-GlcNac and UDP- GalNac). In general, it should be underlined that longer times post injury were often characterized by worsening of the biochemical, metabolic, molecular alterations induced by sTBI.

At this time post injury, treatment with LMW-DS produced a general improvement of cerebral energy metabolism, more evident when drug administration dose was higher than 1 mg/kg b.w. Although differences with controls were recorded even in rats receiving repeat administration of 15 mg/kg b.w. LWM-DS, significantly higher values of nucleotide triphosphates were found in drug treated animals. Of particular relevance is the progressive recovery of the calculated, adimensional value of the ATP/ADP ratio (which is considered as a good indicator of the mitochondrial phosphorylating capacity) that progressively increased by increasing the dose of drug administered to sTBI animals.

Concentrations of energy metabolites (phosphorylated purines and pyrimidines) measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
CMP	13.52 ± 3.44	30.98 ± 3.18	25.41 ± 10.81	55.67 ± 22.97	47.10 ± 20.04	31.21 ± 13.28
UMP	82.30 ± 9.82	139.70 ± 27.06	103.06 ± 19.96	167.18 ± 32.39	181.82 ± 35.22	107.66 ± 20.86
IMP	51.57 ± 4.61	110.07 ± 28.19	80.16 ± 20.53	68.72 ± 17.60	74.70 ± 19.13	141.84 ± 36.32
GMP	82.81 ± 7.82	164.41 ± 77.81	113.06 ± 53.51	101.42 ± 48.00	41.55 ± 19.66	61.86 ± 29.28
UDP-Glc	51.00 ± 10.89	39.28 ± 7.98	63.19 ± 12.84	58.10 ± 11.81	62.97 ± 12.80	61.37 ± 12.47
UDP-Gal	131.00 ± 13.26	112.58 ± 7.74	130.20 ± 8.95	132.66 ± 9.12	137.57 ± 9.46	135.15 ± 9.29
UDP-GlcNac	88.77 ± 19.55	134.24 ± 46.44	85.36 ± 29.53	85.14 ± 29.45	67.47 ± 23.34	86.42 ± 29.89
UDP-GalNac	38.82 ± 9.83	13.08 ± 3.75	15.85 ± 4.54	17.37 ± 4.98	17.91 ± 5.13	16.50 ± 4.73
GDP Glucose	85.35 ± 12.76	90.43 ± 10.58	112.22 ± 13.13	104.76 ± 12.25	101.65 ± 11.89	106.42 ± 12.45
AMP	43.59 ± 9.90	55.86 ± 4.39	43.13 ± 3.39	59.50 ± 4.68	50.50 ± 3.97	43.12 ± 3.39
UDP	23.94 ± 6.75	45.30 ± 6.37	38.59 ± 5.43	44.19 ± 6.22	37.91 ± 5.33	37.12 ± 5.22
GDP	57.40 ± 14.06	112.05 ± 12.80	121.72 ± 13.91	126.82 ± 14.49	122.07 ± 13.95	109.06 ± 12.46
ADP-Ribose	12.69 ± 1.43	22.64 ± 5.68	7.95 ± 1.99	6.76 ± 1.70	19.21 ± 4.82	13.23 ± 3.32
CTP	41.85 ± 10.32	34.12 ± 9.03	81.75 ± 31.55	79.08 ± 14.54	96.44 ± 25.54	92.67 ± 16.27
ADP	222.67 ± 30.99	302.60 ± 40.30	286.78 ± 38.19	289.27 ± 38.52	276.83 ± 36.87	260.32 ± 34.67
UTP	152.64 ± 17.39	108.55 ± 19.01	179.75 ± 31.48	175.02 ± 30.65	127.42 ± 22.32	133.72 ± 23.42
GTP	569.00 ± 45.32	375.24 ± 34.12	438.65 ± 39.88	453.86 ± 41.27	479.98 ± 43.64	466.06 ± 42.38
ATP	2390.14 ± 213.98	1561.36 ± 125.60	1792.01 ± 144.16	1730.92 ± 139.24	1846.63 ± 148.55	1971.17 ± 158.57
ATP/ADP	10.99 ± 2.21	5.23 ± 0.66	6.12 ± 0.78	6.28 ± 0.80	6.76 ± 0.86	7.67 ± 0.97

To better show that drug effects were related to the drug dosage, we graphically reported in FIG. 10 results concerning ATP. It is possible to observe that ATP increase was somehow related to the dosage administered and that drug administration produced significant increases of the most important high energy phosphate at any dose tested.

Effects of Increasing Doses of LMW-DS on Nicotinic Coenzymes

Values of oxidized (NAD+ and NADP+) and reduced (NADH and NADPH) nicotinic coenzymes are summarized in Table 17. Table 17 also reports the calculated, adimensional value of the NAD+/NADH ratio which is suitable to evaluate how much metabolism is dependent on glycolysis or on mitochondrial oxidative phosphorylation.

As formerly observed, profound decrease of nicotinic coenzymes and of the NAD+/NADH ratio was recorded in sTBI rats at 7 days post injury. With the exclusion of the lowest dose, treatment with LMW-DS produced significant improvement of the concentrations of nicotinic coenzymes. Particularly, single and repeat administration of 15 mg/kg b.w. LMW-DS were able to normalize NAD+ level and to restore the correct NAD+/NADH ratio determined in control animals.

Concentrations of nicotinic coenzymes measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
NAD+	485.74 ± 37.06	249.37 ± 35.32	268.14 ± 37.97	293.36 ± 41.55	491.52 ± 69.61	401.73 ± 56.89
NADH	13.57 ± 1.94	8.98 ± 1.55	8.20 ± 1.41	8.83 ± 1.26	11.65 ± 1.63	11.05 ± 1.52
NADP+	23.17 ± 4.58	11.69 ± 4.29	39.94 ± 14.65	24.45 ± 8.97	23.75 ± 8.72	16.56 ± 6.08
NADPH	8.51 ± 0.71	10.66 ± 2.48	18.91 ± 4.39	12.30 ± 2.86	6.66 ± 1.55	11.21 ± 2.60
NAD+/NADH	36.47 ± 5.46	27.51 ± 5.83	33.91 ± 9.32	33.90 ± 7.19	42.51 ± 5.26	37.47 ± 9.46

Effects of Increasing Doses of LMW-DS on CoA-SH Derivatives

Table 18 reports data referring to free CoA-SH and CoA-SH derivatives. Remarkable positive effects of the administration of 5 or 15 mg/kg b.w. (this dose both as a single and repeat administration) were detected both for CoA-SH and Acetyl-CoA, suggesting much more favorable metabolic conditions for the functioning of the TCA cycle.

Concentrations of free CoA-SH and CoA-SH derivatives (Acetyl-CoA and Malonyl-CoA) measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
Malonyl-CoA	15.02 ± 2.38	13.01 ± 2.35	5.43 ± 0.98	6.02 ± 1.09	7.98 ± 1.44	12.56 ± 2.27
CoA-SH	26.31 ± 3.86	26.44 ± 3.39	38.50 ± 4.94	51.86 ± 6.66	64.93 ± 8.33	45.76 ± 5.87
Acetyl-CoA	36.97 ± 5.43	18.28 ± 3.11	27.05 ± 4.61	22.87 ± 3.89	38.60 ± 6.57	37.91 ± 6.46

Effects of Increasing Doses of LMW-DS on Antioxidants and Oxidative/nitrosative Stress Biomarkers

Table 19 shows the concentrations of the main water-soluble brain antioxidants (ascorbic acid and GSH) and of biomarkers of oxidative (MDA) and nitrosative stress (—NO₂^- and —NO₃^-). At 7 days post impact, no recovery in the concentrations of both water-soluble antioxidants occurred in rats experiencing sTBI. Remarkably high levels of signatures of oxidative/nitrosative stress were also recorded. The effects of the administration of the highest single and repeat dose of LWM-DS were particularly beneficial to rescue the concentrations of both ascorbic acid and reduced glutathione (GSH) with evident decrease of cerebral tissue nitrites and nitrates. These effects were also significant when 5 mg kg/b.w. where used.

Concentrations of antioxidants and oxidative/nitrosative stress biomarkers measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
ASCORBIC ACID	3315.38 ± 351.59	2251.89 ± 271.20	2177.22 ± 262.21	2195.87 ± 264.45	2853.35 ± 343.64	2617.09 ± 315.18
GSH	3521.63 ± 275.04	1752.50 ± 231.01	1627.30 ± 214.51	2412.17 ± 317.97	2390.89 ± 315.16	2342.03 ± 308.72
MDA	0.85 ± 0.26	10.70 ± 1.77	32.98 ± 5.44	17.78 ± 2.94	6.23 ± 1.03	4.09 ± 0.67
NO2	142.93 ± 28.19	241.72 ± 52.37	93.04 ± 20.16	59.61 ± 12.91	110.72 ± 23.99	130.69 ± 28.31
NO3	169.51 ± 20.79	315.71 ± 53.92	153.62 ± 26.24	234.45 ± 40.05	161.99 ± 27.67	271.69 ± 46.41

To better appreciate that drug effects were related to the drug dosage, we graphically reported in FIGS. 11 and 12 results concerning Ascorbic acid and GSH.

Effects of Increasing Doses of LMW-DS on De-phosphorylated Purines and Pyrimidines

A further worsening in the majority of the compounds reported in Table 20, originating from the degradation pathways of purine and pyrimidine nucleotides and indirectly connected to cell energy metabolism, were observed in rats receiving sTBI at 7 days post injury. Most of these compounds were positively affected by the drug administration.

Concentrations of de-phosphorylated purines and pyrimidines measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
CYTOSINE	14.14 ± 3.38	21.43 ± 4.60	16.03 ± 3.44	12.67 ± 2.72	13.87 ± 2.98	8.76 ± 1.88
CREATININE	17.12 ± 2.49	7.68 ± 1.36	6.57 ± 1.16	5.48 ± 0.97	5.23 ± 0.92	9.07 ± 1.60
URACIL	10.91 ± 2.27	22.71 ± 4.67	14.78 ± 3.04	18.34 ± 3.77	15.92 ± 3.27	24.13 ± 4.96
β-PSEUDOURIDINE	6.89 ± 1.27	23.36 ± 4.33	14.00 ± 2.60	17.51 ± 3.25	63.77 ± 11.83	16.72 ± 3.10
CYTIDINE	12.76 ± 2.59	29.68 ± 10.44	29.67 ± 10.44	26.51 ± 9.33	33.06 ± 11.63	72.85 ± 25.63
HYPOXANTHINE	7.57 ± 0.93	24.66 ± 7.18	16.97 ± 4.94	13.45 ± 3.91	10.21 ± 2.97	4.10 ± 1.19
GUANINE	3.34 ± 0.87	5.21 ± 2.22	6.86 ± 2.92	7.92 ± 3.37	5.27 ± 2.24	3.32 ± 1.41
XANTHINE	7.61 ± 1.39	13.58 ± 3.84	12.53 ± 3.54	14.33 ± 4.05	12.71 ± 3.60	11.24 ± 3.18
CDP choline	7.97 ± 1.37	7.90 ± 2.54	6.26 ± 2.01	10.37 ± 3.33	10.06 ± 3.23	11.72 ± 3.76
URIDINE	64.08 ± 14.14	84.44 ± 20.01	110.17 ± 26.11	134.60 ± 31.89	134.04 ± 31.76	97.21 ± 23.03
INOSINE	89.43 ± 15.04	139.98 ± 15.70	124.27 ± 13.94	196.61 ± 22.06	104.41 ± 11.72	139.26 ± 15.62
URIC ACID	3.36 ± 0.64	25.06 ± 5.96	7.13 ± 1.70	17.26 ± 4.11	8.60 ± 2.05	7.80 ± 1.86
GUANOSINE	21.10 ± 5.56	31.85 ± 6.64	19.11 ± 3.98	33.42± 6.96	20.91 ± 4.36	19.66 ± 4.10
ADENOSINE	46.71 ± 7.39	69.37 ± 51.38	26.08 ± 19.31	22.24 ± 16.47	55.95 ± 41.44	40.84 ± 30.25

Effects of Increasing Doses of LMW-DS on N-acetylaspartate (NAA)

As previously mentioned, sTBI causes an irreversible modification in NAA homeostasis. Even in this study, we found that at 7 days post sTBI whole brain NAA was about 50% lower than that measured in control rats, see FIG. 13. Interestingly, a dose dependent increase in NAA was detected in rats receiving increasing doses of single LMW-DS or repeat administrations of the maximal dose tested.

Effects of Increasing Doses of LMW-DS on Free Amino Acids Involved in Neurotransmission

Compounds listed in Table 21 are amino acids directly (GLU, GABA) of indirectly (GLN, ASP, AASN, GLY, SER, THR, ALA) involved in neurotransmission. Most of these amino acids had still higher in sTBI rats at 7 days post injury when compared with controls. It is evident from this Table that administration of LMW-DS was effective particularly when the drug was subcutaneously infused at 15 mg/kg b.w., either in a single or in repeat administrations. Particularly relevant is the normalization of GLU, thus indicating that LMW-DS is capable to abolish excitotoxicity cause by excess GLU release after sTBI.

Concentrations of free amino acids with neurotransmitter functions measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
ASP	2.88 ± 0.88	4.14 ± 0.75	4.17 ± 0.67	3.63 ± 0.59	2.29 ± 0.37	2.42 ± 0.39
GLU	9.92 ± 0.83	12.26 ± 1.03	12.14 ± 1.02	11.82 ± 0.99	10.25 ± 0.86	10.78 ± 0.91
ASN	0.10 ± 0.03	0.10 ± 0.02	0.10 ± 0.02	0.10 ± 0.02	0.10 ± 0.02	0.10 ± 0.02
SER	0.64 ± 0.17	1.04 ± 0.18	0.92 ± 0.16	0.83 ± 0.14	0.76 ± 0.12	0.79 ± 0.13
GLN	3.89 ± 0.87	3.97 ± 0.41	4.10 ± 0.42	3.86 ± 0.40	3.73 ± 0.38	3.88 ± 0.40
GLY	0.78 ± 0.13	0.91 ± 0.17	0.98 ± 0.20	0.88 ± 0.15	0.78 ± 0.12	0.78 ± 0.10
THR	0.69 ± 0.18	0.76 ± 0.10	0.71 ± 0.12	0.71 ± 0.15	0.72 ± 0.14	0.77 ± 0.14
ALA	0.41 ± 0.11	0.51 ± 0.05	0.57 ± 0.06	0.44 ± 0.05	0.38 ± 0.04	0.47 ± 0.05
GABA	1.36 ± 0.22	1.78 ± 0.18	1.73 ± 0.18	1.63 ± 0.17	1.43 ± 0.15	1.38 ± 0.14

Effects of Increasing Doses of LMW-DS on Free Amino Acids Involved in the Methyl Cycle

As shown in Table 22, levels of free amino acids involved either in the so called methyl cycle or in the formation of cysteine, were still different in sTBI rats at 7 days post impact, when compared to corresponding values of controls. Increase in MET was observed in animals receiving the highest dose of LWM-DS (both as single or as repeat administrations). As already observed at 2 days post injury, these drug levels produced a significant increase in L-Cystathionine (L-Cystat). Since this compound is an intermediate in the generation of cysteine (CYS), it is conceivable to hypothesize that increase in L-Cystat may produce a consequent increase in CYS. It is worth recalling that determination of CYS requires a specific additional HPLC assay with additional derivatization with F-MOC, a fluorescent compound that reacts with secondary amine and with CYS.

Concentrations of free amino acids involved in the methyl cycle and homeostasis of -SH groups measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
SAH	0.03 ± 0.01	0.05 ± 0.01	0.04 ± 0.01	0.04 ± 0.01	0.04 ± 0.01	0.04 ± 0.04
L-Cystat	0.15 ± 0.03	0.23 ± 0.04	0.24 ± 0.04	0.26 ± 0.04	0.25 ± 0.04	0.44 ± 0.07
MET	0.03 ± 0.01	0.03 ± 0.01	0.03 ± 0.01	0.04 ± 0.01	0.04 ± 0.01	0.05 ± 0.01

Effects of Increasing Doses of LMW-DS on Free Amino Acids Involved in the Generation of Nitric oxide (NO)

Table 23 illustrates concentrations of the free amino acids directly involved in the generation of NO. Animals at 7 days post sTBI showed still concomitant decrease in ARG and increase in CITR, in line with data showing increase in the stable NO end products nitrites and nitrates (Table 8). Administration of LMW-DS was particularly effective when 5 or 15 mg/kg b.w. (single and repeat) was used.

Concentrations of free amino acids involved in nitric oxide formation measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
CITR	0.03 ± 0.01	0.04 ± 0.02	0.03 ± 0.01	0.03 ± 0.01	0.03 ± 0.01	0.03 ± 0.01
ARG	0.17 ± 0.03	0.13 ± 0.02	0.13 ± 0.02	0.15 ± 0.02	0.14 ± 0.02	0.19 ± 0.02
ORN	0.02 ± 0.01	0.01 ± 0.01	0.01 ± 0.01	0.01 ± 0.01	0.01 ± 0.01	0.02 ± 0.01

Effects of Increasing Doses of LMW-DS on Long-chain Free Amino Acids

The free amino acids reported in Table 24, representing a source of carbon skeleton useful to generate α-ketoacids that cells use to replenish the TCA cycle, were practically normal at 7 days post sTBI and any other group of animals treated with the drug of interest.

Concentrations of long chain free amino acids measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
VAL	0.07 ± 0.02	0.07 ± 0.01	0.08 ± 0.01	0.08 ± 0.01	0.10 ± 0.01	0.07 ± 0.01
ILE	0.03 ± 0.01	0.03 ± 0.01	0.04 ± 0.01	0.05 ± 0.01	0.07 ± 0.01	0.03 ± 0.01
LEU	0.04 ± 0.01	0.04 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.07 ± 0.01	0.04 ± 0.01
LYS	0.23 ± 0.03	0.19 ± 0.03	0.19 ± 0.06	0.21 ± 0.04	0.21 ± 0.05	0.23 ± 0.07

Effects of Increasing Doses of LMW-DS on Free Amino Acids Acting as Osmolytes and Aromatic Free amino acids

Results summarized in Table 25 clearly show that sTBI caused the increase in the concentrations of taurine (TAU) at 7 days after injury. LMW-DS administration normalized TAU concentrations and caused the increase in aromatic amino acids.

Concentrations of free amino acids acting as osmolytes and aromatic free amino acids measured in deproteinized brain homogenates of rats sacrificed at 7 days post-sTBI, without and with administration of increasing doses of LWM-DS (single administration of 1, 5 and 15 mg/kg b.w. and repeated administration of 15 mg/kg b.w.). Controls are represented by sham-operated animals. Values are the mean ± S.D. of 12 animals in each group and are expressed as nmol/g w.w.

Compound	Controls	TBI only	LMW-DS 1	LMW-DS 5	LMW-DS 15	LMW-DS 15-R
HYS	0.05 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01
TAU	3.82 ± 0.61	4.36 ± 0.56	4.02 ± 0.51	3.51 ± 0.44	3.38 ± 0.44	3.47 ± 0.44
TYR	0.13 ± 0.03	0.14 ± 0.02	0.13 ± 0.02	0.13 ± 0.02	0.14 ± 0.02	0.14 ± 0.02
TRP	0.02 ± 0.01	0.02 ± 0.01	0.01 ± 0.01	0.02 ± 0.01	0.03 ± 0.01	0.03 ± 0.01
PHE	0.03 ± 0.01	0.04 ± 0.01	0.05 ± 0.01	0.06 ± 0.01	0.07 ± 0.01	0.05 ± 0.01

Discussion

The study conducted to evaluate the effects of increasing doses of LMW-DS on a large panel of brain metabolites in rats experiencing sTBI at different times post injury evidenced that the administration of this compound produces a general amelioration of cerebral metabolism.

LMW-DS was effective in restoring mitochondrial related energy metabolism, profoundly imbalanced in sTBI animals with no treatment, with positive effects on the concentration of triphosphates purine and pyrimidine nucleotides. Particularly, ATP levels, at 7 days post impact, were only 16% lower than the value of controls, whilst in sTBI rats a 35% decrease was found (Table 16 and FIG. 10). Remarkably, NAA concentration in animals treated with LMW-DS at the same time point was only 16% lower than the value of controls, whilst sTBI animals showed 48% lower values of this compound. This finding once again strongly confirms the strict connection between the homeostasis of NAA and correct mitochondrial energy metabolism, and underlines the importance of pharmacological interventions capable to act positively on mitochondrial functioning.

The general amelioration of brain metabolism produced by LMW-DS administration also involved nicotinic coenzymes and metabolism of free CoA-SH and CoA-SH derivatives. This implies that drug treated animals, notwithstanding submitted to sTBI, had quasi-normal coenzymes to ensure correct oxido-reductive reactions and to allow a good functioning of the TCA cycle.

The aforementioned improvement of brain metabolism certainly contributed to the other remarkable drug effect, i.e., the abolishment of GLU excitotoxicity. Additionally, the drug affected sulphur-containing amino acids. Possibly, this effect might be related to the drug molecule that contains S atoms. Increasing the bioavailability of this atom might have produced a net increase in the biosynthesis of these amino acids, one of them (MET) is crucial in the methylation reaction and in the so called methyl cycle.

Further positive effects recorded in this study were the increase in antioxidants and the decrease of biochemical signatures of oxidative/nitrosative stress in sTBI rats receiving administration of LMW-DS. Even this phenomenon might well be connected with the normalization of mitochondrial functions, since dysfunctional mitochondria are the main intracellular source of both ROS and RNS. Of relevance is that the effects of LMW-DS were more evident at 7 than at 2 days post sTBI. This strongly suggests that the general amelioration of brain metabolism caused by the drug administration is not a transitory phenomenon.

This experiment confirmed the positive effects of LMW-DS in protecting mitochondrial function and reducing oxidative stress as seen in, among others, improvement of the recovery of antioxidant status, protection of mitochondrial ATP energy supply by preserving ATP production and metabolism, normalization of mitochondrial phosphorylating capacity all as induced by LMW-DS. As a consequence, LMW-DS is capable of protecting and preserving mitochondrial function in cells exposed to a damage or disease, which is of importance in order to have functional cells that can combat the infectious disease. The metabolic normalization as induced by LMW-DS is of benefit for COVID-19 patients both during the pulmonary phase (stage II) and the recovery phase (stage IV) (FIG. 25).

Example 5

This Example investigated the potential of LMW-DS as an anti-coagulant and anti-thrombotic agent in ALS patients.

Materials & Methods

This was a single-centre, open single-arm study where the safety, tolerability and efficacy of subcutaneous (s.c.) administered LMW-DS was evaluated in patients with ALS. There were 10 planned visits to the clinic: 1 screening visit divided in 2 parts (Visit 1a and Visit 1b), 5 IMP administration visits (Visit 2 to Visit 6) and 3 follow-up visits (Visit 7 to Visit 9). Each individual patient’s study participation was planned to be approximately 4 months, including the screening and follow-up visits.

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

Results

The mean APTT at baseline was 26.5 s, prior to injection of LMW-DS. Following each LMW-DS treatment, transient increases were observed with a maximum at 2 to 2.5 hours post-dosing with a relative change from baseline: +33.5% and +26.5% respectively. The APTT values returned to pre-dose levels at 6 hours post dosing

Discussion

Acute tissue inflammation elevates coagulation Factor VIII, leading to decreased aPTT in blood and microthrombosis in tissue. Experimental data as presented in this Example, shows that LMW-DS increases aPTT in blood and thereby acts as an anti-coagulant and anti-thrombotic agent. Such anti-coagulant and anti-thrombotic effect is beneficial for COVID-19 subject during the hyperinflammation phase (stage III) (FIG. 25).

Example 6

In this study LMW-DS was characterized by profiling in the BioMAP® Diversity PLUS panel. The BioMAP® panel consists of human primary cell-based systems designed to model different aspects of the human body in an in vitro format. The 12 systems in the BioMAP® Diversity PLUS panel (Table 26) allow test agent characterization in an unbiased way across a broad set of systems modeling various human disease states. The BioMA®P systems are constructed with one or more primary cell types from healthy human donors, with stimuli, such as cytokines or growth factors, added to capture relevant signaling networks that naturally occur in human tissue or pathological conditions. Vascular biology is modeled in both a Th1 (3C system) and a Th2 (4H system) inflammatory environment, as well as in a Th1 inflammatory state specific to arterial smooth muscle cells (CASM3C system). Additional systems recapitulate aspects of the systemic immune response including monocyte-driven Th1 inflammation (LPS system) or T cell stimulation (SAg system), chronic Th1 inflammation driven by macrophage activation (IMphg system) and the T cell-dependent activation of B cells that occurs in germinal centers (BT system). The BE3C system (Th1) and the BF4T system (Th2) represent airway inflammation of the lung, while the MyoF system models myofibroblast-lung tissue remodeling. Lastly, skin biology is addressed in the KF3CT system modeling Th1 cutaneous inflammation and the HDF3CGF system modeling wound healing.

Each test agent generates a signature BioMAP® profile that is created from the changes in protein biomarker readouts within individual system environments. Biomarker readouts (7 - 17 per system) are selected for therapeutic and biological relevance, are predictive for disease outcomes or specific drug effects and are validated using agents with known mechanism of action (MoA). Each readout is measured quantitatively by immune-based methods that detect protein, e.g., ELISA, or functional assays that measure proliferation and viability. BioMAP® readouts are diverse and include cell surface receptors, cytokines, chemokines, matrix molecules and enzymes. In total, the BioMAP® Diversity PLUS panel contains 148 biomarker readouts that capture biological changes that occur within the physiological context of the particular BioMAP® system.

Materials and Methods

Four concentrations of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016/076780; 150 nM, 440 nM, 1.3 µM, 4 µM) were investigated in the BioMAP® Diversity PLUS panel by Eurofins.

Methods for Diversity PLUS

Human primary cells in BioMAP systems are used at early passage (passage 4 or earlier) to minimize adaptation to cell culture conditions and preserve physiological signaling responses. All cells are from a pool of multiple donors (n = 2 - 6), commercially purchased and handled according to the recommendations of the manufacturers. Human blood derived CD14+ monocytes are differentiated into macrophages in vitro before being added to the Mphg system. 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).

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

Systems are derived from either single cell types or co-culture systems. Adherent cell types are cultured in 96 or 384-well plates until confluence, followed by the addition of PBMC (SAg and LPS systems). The BT system consists of CD19+ B cells co-cultured with PBMC and stimulated with a BCR activator and low levels of TCR stimulation. Test agents prepared in either DMSO (small molecules; final concentration ≤ 0.1%) or PBS (biologics) are added at the indicated concentrations 1-hr before stimulation, and remain in culture for 24-hrs or as otherwise indicated (48-hrs, MyoF system; 72-hrs, BT system (soluble readouts); 168-hrs, BT system (secreted IgG)). Each plate contains drug controls (e.g., legacy control test agent colchicine at 1.1 µM), negative controls (e.g., non-stimulated conditions) and vehicle controls (e.g., 0.1% DMSO) appropriate for each system. Direct ELISA is used to measure biomarker levels of cell-associated and cell membrane targets. Soluble factors from supernatants are quantified using either HTRF® detection, bead-based multiplex immunoassay 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 cells in suspension. For proliferation assays, individual cell types are cultured at subconfluence and measured at time points optimized for each system (48-hrs: 3C and CASM3C systems; 72-hrs: BT and HDF3CGF systems; 96-hrs: SAg system). Cytotoxicity for adherent cells is measured by SRB (24-hrs: 3C, 4H, LPS, SAg, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, and Mphg systems; 48-hrs: MyoF system), and by alamarBlue staining for cells in suspension (24-hrs: SAg system; 42-hrs: BT system) at the time points indicated.

Data Analysis

Biomarker measurements in a test agent-treated sample are divided by the average of control samples (at least 6 vehicle controls from the same plate) to generate a ratio that is then log₁₀ transformed. Significance prediction envelopes are calculated using historical vehicle control data at a 95% confidence interval.

Profile Analysis

Biomarker activities are annotated when 2 or more consecutive concentrations change in the same direction relative to vehicle controls, are outside of the significance envelope and have at least one concentration with an effect size > 20% (|log₁₀ ratio| > 0.1). Biomarker key activities are described as modulated if these activities increase in some systems, but decrease in others. Cytotoxic conditions are noted when total protein levels decrease by more than 50% (log₁₀ ratio of SRB or alamarBlue levels < -0.3) and are indicated by a thin black arrow above the X-axis. A compound is considered to have broad cytotoxicity when cytotoxicity is detected in 3 or more systems. Concentrations of test agents with detectable broad cytotoxicity are excluded from biomarker activity annotation and downstream benchmarking, similarity search and cluster analysis. Antiproliferative effects are defined by an SRB or alamarBlue log₁₀ ratio value < -0.1 from cells plated at a lower density and are indicated by grey arrows above the X-axis. Cytotoxicity and antiproliferative arrows only require one concentration to meet the indicated threshold for profile annotation.

Benchmark Analysis

Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% in the same direction. Differentiating biomarkers are annotated when one profile has a readout outside of the significance envelope with an effect size > 20%, and the readout for the other profile is either inside the envelope or in the opposite direction. Unless specified, the top non-cytotoxic concentration of both the test agent and benchmark agent are included in the benchmark overlay analysis.

Similarity Analysis

Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% in the same direction. Concentrations of test agents that have 3 or more detectable systems with cytotoxicity are excluded from similarity analysis. Concentrations of test agents that have 1 - 2 systems with detectable cytotoxicity will be included in the similarity search analysis, along with an overlay of the database match with the top concentration of the test agent. This will be followed by an additional overlay of the next highest concentration of the test agent containing no systems with detectable cytotoxicity and the respective database match. To determine the extent of similarity between BioMAP® profiles of compounds run in the Diversity PLUS panel, we have developed a custom similarity metric (BioMAP Z-Standard) that is a combinatorial approach that has improved performance in mechanism classification of reference agents compared to other measures tested (including Pearson’s and Spearman’s correlation coefficients). This approach more effectively accounts for variations in the number of data points, systems, active biomarker readouts and the amplitude of biomarker readout changes that are characteristic features of BioMAP® profiles. A Pearson’s correlation coefficient (r) is first generated to measure the linear association between two profiles that is based on the similarity in the direction and magnitude of the relationship. Since the Pearson’s correlation can be influenced by the magnitude of any biomarker activity, a per-system weighted average Tanimoto metric is used as a filter to account for underrepresentation of less robust systems. The Tanimoto metric does not consider the amplitude of biomarker activity, but addresses whether the identity and number of readouts are in common on a weighted, per system basis. A real-value Tanimoto metric is calculated first by normalizing each profile to the unit vector (e.g.,

$A=\left(\frac{A}{‖A‖}\right)$

and then applying the following formula:

$\frac{A\cdot B}{‖A‖+‖B‖-A\cdot B},$

where A and B are the 2 profile vectors. Then, it is incorporated into a system weighted-averaged real-value Tanimoto metric in this calculation:

$\frac{{\displaystyle \sum W_i\cdot T_i}}{{\displaystyle \sum W_i}}.$

The calculation uses the real-value Tanimoto score for each i^th system (T_i) and the weight of each i^h system (W_i). W_i is calculated for each system in the following formula:

$\frac{1}{1+exp\left(-100\times \left(\right)\right)lr-0.09},$

where Ir is the largest absolute value of the ratios from the 2 profiles being compared. Based on the optimal performance of reference compounds, profiles are identified as having mechanistically relevant similarity if the Pearson’s correlation coefficient (r) ≥ 0.7. Finally, a Fisher r-to-z-transformation is used to calculate a z-score to convert a short tail distribution into a normal distribution as follows:

$z=0.5lo{g}_{10}\frac{1+r}{1-r}.$

Then the BioMAP® Z-Standard, which adjusts for the number of common readouts (CR), is generated according to the following formula: Z-Standard =

$z\cdot \sqrt{CR-3}.$

A larger BioMAP® Z-Standard value corresponds to a higher confidence level, and this is the metric used to rank similarity results.

Cluster Analysis

Cluster analysis (function similarity map) uses the results of pairwise correlation analysis to project the “proximity” of agent profiles from multi-dimensional space into two dimensions. Functional clustering of the agent profiles generated during this analysis uses Pearson correlation values for pairwise comparisons of the profiles for each agent at each concentration, and then subjects the pairwise correlation data to multidimensional scaling. Profiles that are similar with a Pearson’s correlation coefficient (r) ≥ 0.7 are connected by lines. Agents that do not cluster with one another are interpreted as mechanistically distinct. This analysis is performed for projects with 3 or more agents tested. Cytotoxic concentrations are excluded from cluster analysis.

Mechanism HeatMAP Analysis

Mechanism HeatMAP analysis provides a visualization of the test compound and 19 consensus mechanisms allowing comparison of biomarker activities across all compound concentrations and consensus mechanisms. The synthetic consensus profiles used in the Mechanism HeatMAP analysis are representative BioMAP® profiles of the average of multiple compounds from structurally distinct chemical classes. Profiles were calculated by averaging the values for each biomarker endpoint for all profiles selected (multiple agents at different concentrations) to build the consensus mechanism profile. Biomarker activities are colored in the heatmap for consensus mechanisms and compounds when they have expression relative to vehicle controls outside of the significance envelope. Red represents increased protein expression, blue represents decreased expression and white indicates levels that were unchanged or within filtering conditions. Darker shades of color represent greater change in biomarker activity relative to vehicle control. The Mechanism HeatMAP was prepared using R and the gplots package for R.

Assay Acceptance Criteria

A BioMAP® assay includes the multi-parameter data sets generated by the BioMAP® platform for agents tested in the systems that make up the Diversity PLUS panel. Assays contain drug controls (e.g., legacy control test agent colchicine), negative controls (e.g., non-stimulated conditions), and vehicle controls (e.g., DMSO) appropriate for each system. BioMAP® assays are plate-based, and data acceptance criteria depend on both plate performance (% CV of vehicle control wells) and system performance across historical controls for that system. The QA/QC Pearson Test is performed by first establishing the 1% false negative Pearson cutoff from the reference dataset of historical positive controls. The process iterates through every profile of system biomarker readouts in the positive control reference dataset, calculating Pearson values between each profile and the mean of the remaining profiles in the dataset. The overall 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 the one percentile of all values calculated is the 1% false negative Pearson cutoff. A system will pass if the Pearson value between the experimental plate’s negative control or drug control profile and the mean of the historical control profiles in the reference dataset exceeds this 1% false negative Pearson cutoff. Overall assays are accepted when each individual system passes the Pearson test and 95% of all project plates have% CV <20%.

Results

The BioMAP® Diversity PLUS panel contained 12 individual BioMAP human primary cell-based co-culture system as shown in Table 26.

BioMAP® Diversity PLUS panel
System name	Disease/Tissue relevance	Human cell types	Biomarker readouts
3C	Cardiovascular Disease, Chronic Inflammation	Venular endothelial cells	CCL2/MCP-1, CD106/VCAM-1, CD141/Thrombomodulin, CD142/Tissue Factor, CD54/ICAM-1, CD62E/E-Selectin, CD87/uPAR, CXCL8/IL-8, CXCL9/MIG, HLA-DR, Proliferation, SRB
4H	Allergy, Asthma, Autoimmunity	Venular endothelial cells	CCL2/MCP-1, CCL26/Eotaxin-3, CD106/VCAM-1, CD62P/P-Selectin, CD87/uPAR, SRB, VEGFR2
BE3C	COPD, Lung Inflammation	Bronchial epithelial cells	CD54/ICAM-1, CD87/uPAR, CXCL10/IP-10, CXCL11/I-TAC, CXCL8/IL-8, CXCL9/MIG, EGFR, HLA-DR, IL-1α, Keratin 8/18, MMP-1, MMP-9, PAI-I, SRB, tPA, uPA
BF4T	Allergy, Asthma, Fibrosis, Lung Inflammation	Bronchial epithelial cells + Dermal fibroblasts	CCL2/MCP-1, CCL26/Eotaxin-3, CD106/VCAM-1, CD54/ICAM-1, CD90, CXCL8/IL-8, IL-1α, Keratin 8/18, MMP-1, MMP-3, MMP-9, PAI-I, SRB, tPA, uPA
BT	Allergy, Asthma, Autoimmunity, Oncology	B cells + Peripheral blood mononuclear cells	B cell Proliferation, PBMC Cytotoxicity, Secreted IgG, sIL-17A, sIL-17F, sIL-2, sIL-6, sTNF-α
CASM3C	Cardiovascular Inflammation, Restenosis	Coronary artery smooth muscle cells	CCL2/MCP-1, CD106/VCAM-1, CD141/Thrombomodulin, CD142/Tissue Factor, CD87/uPAR, CXCL8/IL-8, CXCL9/MIG, HLA-DR, IL-6, LDLR, M-CSF, PAI-I, Proliferation, Serum Amyloid A, SRB
HDF3CGF	Chronic Inflammation,	Dermal fibroblasts	CCL2/MCP-1, CD106/VCAM-1, CD54/ICAM-1, Collagen I, Collagen III, CXCL10/IP-10,
	Fibrosis		CXCL11/I-TAC, CXCL8/IL-8, CXCL9/MIG, EGFR, M-CSF, MMP-1, PAI-I, Proliferation_72hr, SRB, TIMP-1, TIMP-2
KF3CT	Dermatitis, Psoriasis	Dermal fibroblasts + Keratinocytes	CCL2/MCP-1, CD54/ICAM-1, CXCL10/IP-10, CXCL8/IL-8, CXCL9/MIG, IL-1α, MMP-9, PAI-I, SRB, TIMP-2, uPA
LPS	Cardiovascular Disease, Chronic Inflammation	Peripheral blood mononuclear cells + Venular endothelial cells	CCL2/MCP-1, CD106/VCAM-1, CD141/Thrombomodulin, CD142/Tissue Factor, CD40, CD62E/E-Selectin, CD69, CXCL8/IL-8, IL-1α, M-CSF, sPGE2, SRB, sTNF-α
MyoF	Chronic Inflammation, Fibrosis, Matrix Remodeling, Wound Healing	Lung fibroblasts	bFGF, CD106/VCAM-1, Collagen I, Collagen III, Collagen IV, CXCL8/IL-8, Decorin, MMP-1, PAI-I, SRB, TIMP-1, α-SM Actin
SAg	Autoimmune Disease, Chronic Inflammation	Peripheral blood mononuclear cells + Venular endothelial cells	CCL2/MCP-1, CD38, CD40, CD62E/E-Selectin, CD69, CXCL8/IL-8, CXCL9/MIG, PBMC Cytotoxicity, Proliferation, SRB
Mphg	Cardiovascular Disease, Chronic Inflammation, Restenosis	Macrophages + Venular endothelial cells	CCL2/MCP-1, CCL3/MIP-1α, CD106NCAM-1, CD40, CD62E/E-Selectin, CD69, CXCL8/IL-8, IL-1α, M-CSF, sIL-10, SRB, SRB-Mphg

Biomarker activities were annotated when two or more consecutive concentrations changed in the same direction relative to vehicle controls, were outside of the 95% significance envelope, and had at least one concentration with an effect size > 20% (|log₁₀ ratio| > 0.1). Biomarker key activities were described as modulated if these activities increased in some systems, but decreased in others.

LMW-DS was active with 25 annotated readouts. LMW-DS was not cytotoxic for any of the human primary cells at the concentrations tested in this study. LMW-DS mediated changes in key biomarker activities included inflammation-related activities in the form of decreased vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), soluble tumor necrosis factor alpha (sTNFα), interferon-inducible T cell alpha chemoattractant (I-TAC), monokine induced by gamma interferon (MIG), and interferon gamma-induced protein 10 (IP-10) and increased Eotaxin 3 (Eot3), and interleukin 8 (IL-8). LMW-DS also had immunomodulatory activities in the form of decreased secreted immunoglobulin G (slgG) and macrophage colony-stimulating factor (M-CSF) and increased soluble IL-17A (sIL-17A), and cluster of differentiation 69 (CD69). LMW-DS also showed tissue remodeling activities in the form of increased matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), urokinase plasminogen activator receptor (uPAR) and epidermal growth factor receptor (EGFR), and hemostasis-related activities in the form of increased thrombomodulin (TM). Table 27 summaries the effects of LMW-DS on the 12 different human primary cells in the BioMAP® Diversity PLUS panel.

Summary of BioMAP® Diversity PLUS results
Cell system	Increased biomarker activity	Decreased biomarker activity
3C	IL-8
4H	uPAR
LPS	IL-8	sTNFα
SAg	IL-8
BT	sIL-17A	sIgG, sIL-17F
BF4T	Eot3
BE3C
CASM3C	TM	VCAM-1, MIG
HDF3CGF	EGFR, MMP-1, PAI-1	VCAM-1, IP-10, ITAC, MIG, M-CSF
KF3CT	IL-8	MCP-1
MyoF	IL-8
Mphg	IL-8, CD69

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

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

Discussion

In study LMW-DS was characterized by profiling in the BioMAP® Diversity PLUS panel of human primary cell-based assays modeling complex tissue and disease biology of organs (vasculature, immune system, skin, lung) and general tissue biology. The BioMAP® Diversity PLUS panel evaluated the biological impact of LMW-DS in conditions that preserve the complex crosstalk and feedback mechanisms that are relevant to in vivo outcomes.

LMW-DS was active and noncytotoxic at the concentrations tested in this study. LMW-DS was modestly and selectively antiproliferative to human primary endothelial cells at the top concentration only (4 µM). LMW-DS profiles had 25 annotated readouts indicating modulation of immune and inflammation-related readouts as well as matrix related biomarkers. Specific activities included decreased inflammation-related sTNFα, VCAM-1, IP-10 (CXCL10), MIG (CXCL9), I-TAC (CXCL11), and MCP-1, and as well as increased IL-8. Modestly increased Eotaxin-3 was observed in the BF4T system at the lower concentrations only. Immunomodulatory activities included decreased slgG and IL-17A and IL-17F in the BT system, but without any antiproliferative effects on B cells. Decreased M-CSF and increased CD69, sIL-17A and sIL-17F were also identified. LMW-DS also modulated tissue remodeling biomarkers including increased MMP-1, PAI-1, uPAR, EGFR, and the hemostasis-related TM. Key inflammation biomarkers including MIG, VCAM, IP-10 and ITAC were decreased over all tested concentrations in the CASM3C and HDF3CGF systems, while an increase in the chemotactic factor IL-8 was noted in multiple systems. Together these data indicate that LMW-DS plays a role in regulating immune activation and/or immune resolution responses in the context of inflammation and wound healing biology.

The modulations of the inflammatory markers indicate utility of LMW-DS in treating multiple chronic and acute inflammatory conditions and diseases including inflammatory components.

Initially after injury, the innate/proinflammatory response and selected components of the acquired immune response are up-regulated to maintain a defense against foreign pathogens, clear tissue debris present at the injury site, and orchestrate tissue remodeling, cell proliferation and angiogenic processes associated with the wound response. However, for proper wound healing to progress, this initial inflammatory response has to be regulated or shut down so as to allow for the reestablishment of matrix, recellularization and tissue remodeling. Such immune resolving activities were induced by LMW-DS, including activation of MMP-1, PAR-1 and uPAR, indicating an induced immune resolution having utility in treating tissue damaged in COVID-19 and which otherwise would result in deleterious fibrosis formation.

LMW-DS modulated a lot of biomarker activities in the HDF3CGF system but merely IL-8 in the MyoF system. Both systems include fibroblasts but HDF3CGF models wound healing and matrix remodeling in connection with such wound healing, whereas MyoF is more a fibrosis model of collagen deposition. The results thereby indicate that LMW-DS had immunomodulatory and tissue remodeling activities but without inducing undesired collagen fibrosis, which could result in deleterious fibrosis deposition.

In conclusion, LMW-DS seems to normalize and resolve the inflammation present in tissue after trauma or a disease and these results are thereby consistent with the effects of LMW-DS seen in foregoing Examples.

Hence, LMW-DS modulated secretion of pro-inflammatory cytokines and chemokines to thereby suppress inflammatory signaling in disease compromised tissues. These findings are of relevance in suppressing the deleterious inflammatory responses, including ARDS and SIRS and septic shock, as seen in the most severely affected COVID-19 subjects. Furthermore, the tissue remodeling effects of LMW-DS would be beneficial to target pulmonary fibrosis, kidney fibrosis and cardiomyopathy in COVID-19 subjects.

Example 7

An analysis of changes in gene-expression induced by LMW-DS was investigated in cell lines.

Materials & Methods

Experimental Design

For each cell line, n=8 × 25 cm² culture flasks were set up. Two flasks were harvested for each cell type on the day of treatment (24 hours after seeding). This represents the Day0 time point. From the remaining flasks, three flasks were treated with Control Medium and three were treated with Culture Medium (CM) containing LMW-DS to give a final concentration of 0.01 mg/ml. Cells from the treated flasks were collected after 48 hours. Therefore the collected data represent (a) untreated cells (Day0 Controls and Day2 Controls) and (b) cells treated with LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016/076780) for 48 hours (Day2 LMW-DS treated).

Coating of Tissue Culture Plates for All Cells

25 cm² flasks were coated by adding 2 ml per flask of a solution of 50 µg/ml poly-d-lysine in Hank’s balanced salt solution (HBSS) and incubating overnight at 37° C. in the dark. Flasks were washed with cell culture water and air-dried for 30 min in the dark. Flasks were coated by adding 1 ml per flask of a solution of 25 µg/ml laminin in phosphate-buffered saline (PBS) and incubating for 2 hour at 37° C. in the dark. The laminin flasks were washed with PBS three times before plating cells.

Human Umbilical Vein Endothelial Cells (HUVECs)

Medium 200 + Large Vessel Endothelial Supplement (M200+LVES) additive (1:50) was prepared and pre-warmed to 37° C. Cells were thawed in a 37° C. water bath for no longer than 2 min and gently transferred into a 50 ml tube containing 20 ml Dulbecco’s Modified Eagle Medium, Nutrient Mixture F-12 (DMEM-F12). The cell suspension was mixed by inverting the tube carefully twice. Cells were spun at 400 × g for 10 minutes. Supernatant removed and cells were re-suspended in 10 ml of culture media (M200+LVES additive).

Cells were counted with the Cellometer. 1,000,000 cells/flask were seeded in 25 cm² flasks (n=8) and medium was topped up to a total of 5 ml per flask. Cells were incubated at 37° C. with 5% CO₂. Cells were allowed to settle for 24 hours before LMW-DS treatment.

Human Schwann Cells

Schwann cells growth medium was prepared by adding 10% of 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 longer than 2 min.

Cells from 12 vials were each gently transferred to a tube containing 10 ml of high-glucose DMEM medium and centrifuged at 400 relative centrifugal field (RCF) for 10 min. Pellet was re-suspended in culture medium. The cells from the 12 vials were mixed and distributed equally into the previously coated 25 cm² flasks (n=8). Cells were incubated at 37° C. with 5% CO₂. Cells were allowed to settle for 24 hours before LMW-DS treatment.

Mouse Cortical Neurons (Lonza)

Medium was prepared by adding 10 ml B-27 Serum-Free Supplement and 2.5 ml GlutaMAX™-1 Supplement to 500 ml of Neurobasal medium. The medium was pre-warmed to 37° C. Cells from 12 vials were thawed sequentially in a 37° C. water bath for no longer than 2 min and gently transferred into a 15 ml tube. 9 ml of medium was gently added drop-wise to each. The cell suspension was mixed by inverting the tubes carefully twice.

The cells were centrifuged for 5 minutes at 200 x g. Supernatant was removed (to the last 0.5 ml) and cells were gently re-suspended by trituration. The cells from the 12 vials were mixed and distributed equally into the previously coated 25 cm² flasks (n=8). Cells were incubated at 37° C. with 5% CO₂ for 24 hours.

Mouse Motor Neurons (Aruna)

The culture medium was prepared according to Table 28.

Preparation of culture medium
Component	Stock concentration	Final concentration	For 50 ml
Advanced DMEM/F12			25 ml
AB2™ Basal Neural Medium			25 ml
Knockout Serum Replacement			5 ml
L-Glutamate	100 ×	1 ×	0.5 ml
Penicillin/Streptomycin	100 ×	1 ×	0.5 ml
B-mercaptoethanol	1 M (diluted in PBS)	0.1 mM	5 µl
Glial cell-derived neurotrophic factor (GDNF)	100 µg/ml in H2O	10 ng/ml	5 µl
Ciliary neurotrophic factor (CNTF)	100 µg/ml in PBS with 0.1% BSA	10 ng/ml	5 µl

Medium (see Table 28) was pre-warmed to 37° C. Cells were thawed in a 37° C. water bath for no longer than 2 min. 9 ml of media was gently added drop-wise. The cell suspension was mixed by inverting the tube carefully twice. The cells were counted with a Cellometer. The cells were centrifuged for 5 minutes at 200 x g. Supernatant was removed (to the last 0.5 ml) and cells were gently re-suspended by trituration. The cells from the 8 vials were mixed and distributed equally into the previously coated 25 cm² flasks (n=8). Cells were incubated at 37° C. with 5% CO₂ for 24 hours before treatment.

Drug Treatment

LMW-DS was provided at a stock concentration of 20 mg/ml and was kept in a temperature monitored refrigerator at 4° C. A fresh 100x LMW-DS stock (1.0 mg/ml) was prepared in sterile DMEM-F12. The concentrated drug stock was sterile filtered and added to the respective culture media (19.6 ml CM and 0.4 ml LMW-DS stock solution). The Control was made using 19.6 ml CM and 0.4 ml of DMEM-F12. LMW-DS and CM were added to the respective flasks (5 ml each) to reach the 0.01 mg/ml concentration of LMW-DS in each dish with a total of 10 ml CM each.

Culture Collection and Cell Lysis

CM was aspirated into a clean and labelled 15 ml Falcon tube. The flasks (without culture medium) were placed into the -80° C. freezer for 30 minutes. The CM in the Falcon tubes was spun at 3000 x g for 5 minutes. Supernatant was removed and the small pellet was re-suspended in 2.5 ml Trizol:Water (4:1) solution at room temperature (RT, ~22° C.).

The frozen flasks were removed one-by one from the freezer and the Trizol-Water from the appropriate tubes was moved to the flask. Flasks were left at RT for 5 minutes before the content was aspirated back into the 15 ml Falcon tube (after washing the bottom of the flask with the solution thoroughly). The flasks were inspected under the microscope to ensure full removal of cells. The collected lysates in the 15 ml Falcon tubes were placed into the -80° C. freezer.

RNA Extraction

Falcon tubes containing the homogenates were removed from the freezer and stored for 5 minutes at RT to permit the complete dissociation of nucleoprotein complexes.

Two aliquots of 1 ml lysate were removed from each sample and 200 µl of chloroform was added to each (0.2 ml of chloroform per 1 ml of TRIzol Reagent used during the cell lysis step) and the tube was shaken vigorously. Samples were stored at RT for 2-3 minutes and subsequently centrifuged at 12,000 x g for 15 minutes at 4° C.

The mixture separated into three layers: a lower red phenol-chloroform phase, an interphase and a colorless upper aqueous phase. The RNA remained in the top aqueous phase, DNA in the white middle (interphase) phase and protein in the pink bottom (organic) phase. The top ¾ of the aqueous phase was transferred to a new clean Eppendorf tube.

The RNA was precipitated from the aqueous phase by adding an equal amount of 100% ethanol. The precipitated RNA was fixed onto a Spin Cartridge, washed twice and dried. The RNA was eluted in 50 µl warm RNase-Free Water. The amount and quality of the purified RNA was measured by Nanodrop. The RNA was stored at -80° C. before transfer to Source Bioscience for Array analysis.

Analysis Plan for Expression Data

The expression data were downloaded into separate files for each cell line. The ‘Background corrected’ expression is the data from the “gProcessedSignal” of the arrays that is the result of the background signal extracted from the actual signal of the relevant probe. This is the most often used variable in array analysis. The background corrected signal was log2 transformed for all samples for statistical analysis. To reduce the false discovery rate in the samples, the signals that were below ‘expression level’ were removed. The ‘below expression’ level was set at 5 for the log2 transformed expression values.

Statistical Analysis

Based on the expression pattern of the Control probes on each array it was decided to carry out Median Centering for all arrays before analysis to reduce the variability of the results. Data were grouped by cell type and each cell type was analyzed using the following algorithms:

• Comparison of D0 control to D2 control samples - expression changes seen in the cells in normal cultures
• Comparison of D0 control to D2 LMW-DS treated samples - expression changes seen in the cells in the LMD-DS treated cultures
• Comparison of D2 control to D2 LMD-DS treated samples - differential expression induced by LMW-DS in the culture.

A preliminary analysis was carried out to screen out genes that were not differentially expressed between any combination of the three datasets. Simple, non-stringent ANOVA (p<0.05) was carried out to look for patterns of expression. Probes with no changes across the three datasets were eliminated. The remaining probe sets were analyzed for fold change and significance using Volcano plots. More than 20% change in the expression of a probe (fold change (FC) ≥ 1.2 or FC ≤ 0.84) was regarded as significant in the first instance to allow the detection of expression patterns.

Quality Parameters

Seeding densities were calculated from the cell counts retrieved from the cell stocks for the Schwann cells. The HUVECS were seeded at their optimum density.

The additional quality control from the Array service provider indicated that the RNA was high quality (no degradation) and the amounts were within the parameters of the Low input RNA microarray from Agilent.

The analysis of the raw data indicated that, as expected, there were significant differences between arrays. These differences (reflected by differences in the same control samples included on all arrays), were, however, easily eliminated by normalization techniques. The chosen median centering of the data that eliminates the array-to-array variation did not affect the overall differences expected to be seen between the controls representing different concentrations of RNA.

Expression Analysis of Schwann Cells

As described in the foregoing, genes not expressed in the Schwann cells were removed prior to data analysis. The ‘below expression’ level was set at 5 for the log2 transformed expression values. This left 15,842 unique probes to analyze in the Schwann cell cultures. In the next step of the analysis, three sets of data (comparison of D0 control to D2 control samples; comparison of D0 control to D2 LMW-DS treated samples; comparison of D2 control to D2 LMD-DS treated samples) were analyzed to establish the effect of the CM on the cells and the relative changes induced by LMW-DS.

585 genes were differentially expressed in Schwann cell cultures when comparing the D0 control to the D2 control samples. The molecular functions influenced by these genes relate to cellular movement (1.14E-07-2.49E-03); cell morphology (5.56E-07-2.36E-03); cellular development (7.3E-06-2.48E-03); cellular growth and proliferation (7.3E-06-2.48E-03); cellular assembly and organization (1.23E-05-2.36E-03); cellular function and maintenance (1.23E-05-2.47E-03); cell death and survival (1.53E-05-2.51E-03); lipid metabolism (8.14E-05-1.6E-03); small molecule biochemistry (8.14E-05-1.6E-03); molecular transport (1.18E-04-2.29E-03); protein trafficking (1.62E-04-1.6E-03); carbohydrate metabolism (3.22E-04-1.78E-03); gene expression (3.98E-04-2.2E-03); cell signaling (4.39E-04-2.25E-03); cell-to-cell signaling and interaction (5.05E-04-2.48E-03); cellular compromise (7.69E-04-1.58E-03); cell Cycle (1.12E-03-1.8E-03); amino acid metabolism (1.6E-03-1.6E-03); and nucleic acid metabolism (1.6E-03-1.6E-03).

The values presented above are p-values representing the statistical significance of the association of these genes with the different pathways. The two p values represent the lower and upper limits of the statistical significance observed (p<0.05 is significant).

LMW-DS induced differential expression in Schwann cell culture of 1244 genes as assessed when comparing the D0 control to the D2 LMW-DS treated samples. The molecular functions influenced by these genes relate to cell morphology (1.43E-08-8.39E-04); cellular movement (1.4E-07-9.6E-04); post-translational modification (3.93E-07-6.71E-05); protein synthesis (3.93E-07-1.08E-04); protein trafficking (3.93E-07-1.26E-06); cell death and survival (2.13E-06-8.65E-04); cellular assembly and organization (7.46E-06-8.24E-04); DNA replication, recombination, and repair (7.46E-06-7.46E-06); cellular function and maintenance (9.53E-06-6.46E-04); gene expression (1.27E-05-4.92E-04); cellular development (1.29E-05-9.06E-04); cellular growth and proliferation (1.29E-05-9.06E-04); cell-to-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).

LMW-DS induced differential expression in Schwann cell culture of 700 genes as assessed when comparing the D2 control to the D2 LMW-DS treated samples. The molecular functions influenced by these genes relate to cell morphology (1.49E-07-5.62E-03); cellular assembly and organization (1.49E-07-5.95E-03); cellular movement (7.24E-07-6.06E-03); cell death and survival (9.41E-06-5.95E-03); amino acid metabolism (2.56E-05-3.7E-03); post-translational modification (2.56E-05-1.05E-03); small molecule biochemistry (2.56E-05-3.7E-03); cell-to-cell signaling and interaction (5.05E-05-5.76E-03); gene expression (7.18E-05-4.94E-03); cell cycle (1.06E-04-5.95E-03); cellular development (1.06E-04-5.95E-03); cellular function and maintenance (1.96E-04-5.95E-03); cellular growth and proliferation (2.35E-04-5.95E-03); DNA replication, recombination and repair (2.75E-04-5.95E-03); cell signaling (5.92E-04-2.54E-03); cellular comprise (6.26E-04-6.26E-04); lipid metabolism (6.26E-04-1.85E-03); molecular transport (6.26E-04-5.95E-03); protein synthesis (1.05E-03-1.93E-03); cellular response to therapeutics (1.85E-03-1.85E-03); protein trafficking (2.66E-03-5.95E-03); and RNA post-transcriptional modification (4.32E-03-4.32E-03).

The mechanistic molecular network model simulates the effect of the differentially regulated molecules by LMW-DS enabling the functional consequences of these changes to be evaluated. The in silico model indicated that LMW-DS inhibits neuronal cell death; apoptosis; and synthesis of protein and activates angiogenesis; migration of cells; cell viability; cell survival; cell movement; proliferation of cells; differentiation of cells; cellular homeostasis; cell cycle progression; cell transformation; and expression of RNA.

Table 29 summarizes the results of the gene expression changes in the cultured Schwann cells.

Overall pattern of gene expression changes in Schwann cells
	abolished nutrient effect	enhanced response to nutrients	new effect induced by LMW-DS	not different from control	total
no effect	21				21
significant downregulation	1	122	352	42	517
significant upregulation	13	441	74	373	901
total	35	563	426	415	1439

21 genes that have altered expression in the Control cultures in the two days did not show any changes at all in the LMW-DS treated cultures during the same two days. 1 gene that had increased expression in the control cultures was downregulated in the LMW-DS treated cultures during the same two days. 13 genes that were downregulated in the control cultures were upregulated in the LMW-DS treated cultures during the two days. 122 genes were significantly downregulated by growth factors in the culture medium and this downregulation was even stronger in the LMW-DS treated cultures. 441 genes were upregulated in the Control cultures and the addition of LMW-DS made this upregulation significantly stronger.

Expression Analysis of HUVECs

As described in the foregoing, genes that are not expressed in the HUVECs have been removed before attempting any analysis. The ‘below expression’ level was set at 5 for the log2 transformed expression values. This left 15,239 unique probes to analyze in HUVEC cultures. In the next step, the three sets of data were analyzed to establish the effect of the CM on gene expression in the cells and the differences induced by LMW-DS. A preliminary analysis was carried out to screen out genes that were not differentially expressed between any combination of the three datasets. Simple, non-stringent ANOVA (p<0.05) was carried out to look for patterns of expression. Genes with no changes across the three datasets were eliminated, leaving a total of 12,313 probes (10,368 genes) to analyze.

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

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

LMW-DS induced differential expression in HUVEC culture of 76 genes as assessed when comparing the D2 control to the D2 LMW-DS treated samples. The molecular functions influenced by these genes relate to DNA replication, recombination, and repair (9.62E-05-2.57E-02); cell cycle (1.22E-04-2.4E-02); cellular development (1.59E-04-2.67E-02); cell morphology (4.64E-04-2.42E-02); cellular function and maintenance (4.64E-04-2.57E-02); lipid metabolism (9.49E-04-1.07E-02); molecular transport (9.49E-04-1.61E-02); small molecule biochemistry (9.49E-04-1.87E-02); cellular compromise (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); carbohydrate metabolism (2.7E-03-1.07E-02); cell-to-cell signaling and interaction (2.7E-03-2.4E-02); cellular assembly and organization (2.7E-03-2.57E-02); cellular growth and proliferation (2.7E-03-2.4E-02); cellular movement (2.7E-03-2.4E-02); energy production (2.7E-03-2.7E-03); nucleic acid metabolism (2.7E-03-1.07E-02); post-translational modification (2.7E-03-1.61E-02); gene expression (5.39E-03-2.36E-02); RNA post-transcriptional modification (5.39E-03-2.4E-02); drug metabolism (8.07E-03-1.61E-02); vitamin and mineral metabolism (8.07E-03-8.07E-03); protein synthesis (1.07E-02-1.07E-02); RNA trafficking (1.07E-02-1.07E-02); cellular response to therapeutics (1.24E-02-1.24E-02); and free radical scavenging (1.43E-02-1.43E-02).

Although the overall difference between Control and LMW-DS-treated cultures after 2 days of treatment at first hand does not appear to be large, the effects of LMW-DS on gene expression changes were significant, in particular when considering the modulation of growth factor induced gene expression by LMW-DS.

Using the mechanistic molecular network model it is possible to simulate the effect of the genes differentially regulated by LMW-DS to look for the functional consequences of these changes. The in silico model indicated that LMW-DS inhibits neuronal cell death; apoptosis; and synthesis of protein and activates angiogenesis; migration of cells; cell viability; cell survival; cell movement; proliferation of cells; differentiation of cells; cellular homeostasis; cell cycle progression; cell transformation; and expression of RNA.

The HUVEC control cultures comprise growth factors. In the treated cultures, LMW-DS was added to the culture medium that already contained growth factors.

Table 30 summarizes the results of the gene expression changes in the cultured HUVECs. 67 genes that have altered expression in the Control cultures in the two days (under the effect of the growth factors) did not show any changes at all in the LMW-DS treated cultures during the same two days. 4 genes that had increased expression in the control cultures with the growth factors were downregulated in the LMW-DS treated cultures during the same two days. 11 genes that were downregulated by the growth factors in the control cultures were upregulated in the LMW-DS treated cultures during the two days. 120 genes were significantly downregulated by growth factors and this downregulation was even stronger in the LMW-DS treated cultures. 229 genes were upregulated in the Control cultures and the addition of LMW-DS made this upregulation significantly stronger.

Overall pattern of gene expression changes in HUVECs
	abolished nutrient effect	enhanced response to nutrients	not different from control	total
no effect	67			67
significant downregulation	4	120	167	291
significant upregulation	11	229	1326	1566
total	82	349	1493	1924

The effect of LMW-DS on several molecular pathways that are important for different disease conditions and therapeutic applications were analyzed. For the analysis, the effects of adding LMW-DS on gene expression was compared to that seen in cells in CM and the functional effects were predicted based on the observed changes in the expression patterns.

Expression Analysis of Motor Neurons

As described in the foregoing, genes that are not expressed in the motor neurons have been removed before attempting any analysis. The ‘below expression’ level was set at 5 for the log2 transformed expression values. This left 12,240 unique probes where the expression threshold was met by at least three samples in the series. In the next step, the three sets of data were analyzed to establish the effect of the CM on the cells and the differences induced by the LMW-DS.

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

485 genes were differentially expressed in motor neuron cultures when comparing the D0 control to the D2 control samples. The molecular functions influenced by these genes relate to cell death and survival (1.99E-17-1.98E-04); cellular movement (1.14E-16-1.91E-04); cellular assembly and organization (1.22E-16-1.93E-04); cellular function and maintenance (1.22E-16-1.95E-04); cell morphology (6.46E-16-1.74E-04); cell-to-cell signaling and interaction (3.16E-12-1.95E-04); cellular development (1.59E-10-1.93E-04); cellular growth and proliferation (1.59E-10-1.9E-04); molecular transport (4.27E-10-1.89E-04); protein synthesis (9.85E-09-5.03E-05); lipid metabolism (1.08E-08-1.61E-04); small molecule biochemistry (1.08E-08-1.89E-04); gene expression (8.45E-08-3.8E-05); cell cycle (4.55E-07-1.09E-04); free radical scavenging (7.12E-07-1.65E-04); cell signaling (1.23E-05-1.89E-04); vitamin and mineral metabolism (1.23E-05-1.89E-04); protein degradation (3.07E-05-1.31E-04); carbohydrate metabolism (3.32E-05-1.61E-04); drug metabolism (4.16E-05-4.16E-05); post-translational modification (7.1E-05-1.31E-04); and protein folding (7.1E-05-7.1E-05).

LMW-DS induced differential expression in motor neurons of 315 genes as assessed when comparing the D0 control to the D2 LMW-DS treated samples. The molecular functions influenced by these genes relate to cell death and survival (6.54E-08-9.06E-03), cellular movement (8.21E-08-5.42E-03); cellular assembly and organization (8.36E-08-9.01E-03); cellular function and maintenance (8.36E-08-9.01E-03); cell morphology (2.9E-06-8.75E-03); cellular development (1.04E-05-9.01E-03); cellular growth and proliferation (1.04E-05-7.83E-03); DNA replication, recombination, and repair (2.79E-05-8.01E-03); cell-to-cell signaling and interaction (8.18E-05-7.11E-03); post-translational modification (1.32E-04-7.56E-03); protein degradation (1.32E-04-4.35E-03); protein synthesis (1.32E-04-5.09E-03); gene expression (1.9E-04-9.01E-03); cellular compromise (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 molecule biochemistry (7.67E-04-9.01E-03); vitamin and mineral metabolism (7.67E-04-1.13E-03); lipid metabolism (1.05E-03-9.01E-03); molecular transport (1.05E-03-9.01E-03); cell signaling (1.13E-03-5.09E-03); and carbohydrate metabolism (4.71E-03-4.71E-03).

LMW-DS induced differential expression in motor neurons of 425 genes as assessed when comparing the D0 control to the D2 LMW-DS treated samples. The molecular functions influenced by these genes relate to cell death and survival (2.87E-08-6.27E-03); cellular movement (4.73E-07-6.47E-03); cell morphology (4.95E-07-7.47E-03); cellular development (1.02E-06-7.13E-03); cellular growth and proliferation (1.02E-06-7.48E-03); cellular assembly and organization (7.03E-06-7.47E-03); cellular function and maintenance (7.03E-06-7.47E-03); gene expression (1.95E-05-6.18E-03); cell cycle (2.88E-05-7.48E-03); DNA replication, recombination, and repair (3.39E-05-5.16E-03); amino acid metabolism (7.75E-05-4.68E-03); small molecule biochemistry (7.75E-05-4.68E-03); cellular compromise (8.23E-05-4.61E-03); cell-to-cell signaling and interaction (3.27E-04-7.48E-03); vitamin and mineral metabolism (3.27E-04-3.27E-04); protein synthesis (8.94E-04-5.29E-03); post-translational modification (9.67E-04-9.67E-04); molecular transport (9.7E-04-4.68E-03); protein trafficking (9.7E-04-9.7E-04); carbohydrate metabolism (1.44E-03-1.92E-03); cellular response to therapeutics (1.92E-03-1.92E-03); and lipid metabolism (4.68E-03-4.68E-03).

Overall pattern of gene expression changes in motor neurons
	abolished nutrient effect	enhanced response to nutrients	new effect induced by LMW-DS	not different from control	total
no effect	177		108		285
significant downregulation	47	36	375	104	562
significant upregulation	40	103	71	75	289
total	264	139	554	179	1136

Expression Analysis of Cortical Neurons

As described in the foregoing, genes that are not expressed in the motor neurons have been removed before attempting any analysis. The ‘below expression’ level was set at 5 for the log2 transformed expression values. This left 10,653 unique probes where the expression threshold was met by at least three samples in the series. In the next step, the three sets of data were analyzed to establish the effect of the CM on the cells and the differences induced by the LMW-DS.

The changes in gene expression under normal culture conditions mimic the normal developmental processes of the cortical neurons, when from a dissociated set of cells they develop a cortical neuron phenotype. The growth factors in the normal culture medium are those necessary for these cells to differentiate. The stress factor present in these cultures is the oxidative stress (normal for tissue culture conditions).

1101 genes were differentially expressed in motor neuron cultures when comparing the D0 control to the D2 control samples. The molecular functions influenced by these genes relate to cellular assembly and organization (3.57E-25-6.65E-04); cellular function and maintenance (3.57E-25-6.65E-04); cell morphology (4.28E-22-6.36E-04); cellular development (4.28E-22-6.53E-04); cellular growth and proliferation (4.28E-22-6.6E-04); cell-to-cell signaling and interaction (2.16E-13-6.65E-04); molecular transport (5.18E-12-4.95E-04); cellular movement (1.86E-11-6.65E-04); cell death and survival (3.37E-11-6.41E-04); gene expression (1.27E-08-8.96E-05); protein synthesis (3.84E-07-8.69E-05); small molecule biochemistry (6.65E-07-5.18E-04); cellular compromise (7.12E-06-4.54E-04); protein degradation (1.62E-05-1.62E-05); amino acid metabolism (2.11E-05-4.25E-04); protein trafficking (3.4E-05-3.4E-05); cell signaling (8.69E-05-3E-04); post-translational modification (8.69E-05-2.15E-04); protein folding (2.15E-04-2.15E-04); cell cycle (2.69E-04-3.07E-04); DNA replication, recombination, and repair (2.69E-04-4.77E-04); nucleic acid metabolism (2.69E-04-2.69E-04); lipid metabolism (3.12E-04-5.18E-04); and carbohydrate metabolism (5.18E-04-5.18E-04).

LMW-DS induced differential expression in motor neurons of 609 genes as assessed when comparing the D0 control to the D2 LMW-DS treated samples. The molecular functions influenced by these genes relate to cellular assembly and organization (3.91E-15-1.83E-03); cellular function and maintenance (3.91E-15-1.83E-03); cell morphology (2.53E-13-1.43E-03); cellular development (2.53E-13-1.81E-03); cellular growth and proliferation (2.53E-13-1.83E-03); cellular movement (4.95E-09-1.2E-03); cell-to-cell signaling and interaction (5.96E-09-1.47E-03); cell death and survival (2.25E-08-1.77E-03); molecular transport (7.08E-08-1.79E-03); DNA replication, recombination, and repair (3.03E-06-1.71E-03); cellular compromise (9.23E-06-7.65E-04); amino acid metabolism (1.75E-05-1.64E-03); cell cycle (1.75E-05-1.77E-03); small molecule biochemistry (1.75E-05-1.79E-03); protein synthesis (2.77E-05-1.5E-03); protein trafficking (2.77E-05-1.9E-04); cell signaling (7.65E-05-1.73E-03); post-translational modification (3.01E-04-1.4E-03); gene expression (3.65E-04-1.15E-03); drug metabolism (6.49E-04-6.49E-04); carbohydrate metabolism (6.95E-04-7.69E-04); vitamin and mineral metabolism (1.09E-03-1.09E-03); and nucleic acid metabolism (1.44E-03-1.73E-03).

LMW-DS induced differential expression in motor neurons of 247 genes as assessed when comparing the D0 control to the D2 LMW-DS treated samples. The molecular functions influenced by these genes relate to cell morphology (6.01E-08-1.01E-02); cellular development (7.46E-08-1.01E-02); cellular growth and proliferation (7.46E-08-1.01E-02); cell death and survival (4.23E-07-1.01E-02); cellular movement (2.69E-06-9.91E-03); cellular assembly and organization (1.57E-05-1.01E-02); cellular function and maintenance (1.57E-05-1.01E-02); cell cycle (1.01E-04-1.01E-02); cell-to-cell signaling and interaction (1.01E-04-1.01E-02); lipid metabolism (1.56E-04-1.01E-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 compromise (4.47E-04-2.2E-03); protein synthesis (2.66E-03-7.29E-03); protein trafficking (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).

Overall pattern of gene expression changes in cortical neurons
	abolished nutrient effect	enhanced response to nutrients	new effect induced by LMW-DS	not different from control	total
no effect	572		19		591
significant downregulation	7	158	22	95	282
significant upregulation	33	43	7	221	304
total	612	612	48	316	1177

The Effect of LMW-DS on Oxidative Stress Pathways in Mitochondria

The oxidative stress pathways occurring in mitochondria are important for various infectious diseases and also for ageing and age-related degenerative diseases. Normal growth conditions trigger a certain amount of oxidative stress in cells, which contributes to both the in vivo and the in vitro ageing process.

In Schwann cells cultured in normal conditions, Complex I (NADH dehydrogenase) was inhibited while Complex IV (cytochrome c oxidase) was activated. When LMW-DS was added to the cultures Complex III (cytochrome bc1) was inhibited. The inhibition of Complex III inhibits the oxidative stress phenomena that are involved in the pathogenesis of cancer and neurological diseases.

Complex III, sometimes referred to as coenzyme Q : cytochrome c - oxidoreductase or the cytochrome bc1 complex, is the third complex in the electron transport chain (EC 1.10.2.2), playing a critical role in biochemical generation of ATP (oxidative phosphorylation). Complex III is a multi-subunit transmembrane protein encoded by both the mitochondrial (cytochrome b) and the nuclear genomes (all other subunits). Complex III is present in the mitochondria of all animals and all aerobic eukaryotes and the inner membranes of most eubacteria. Mutations in Complex III cause exercise intolerance as well as multisystem disorders. The bc1 complex contains 11 subunits, 3 respiratory subunits (cytochrome B, cytochrome C1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins.

In HUVECs no significant modulation of the effects of oxidative stress on mitochondria was detected following treatment with LMW-DS.

In normal culture conditions the motor neurons appear to suffer from significant oxidative stress. This leads to the activation of some apoptotic mechanisms and involving activation of cytochrome C, AIF, Caspase 3, 8 and 9. In addition, the motor neurons are characterized by production of amyloid-β in the cells further exacerbating oxidative stress and mitochondrial fragmentation, via FIAS1, as well as the oxidation of fatty acids. Furthermore, Complex V was activated.

The addition of LMW-DS to the cultures ameliorated these negative effects by preventing and inhibiting apoptosis by preventing amyloid-β production and its negative effects on mitochondrial fragmentation and dysfunction and subsequent damage and by inhibiting fatty acid oxidation. LMD-DS also inhibited the reaction path involving TRAK1 and PINK1, thereby contributing to improved mitochondrial function. LMW-DS further reduced the level of H₂O₂. A further effect was the inhibition of HtrA2 contributing to inhibition of apoptosis.

In normal culture conditions the cortical neurons are exposed to significant oxidative stress leading to the production of amyloid-β and Lewy body formation and involving activation of Synuclein α and increased levels of ROS; apoptosis; mitochondrial fragmentation; and reduction of mitochondrial function and involving C161. The addition of LMW-DS to the cultures was able to prevent and reverse most of these deleterious effects, such as the accumulation of the amyloid-β and Lewy body pathology, mitochondrial dysfunction. Some apoptosis inducing mechanisms remain active probably due to strong activation in the cultures.

The 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 functions. However, too much glutamate is also associated with excitotoxicity, leading to neuronal death. This later phenomenon is hypothesized to be involved in the neuronal death triggered in chronic neurodegenerative conditions but also in TBI. The genes involved in glutamate signaling are not expressed in HUVECs but are present in the Schwann and neuron cell lines used in this study.

Glutamate production was inhibited by the baseline conditions in the motor neuron cultures. The inhibition was not affected by LMW-DS. Glutamate production was elevated in the cortical neurons at baseline. The addition of LMW-DS did not alter the glutamate production in these cells.

The addition of LMW-DS to the CM of the Schwan cells induced the expression of a protein complex (CALM, Gβγ, GRM7, PICK1). More importantly, LMW-DS increased activity and/or levels of glutamate transporters in the Schwann cells, and in particular of SLC1A⅔, thereby leading to a scavenging of glutamate produced by and released from the presynaptic neuron. Accordingly, LMW-DS induced the Schwann cells to remove the toxic glutamate from the synaptic cleft, thereby preventing it from exerting its excitotoxicity.

SLC1A3, solute carrier family 1 (glial high-affinity glutamate transporter), member 3, is a protein that, in humans, is encoded by the SLC1A3 gene. SLC1A3 is also often called the GLutamate ASpartate Transporter (GLAST) or Excitatory Amino Acid Transporter 1 (EAAT1). SLC1A3 is predominantly expressed in the plasma membrane, allowing it to remove glutamate from the extracellular space. It has also been localized in the inner mitochondrial membrane as part of the malate-aspartate shuttle. SLC1A3 functions in vivo as a homotrimer. SLC1A3 mediates the transport of glutamic and aspartic acid with the cotransport of three Na+ and one H+ cations and counter transport of one K+ cation. This co-transport coupling (or symport) allows the transport of glutamate into cells against a concentration gradient. SLC1A3 is expressed throughout the CNS, and is highly expressed in astrocytes and Bergmann glia in the cerebellum. In the retina, SLC1A3 is expressed in Muller cells. SLC1A3 is also expressed in a number of other tissues including cardiac myocytes.

SLC1A2, solute carrier family 1 member 2, also known as excitatory amino acid transporter 2 (EAAT2) and glutamate transporter 1 (GLT-1), is a protein that in humans is encoded by the SLC1A2 gene. SLC1A2 is a member of a family of the solute carrier family of proteins. The membrane-bound protein is the principal transporter that clears the excitatory neurotransmitter glutamate from the extracellular space at synapses in the CNS. Glutamate clearance is necessary for proper synaptic activation and to prevent neuronal damage from excessive activation of glutamate receptors. SLC1A2 is responsible for over 90% of glutamate reuptake within the brain.

These findings indicate that LMW-DS may be useful for the prevention of glutamate excitotoxicity in conditions where its high extracellular levels are harmful, like after TBI.

The Effect of LMW-DS on Cell Adhesion

One of the strong noticeable phenotypic effects of LMW-DS was the effect on cell adhesion, which was cell type specific. Cell adhesion was affected in neurons most strongly, then in Schwann cells, while HUVECs were not affected.

The analysis of gene expression indicated that this is due to the effect of LMW-DS on the expression of enzymes that regulate cell attachment including metallopeptidases, also referred to as matrix metalloproteinases (MMPs), see Table 33.

The aggregate effect of these molecules on the pathways regulating cell movement and attachment in Schwann cells (17 molecules, see Table 33) was such that cell adhesion would be inhibited while cell movement would be activated, while in HUVECs (1 molecule, ADAM11) adhesion would not be affected but angiogenesis would be activated.

Molecules of the pathway regulating cell movement and attachment in Schwann cells
Symbol	Entrez gene name	Location	Type(s)
A2M	alpha-2-macroglobulin	Extracellular Space	transporter
ADAM10	ADAM metallopeptidase domain 10	Plasma Membrane	peptidase
ADAM23	ADAM metallopeptidase domain 23	Plasma Membrane	peptidase
ADAMTS9	ADAM metallopeptidase with thrombospondin type 1 motif 9	Extracellular Space	peptidase
CDH11	cadherin 11	Plasma Membrane	other
CSF3	colony stimulating factor 3	Extracellular Space	cytokine
FAS	Fas cell surface death receptor	Plasma Membrane	transmembrane receptor
HIF1A	hypoxia inducible factor 1 alpha subunit	Nucleus	transcription regulator
IL6	interleukin 6	Extracellular Space	cytokine
IL15	interleukin 15	Extracellular Space	cytokine
LUM	lumican	Extracellular Space	other
MMP3	matrix metallopeptidase 3	Extracellular Space	peptidase
POSTN	periostin	Extracellular Space	other
RECK	reversion inducing cysteine rich protein with kazal motifs	Plasma Membrane	other
SERPINA3	serpin family A member 3	Extracellular Space	other
TNC	tenascin C	Extracellular Space	other
VCAM1	vascular cell adhesion molecule 1	Plasma Membrane	transmembrane receptor

The effect of differential gene expression induced by LMW-DS in neurons was analyzed. In the motor neurons the same metallopeptidase-dependent pathways could be responsible for the cell detachment seen in the Schwann cells, see Table 34.

Molecules of the pathway regulating cell movement and attachment in motor neurons
Symbol	Entrez Gene Name	Location	Type(s)
ADAM11	ADAM metallopeptidase domain 11	Plasma Membrane	peptidase
ADAM19	ADAM metallopeptidase domain 19	Plasma Membrane	peptidase
ADAMTS7	ADAM metallopeptidase with thrombospondin type 1 motif 7	Extracellular Space	peptidase
ADORA1	adenosine A1 receptor	Plasma Membrane	G-protein coupled receptor
AGT	angiotensinogen	Extracellular Space	growth factor
APP	amyloid beta precursor protein	Plasma Membrane	other
CD44	CD44 molecule (Indian blood group)	Plasma Membrane	other
F2R	coagulation factor II thrombin receptor	Plasma Membrane	G-protein coupled receptor
FAS	Fas cell surface death receptor	Plasma Membrane	transmembrane receptor
FGF2	fibroblast growth factor 2	Extracellular Space	growth factor
FN1	fibronectin 1	Extracellular Space	enzyme
HBEGF	heparin binding EGF like growth factor	Extracellular Space	growth factor
ITGAM	integrin subunit alpha M	Plasma Membrane	transmembrane receptor
JUN	Jun proto-oncogene, AP-1 transcription factor subunit	Nucleus	transcription regulator
KDR	kinase insert domain receptor	Plasma Membrane	kinase
MMP15	matrix metallopeptidase 15	Extracellular Space	peptidase
MMP17	matrix metallopeptidase 17	Extracellular Space	peptidase
NREP	neuronal regeneration related protein	Cytoplasm	other
PLAT	plasminogen activator, tissue type	Extracellular Space	peptidase
PPIA	peptidylprolyl isomerase A	Cytoplasm	enzyme
PSEN1	presenilin 1	Plasma Membrane	peptidase
SDC1	syndecan 1	Plasma Membrane	enzyme
SERPINE2	serpin family E member 2	Extracellular Space	other
SNAP23	synaptosome associated protein 23	Plasma Membrane	transporter
STX12	syntaxin 12	Cytoplasm	other
TIMP3	TIMP metallopeptidase inhibitor 3	Extracellular Space	other
TIMP4	TIMP metallopeptidase inhibitor 4	Extracellular Space	other
TPSAB1/ TPSB2	tryptase alpha/beta 1	Extracellular Space	peptidase

However, none of the MMP-related genes were found to be differentially expressed in the cortical neurons.

This finding led to the re-assessment of all molecular interactions that affect cell attachment and adhesion related molecules and their effect on cellular attachment in the four different cultures. The full list of the 217 attachment-related molecules (197 genes and 20 drugs) is presented 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, chondroitin sulfate, CLEC4M, colchicine, Collagen type I, Collagen(s), COMP, CRK, CRP, CSF1, CSF2RB, CTGF, curcumin, CXCL12, cyclic AMP, DAB2, DAG1, DCN, DDR1, desferriexochelin 772SM, DOCK2, DSG2, DSG4, durapatite, Efna, EFNA1, EFNB, EFNB1, EGF, EGFR, EGR1, ELN, ENG, EP300, Eph Receptor, EPHA8, EPHB1, eptifibatide, ethylenediaminetetraacetic acid, ETS1, F11R, F3, FBLN5, FBN1, Fc receptor, FCN2, FERMT2, FES, FGF2, 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 beta 1, Integrina, IPO9, ITGA1, ITGA2, ITGA3, ITGA5, ITGA6, ITGB1, ITGB2, ITGB3, ITGB5, JAK2, Jnk, KP-SD-1, LAMC1, Laminin, Laminin1, levothyroxine, LGALS3, LIF, lipopolysaccharide, LOX, LRP1, LRPAP1, MAD1L1, mannose, MAPK7, MBL2, MERTK, metronidazole, MGAT5, MMP2, Mn²⁺, NCK, NEDD9, NRG1, okadaic acid, OLR1, P38 MAPK, PDGF BB, phosphatidylinositol, PKM, platelet activating factor, PLD1, PLG, PMP22, PODXL, POSTN, PRKCD, PTAFR, PTEN, PTGER2, PTK2, PTK2B, PTN, PTPN11, PTPRZ1, pyrrolidine dithiocarbamate, Rac, RALB, RANBP9, RHOA, RHOB, RPSA, SDC3, SELE, Selectin, SELL, SEMA3A, simvastatin, SIRPA, SPARC, sphingosine-1-phosphate, SPI1, SPP1, SPRY2, SRC, STARD13, SWAP70, TEK, TFPI, TFPI2, TGFA, TGFB1, TGFBI, TGM2, THBS2, THY1, thyroid hormone, TIMP2, tirofiban, TLN1, TLN2, TNF, TP63, tretinoin, VAV1, VCAM1, VCAN, Vegf, VHL, VTN, VWF, and WRR-086.

Of the 197 genes regulating cell attachment none are differentially regulated by LMW-DS in HUVECs. In the Schwann cell cultures, the 17 molecules differentially expressed lead to an overall slightly increased attachment. However, in the neurons the expression patterns lead to significant inhibition of cellular attachment in these cells.

Upstream Regulator Pathways Affected by LMW-DS

In Schwann cells, the upstream regulator analysis revealed that LMW-DS modulated the effect of several growth factors by either increasing their activation or reducing their inhibition in the system as shown in Table 35.

Upstream regulator comparison in Schwann cells
Analysis	Upstream regulator	Predicted activation state relative D2 control	Activation z-score	p-value of overlap
D2 control	ANGPT2		1.062	0.003
D2 LMW-DS treatment	Activated	1.283	0.00373
D2 control	BMP2		0.674	0.0126
D2 LMW-DS treatment	Activated	1.395	0.00326
D2 control	BMP4		-0.272	0.00253
D2 LMW-DS treatment	Activated	0.927	0.000663
D2 control	BMP7		1.45	0.0346
D2 LMW-DS treatment	Activated	1.86	0.0225
D2 control	EGF		-0.015	0.0000927
D2 LMW-DS treatment	Activated	2.059	0.00735
D2 control	FGF2		1.366	0.0000142
D2 LMW-DS treatment	Activated	2.37	0.000395
D2 control	GDF2		1.556	0.000299
D2 LMW-DS treatment	Activated	2.561	0.000106
D2 control	HGF		-0.823	0.0114
D2 LMW-DS treatment		Activated	1.432	0.0161
D2 control	IGF1		0.365	0.00883
D2 LMW-DS treatment	Activated	1.332	0.0132
D2 control	NRG1		1.073	0.0473
D2 LMW-DS treatment	Activated	1.768	0.143
D2 control	NRTN			0.0118
D2 LMW-DS treatment	Activated	0.958	0.0149
D2 control	PGF		0	0.00185
D2 LMW-DS treatment	Activated	0.254	0.00871
D2 control	TGFβ1		-1.239	0.0000354
D2 LMW-DS treatment	Less inhibited	1.05	0.0000691
D2 control	VEGFA		1.909	0.00981
D2 LMW-DS treatment	Activated	3.4	0.00186
D2 control	WISP2		-1.067	0.0323
D2 LMW-DS treatment	Less inhibited	-0.896	0.0349

In HUVECs, the number of growth factors whose effect was enhanced by LMW-DS was relatively smaller but still highly significant, see Table 36.

Upstream regulator comparison in HUVECs
Analysis	Upstream regulator	Predicted activation state relative D2 control	Activation z-score	p-value of overlap
D2 control	HGF		2.602	0.0000181
D2 LMW-DS treatment	Activated relative to control	3.194	0.00000793
D2 control	TGFβ1		0.682	0.00328
D2 LMW-DS treatment	Activated relative to control	1.429	0.0338
D2 control	VEGF		3.113	2.78E-08
D2 LMW-DS treatment	Activated relative to control	3.432	6.33E-09

In the motor neurons, the upstream regulator analysis revealed that LMW-DS affected the effect of several growth factors either increasing their activation or reducing the inhibitions present in the system as shown in Table 37.

Upstream regulator comparison in motor neurons
Analysis	Upstream regulator	Predicted activation state relative D2 control	Activation z-score
D0 to D2 control	AGT	Activated	2.292
D0 to LMW-DS treatment	Activated	2.631
D0 to D2 control	BMP4		0.798
D0 to LMW-DS treatment	More activated relative to control	0.972
D0 to D2 control	BMP6		-0.269
D0 to LMW-DS treatment	More activated relative to control	0.13
D0 to D2 control	BMP7		-0.862
D0 to LMW-DS treatment	More activated relative to control	1.092
D0 to D2 control	INHA		2.292
D0 to LMW-DS treatment	More activated relative to control	0.588

In cortical neurons, in normal culture conditions, most growth factor dependent pathways were significantly activated by the normal culture medium. In most instances this activation was not altered by LMW-DS. However, LMW-DS activated molecules that are the downstream effector of GDF7 indicating that the effect of this growth factor was enhanced by LMW-DS. As GDF7 is a powerful differentiation factor for neurons, and the additional activation of these growth factors, to the activation of BDNF and NT3, provide a good explanation for the enhanced differentiation of these cells in culture.

Discussion

The normal culture conditions for HUVECs mimics the environment following tissue hypoxia and reperfusion, containing a high nutrient content and growth factors also supplemented with heparin. The LMW-DS-treated cultures mimicked the effect of LMW-DS added after 24 hours of hypoxia and reperfusion. The real life scenario this relates to is that of angiogenesis following ischemic conditions, such a fibrosis condition.

In Schwann cells, the control cultures, with high nutrient content and glucose, recapitulate the activation of Schwann cells. The LMW-DS-treated cultures mimicked the effect of LMW-DS added after 24 hours of glial activation. The real life scenario that this recapitulates is glial activation following damage to the nervous system.

The normal culture conditions for the neurons, both motor neurons and cortical neurons, with high nutrient content and growth factors mimic the environment during normal neuronal differentiation. The only negative effect in these cultures is the oxidative stress the cells suffer. The real life scenario this relates to is the degenerative conditions driven by oxidative stress in the presence of ample growth and differentiation factors. This corresponds to an early stage of a neurodegenerative disease or condition where oxidative stress plays a pivotal role.

It is clear from the cell types that the molecular effects seen in Schwann cells and in HUVECs support a role for LMW-DS in protection against apoptosis; induction of angiogenesis; increased migration and movement of cells; increased cell viability and survival; and induction of cellular differentiation. The analysis of pivotal molecular pathways indicated that in neurons LMW-DS will reduce the effect of oxidative stress on mitochondria and will reduce neurodegeneration-related molecules, such as amyloid-β and Lewy bodies.

Accordingly, the results from the HUVEC cell model indicates that LMW-DS can protect against cell damage and promotes the development of new blood vessels in injured or diseased tissue, such as following stroke. The results from the Schwann cells indicate that LMW-DS can protect against cell loss in a diseased or damaged nervous system.

The analysis of pivotal molecular pathways indicated that in Schwann cells LMW-DS reduced the effect of oxidative stress on mitochondria and increased the uptake of glutamate. The results in Schwann cells indicate that LMW-DS can protect against cell loss that occurs due to oxidative stress and glutamate excitotoxicity in the diseased or damaged nervous system.

Of particular importance, LMW-DS increased the glutamate uptake in glia cells, as presented by Schwann cells. However, LMW-DS did not alter the production of glutamate by neurons. This is important since glutamate is needed for LTP, learning and memory. Thus, it is beneficial that LMW-DS did not alter production of glutamate by neurons since this glutamate is needed for the normal neurotransmission in the above mentioned processed. However, the increased levels of glutamate released from damaged or dying cells will be effectively taken up by surrounding glial cells due to the effects of LMW-DS. Thus, the activation of glutamate transporters in the glial cells caused by LMW-DS effectively removed the glutamate released by the damaged or dying neurons from the neural cleft. This in turn prevented the glutamate from exerting its excitotoxicity and thereby damaging further neurons. Accordingly, LMW-DS induced the uptake of the potentially harmful neurotoxic amounts of glutamate by the glial cells.

The results in the neurons therefore confirm the potential therapeutic usefulness of LMW-DS in reducing secondary tissue damage due to oxidative stress, promoting repair, and reducing degeneration-related protein accumulation.

Taken together the results support the role of LMW-DS in protection against apoptosis in general and protection against neuronal cell death in particular, induction of angiogenesis, increased migration and movement of cells, increased cell viability and survival, induction of cellular differentiation, reduction of the effects of oxidative stress, reduction of glutamate excitotoxicity and reduction of the production of degeneration-related protein products, such as amyloid-β and Lewy bodies.

Cell adhesion was affected mainly in neurons and Schwann cells, where LMW-DS promoted cell detachment and movement. In HUVECs, cell adhesion was not affected. The effect on cell adhesion was mainly due to the expression of metalloproteinase-type enzymes, but the modulation of other adhesion molecules contributed to this effect as well.

Scarring as a pathological reaction is driven by TGFβ. TGFβ induces a large interconnected network of 171 molecules causing adhesion of immune cells, activation of cells, cell movement, aggregation of cells, fibrosis and induction of TGFβ. Administration of LMW-DS totally abolished the TGFβ-induced effect in adhesion of immune cells, activation of cells, aggregation of cells, fibrosis and self-activation of TGFβ. These inactivating effects of LMW-DS on the molecular networks driven by TGFβ in Schwann cells are also seen even when TGFβ is activated, i.e., even in the presence of excessive TGFβ.

These studies therefore confirm the potential therapeutic usefulness of LMW-DS in suppressing signaling by the pro-inflammatory fibrogenic cytokine TGFβ, which in turn suppresses tissue fibrosis, which otherwise is a severe component in COVID-19 subjects.

Example 8

This Example showed that LMW-DS resolved inflammatory scarring and activated matrix remodelling in rodent and human fibrotic disease models to enable functional tissue regeneration.

Results

POAG is an example of a chronic ocular fibrotic disease. In the eye, TGFβ is known to increase deposition of ECM in the TM, most notably, fibronectin. Fibronectin is found within the sheath material linking the inner most region of the TM with Schlemm’s canal and helps to regulate the tissue’s contractile properties and consequently AqH drainage and IOP. Higher levels of TGFβ and fibronectin have been detected in patients with glaucoma. Here we demonstrated that LMW-DS significantly reduced fibronectin levels in cultured human trabecular meshwork cells treated with TGFβ2 (1136 ± 155 vs 1579 ± 210; P<0.05; FIGS. 15A, 15B). Following this, we assessed the effects of subcutaneous (SC) injections of LMW-DS in a rodent model of anterior segment fibrosis, an experimental model of glaucoma, in which fibrosis is induced through bi-weekly intracameral (IC) injections of TGFβ1 (FIG. 16A); twice weekly IC TGF-β1 injections significantly raised IOP to 13 mmHg by day 14 by establishing TM fibrosis, at which point daily SC injections of LMW-DS or saline commenced. The IOP continued to rise in control rats receiving IC TGFβ1 plus SC injections of saline, reaching 17.6 ± 2.7 mmHg on day 28 (FIG. 16B). By contrast, IC TGFβ1 with SC LMW-DS injections reduced IOP to 11.4 ± 0.2 mmHg by day 28 (P<0.0001; FIG. 16B), which was within the normal range (10-12 mmHg). Immunohistochemical analysis of fibrosis in the TM showed significant reductions in the ECM molecules laminin (25.6 ± 3.5%, P<0.001; FIG. 16C) and fibronectin (40.2 ± 4.0%, P<0.01, FIG. 16D) compared to the TM in SC saline treated control rats (63.7 ± 6.8% and 63.6 ± 2.9%, respectively). This was accompanied by significantly enhanced numbers of surviving retinal ganglion cells (RGC) (12.6 ± 0.4 RGC/mm in SC LMW-DS-injected rats compared to 9.0 ± 0.3 RGC/mm in control SC saline-injected rats; P<0.01; FIG. 16E) and preserved retinal nerve fibre layer (RNFL) thickness (59.9 ± 1.2 µm in SC LMW-DS-injected rats compared to 44.0 ± 1.6 µm in control SC saline-injected rats; P<0.01; FIG. 16F) as measured by Optical Coherence Tomography (OCT).

Discussion

As inflammation precedes fibrosis in fibroproliferative conditions, it was important to understand how LMW-DS might modulate inflammatory and tissue remodeling pathways in human cells. Protein and gene expression data from relevant cultured human cells suggest that, through reprogramming immune activation, inflammation resolution and tissue remodeling (Example 7), LMW-DS modulates key innate and adaptive responses leading to improved healing and functional remodeling of diseased and damaged human tissues.

In Example 6, the mechanistic profile of LMW-DS at the protein level was explored using the BioMAP® Diversity Plus assay, which comprised 12 different validated human primary cell culture systems that modelled inflammation and wound healing responses in different human tissue types. The data demonstrated that LMW-DS had a distinct phenotypic profile with multi-modal actions; having effects in different cell types and following stimulation with different inflammatory mediators. In particular LMW-DS reduced levels of 3 chemokines, CXCL9, CXCL10 and CXCL11, which are all structurally related, signal through the CXC receptor 3 (CXCR3) and are induced by interferons and TNFα. CXCR3 and its associated chemokines play an important role in recruitment and function of immune cells and have been implicated in numerous inflammatory and autoimmunity diseases. In addition, they have been used as prognostic and predictive biomarkers of disease. The CXCR3 pathway plays an important role in the aetiology of glaucoma, with expression levels correlating 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.

Both the gene expression data (Example 7) and the results from the glaucoma model support a unique multi-modal influence of LMW-DS, due to its actions on the TGFβ signaling pathway as well as on other ECM remodeling signals. In POAG, TGFβ has a role in the progressive accumulation of ECM proteins in the TM, preventing normal outflow of AqH leading to elevated IOP. The rodent model of glaucoma used in this Example models TGFβ induced anterior segment fibrosis and provides a robust, clinically relevant means of evaluating anti-fibrotic therapies. Here we demonstrate that daily SC injections of LMW-DS resolved inflammatory signaling, reversed established TM fibrosis and lowered IOP, thereby rescuing RGC from progressive death, supporting the potential for LMW-DS to modify 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 displays similar anti-fibrotic efficacy in experimental models of eye, spinal cord and brain fibrosis. Interestingly, LMW-DS may act in part to modulate inflammation and promote tissue remodeling by increasing decorin expression, as shown in the gene expression analysis (Example 7).

In conclusion, this is the first study to demonstrate that s.c. injections of LMW-DS can resolve inflammation and nascent/established fibrosis in both rodent and human models of inflammation and fibrosis, thereby supporting functional tissue regeneration. As a consequence, in the ocular disease model evaluated, inflammatory fibrosis was resolved, IOP was rapidly normalized and compromised retinal cells were protected from progressive death. We have also provided insights into the multi-modal mechanism of action of LMW-DS. Taken together, our studies provide 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 anti-fibrotic and fibrolytic effects of LMW-DS are advantages to prevent or at least inhibit scarring in COVID-19 subjects during the recovery phase (stage IV) and following the hyperinflammation phase (stage III) (FIG. 25). LMW-DS can thereby not only reduce the formation of deleterious tissue scarring in, for instance, lungs, liver and heart, but may additionally resolve such scars.

Materials & Methods

LMW-DS was provided by Tikomed AB, Viken, Sweden (WO 2016/076780) at a stock concentration of 20 mg/ml and was kept in a temperature monitored refrigerator. Immediately prior to use LMW-DS aliquots were diluted to the appropriate concentration in sterile saline and administered by subcutaneous injection.

Culture of TM Cell Populations

Human trabecular meshwork cells (TMC-6590; ScienCell Research Laboratories, Carlsbad, CA) were cultured in TMC (6591; ScienCell Research Laboratories) complete medium and maintained at 37° C. in 5% CO₂ until reaching 80-90% confluency. Cells were plated at densities of 5000-10000 cells per well in Greiner-Bio Cell 96 Well black Cell Culture Microplates (655090, Sigma) and left to adhere overnight. The following 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 hrs.

Immunostaining and Confocal Imaging for TM Cells

After 72 hrs treatments, cells were fixed using 4% PFA in PBS (28908, Thermo Fisher Scientific) for 10 minutes, washed with PBS and then washed with PBS containing saponin-based permeabilization buffer (00-8333-56, Thermo Fisher Scientific). Cells were then immunostained with anti-human Fibronectin Alexa Fluor® 488 (53-9869-82, Thermo Fisher Scientific) and nuclear stain Hoeschst 33342 (H21492, Thermo Fisher Scientific) in permeabilizing buffer in the dark for 1 hr at room temperature. Finally, cells were washed twice in PBS containing saponin-based permealization buffer and once in PBS prior to image analysis by confocal microscopy where images were acquired on a Yokogawa CQ1 spinning-disc microscope using a 40x objective and appropriate excitation/emission settings for Hoechst and Alexa Fluor® 488. Z-series were acquired and are displayed as maximum intensity z-projections. Images were analyzed using the Yokogawa image analysis software.

Experimental Design for Ocular Hypertension Model

Before induction of raised IOP, rats were randomly assigned into two groups (for saline vehicle and LMW-DS treatment). Induction of raised IOP was induced 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). At 0 d, a 15° disposable blade was used to make a self-sealing tunnel through the cornea and this access point was employed for all subsequent twice weekly 3.5 µl IC injections of 5 ng/ml TGFβ1 (Peprotech, London, UK), carried out using sterile glass micropipettes (Harvard Apparatus, Kent, UK) over the 28 d experiment. IOP was also measured twice-weekly (immediately prior to the IC injection) throughout the 28 d experiment. At 14 d, non-responders to TGFβ (i.e., their IOP did not increase above baseline at any time point) were excluded from further analysis to yield an inclusion of n= 7 rats (14 eyes) for LMW-DS and n= 5 rats (10 eyes) for saline vehicle treatments. At 14 d, rats received either subcutaneous injections of 15 mg/kg LMW-DS (Tikomed AB) or an equivalent saline volume, which continued daily for 14 d. OCT measurements of the RFNL were taken at 28 d as a surrogate for RGC density and ocular tissues from both groups were processed for immunohistochemistry to assess levels of TM fibrosis and RGC survival.

Animals and Surgery

The rodent study was performed at the Biomedical Services Unit at the University of Birmingham (UK) in accordance with the Home Office guidelines set out in the 1986 Animal Act (UK), adhering to the ARRIVE guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research under UK Home Office License number 70/8611.

Adult 10 week old) male Sprague Dawley rats (Charles River, Kent, UK) were housed with free access to food and water under a 12 h dark/light cycle. Surgery was performed at the Biomedical Services Unit at the University of Birmingham (UK) in accordance with the Home Office guidelines set out in the 1986 Animal Act (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 taken whilst rats were under inhaled anesthesia using between 2% - 5% isofluorane / 98% - 95% O₂ (National Vet Supplies, UK). The welfare of all rats was monitored closely at all times.

IOP Measurements

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 d experiment to avoid confounding circadian variabilities in IOP. Immediately after induction of anesthesia, six rebound measurements were taken with the tonometer from the central cornea and averaged to give a single reading (mmHg). Each graphical data point represents the mean ± SEM of three readings (6 rebounds) taken from each rat sequentially to ensure accurate measurements.

Optical Coherence Tomography

Retinal nerve fiber layer (RNFL) measurements were taken at 28 d using a Spectralis HRA3 confocal scanning laser ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). Images were obtained from rats whilst under inhalational anesthesia (as described in the ‘Animals and Surgery’ section above). OCT images were taken of the retina around the optic nerve head and in-built software was used to segment images and quantify the RNFL thickness.

Tissue Preparation for Immunohistochemistry

Rats were euthanized by an intraperitoneal injection of sodium pentobarbital (National Veterinary Supplies) on 28 d and intracardially perfused with 4% paraformaldehyde (PFA; TAAB, Aldermaston, UK) in PBS whilst under terminal anesthesia. Eyes were removed and immersion fixed in 4% PFA in PBS for 2 hours at room temperature and then cryoprotected using increasing concentrations of (10%, 20% and 30% sucrose) for 24 hours each at 4° C. Following this, eyes were embedded in optimal cutting temperature (OCT) embedding medium (Thermo Shandon, Runcorn, UK) in a peel-a-way mold container (Agar Scientific, Essex, UK) and rapidly frozen using 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 variation) at -22° C. using a Bright cryostat microtome (Bright, Huntingdon, UK) and mounted on positively charged glass slides (Superfrost Plus, Fisher Scientific, USA) and left to dry for 2 h at 37° C. and stored at -20° C.

Immunohistochemistry

All reagents were purchased from Sigma (Poole, UK) unless otherwise specified. Frozen tissue sections were left for 30 min to equilibrate to room temperature and then hydrated in PBS 3 × 5 min before being immersed in 0.1% Triton X-100 for 20 min to permeabilise the sections. Following a further PBS 3 × 5 min wash and eye sections isolated with a hydrophobic PAP pen (Vector Laboratories, Peterborough, UK). Non-specific antibody binding sites were blocked using 0.5% BSA, 0.3% TWEEN 20® and 15% Normal goat serum in PBS. Sections were then placed in primary antibody (Laminin L9393, Fibronectin F3648, Sigma, Brn3a SC-31984, Santa Cruz) and left overnight at 4° C. before being washed 3 × 5 min in PBS and incubating for 1 hr at room temperature with secondary antibody (Goat-anti rabbit Alexa Fluor 594 or Goat-anti mouse Alexa Fluor 488). Before mounting with Vectashield containing DAPI (Vector Laboratories), sections were washed 3 × 5 min in PBS. Control tissue sections incubated without primary antibodies but with secondary antibodies were all negatively stained (not shown).

Microscopy and Analysis

Fluorescently stained sections were analyzed by on operator masked to treatment groups using randomized numbers using a Zeiss Axioplan 2 fluorescent microscope (Carl Zeiss Ltd, Oberkochen, Germany) as previously described (Hill, L. J. 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 at the same exposure (250 ms) to assess pixel intensity for ECM levels. The percentage of immunofluorescent pixels above the threshold within the region of interest was measured using image J software.

Example 9

This Example investigated the ability of LMW-DS to impact stimulated release of IL-6 from human PBMC in vitro.

Human PBMC can be stimulated in vitro by a variety of agents that will activate directly and indirectly various cell subsets. Monitoring cytokine release allows the potential impact of drugs to be investigated to predict action in patients. IL-6 is an archetypical pro-inflammatory cytokine that has been shown to be associated with numerous pathologies including neurodegenerative conditions like ALS, inflammatory diseases like arthritis and severe consequences of viral infection, such as COVID-19

Materials & Methods

Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors through Ficoll-Paque PLUS (GE Healthcare; 11778538) density centrifugation. PBMC were cultured at 2×10⁵ cells/well in the absence (unstimulated PBS vehicle controls) or presence of stimulation (LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, or Cytostim) in the absence (vehicle) or presence of LMW-DS (Tikomed AB, Viken, Sweden, WO 2016/076780) at three concentrations; 60 µg/ml, 200 µg/ml and 600 µg/ml for 24 hours at 37° C., 5% CO₂. Following centrifugation, cell culture supernatants were removed and stored at -20° C. awaiting analysis for IL-6 by ELISA. Levels of IL-6 were quantified in the supernatant by ELISA (R&D systems; DY206) according to manufacturer’s instructions.

Results

Historical internal data guided selection of stimulus concentration to use sub-maximal concentrations of LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15 and Cytostim. With the PBMC mix, all the stimulations increased IL-6 release into the cell culture supernatant. With promising results generated from the cells from the first six donors with respect to the impact of LMW-DS upon LPS stimulation, it was decided to extend the LPS studies in a further six donors. Furthermore, relative to unstimulated PBMC, the original selected concentrations of CpG + IL-15 and Cytostim gave relatively low increases in IL-6 release and therefore in the cells from the additional six donors, higher concentrations of CpG + IL-15 and Cytostim were investigated. These higher concentrations evoked a greater increase in IL-6 release relative to the unstimulated cells.

LPS

LPS (lipopolysaccharide) is a toll-like receptor (TLR) 4 agonist. In a human PBMC mix, the main cell type activated directly by LPS is monocytes, which express TLR4. Monocytes are part of the innate immune system; these myeloid cells can also be used to model responses to other myeloid cells such as macrophages and microglia. In the present studies, LPS evoked a substantial increase in the release of IL-6 into the cell culture supernatant (FIG. 17A); LMW-DS caused a concentration dependent and statistically significant reduction in IL-6 concentrations (FIG. 17A). This indicates that LMW-DS displays potential to reduce the pro-inflammatory consequences of IL-6 following TLR4 activation of monocytes.

Peptidoglycan

Peptidoglycan is a TLR2 agonist, which in a PBMC mix is expressed predominantly by monocytes and B lymphocytes; the latter are a component of the acquired immune system best known for displaying an integral role in the generation of specific antibodies to antigens. Peptidoglycan evoked a substantial increase in the release of IL-6 into the cell culture supernatant but overall from experiments with cells from six donors there was little evidence of LMW-DS, even at the highest concentration tested, of causing a general reduction in IL-6 release (FIG. 17B).

Pokeweed Mitogen

Pokeweed mitogen is a lectin purified from Phytolacca americana. It evokes a T lymphocyte-dependent activation of B lymphocytes. In the present studies, pokeweed mitogen evoked a large increase in IL-6 release by the PBMC mix but this release was not impacted generally by LMW-DS (FIG. 17C).

Pha-l

PHA-L (phytohemagglutinin-L) is the L-type subunit lectin from Phaseolus vulgaris (red kidney beans), which crosslinks T lymphocyte surface receptors resulting in their activation. PHA-L induced a relatively modest increase in IL-6 release from the PBMC mix and this was inhibited significantly in a concentration-dependent manner by LMW-DS (FIG. 17D).

CpG + IL-15

CpG-ODN are short single-stranded DNA molecules that activate TLR9, which within a PBMC mix is expressed mainly by monocytes and B cells. IL-15 synergises with CpG in the stimulation of B lymphocytes. Unlike pokeweed mitogen, CpG + IL-15 activates B lymphocytes directly, i.e., is T lymphocyte independent. In the first round of experiments with PBMC from six donors, the concentration of CpG-ODN + IL-15 selected evoked only small increases in IL-6 release into the cell culture supernatant. Whilst this relatively low level of IL-6 release was not generally impacted by LMW-DS (FIG. 17E), with the additional experiments performed with extra donors to increase the n number for the LPS stimulation, these same extra donors were subject to application of a higher concentration of CpG + IL-15 in an attempt to evoke a more robust release of IL-6 above that evident from the unstimulated cells; whilst this was achieved there was still no general impact of LMW-DS (FIG. 17F).

Cytostim

Cytostim is an antibody-based activator of T lymphocytes. It binds to the T cell receptor (TCR) and cross links this to the major histocompatibility complex (MHC) molecule of an antigen-presenting cell. Thus, Cytostim stimulates both CD4 and CD8 T lymphocytes. In the present study, the first round of experiments with PBMC from six donors used a concentration of Cytostim that led only to small relatively small increases in IL-6 release into the cell culture supernatant, which was not generally impacted by LMW-DS (FIG. 17G). PBMC from these same extra donors were investigated with a higher concentration of Cytostim to evoke a greater release of IL-6 relative to the IL-6 release from the unstimulated cells yet there was still no general impact of LMW-DS (FIG. 17H).

Whilst all the stimuli investigated in the present study were able to increase IL-6 release from cells in a PBMC mix, the targeted action of LMW-DS (to LPS and PHA-L) upon IL-6 release suggests a refined mode of action rather than a general ability to reduce IL-6 release.

Example 10

Monocytes are circulating innate immune cells that are a key component of the immune system. In addition, as they are readily accessible, they can be used as cells to model other myeloid cells that are less easy to access such as macrophages and microglia.

Like various myeloid cells, monocytes can be activated via Toll-Like receptor (TLR) agonists, like the TLR2 and the TLR4 receptor agonists, peptidoglycan and lipopolysaccharide (LPS), respectively. Activation can be monitored by the expression of activation markers (by flow cytometry) and/or secretion of cytokines such as the pro-inflammatory cytokine, interleukin-6 (IL-6). In Example 9, LMW-DS reduced the secretion of IL-6 in a human peripheral blood mononuclear cell (PBMC) preparation in response to LPS stimulation. However, the multiplicity of cell types in PBMC mix prevents interpretation of the precise cell type(s) mediating this response. The present study investigated the ability of LMW-DS to modify LPS-stimulated IL-6 release from human purified monocytes in an attempt to identify a precise cellular target for LMW-DS. IL-6 is an archetypical pro-inflammatory cytokine that is associated with numerous pathologies including neurodegenerative conditions like ALS, inflammatory diseases such as arthritis and the severe consequences that can result as a consequence of viral/bacterial infections, such as COVID-19.

Materials & Methods

Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors through Ficoll-Paque PLUS (GE Healthcare; 11778538) density centrifugation. Monocytes were purified using the EasySep™ human monocyte enrichment kit (StemCell) that purifies monocytes ‘untouched’ to maintain their phenotype.

Monocytes were cultured in the absence (unstimulated PBS vehicle controls) or presence of stimulation (LPS or peptidoglycan) in the absence (vehicle) or presence of LMW-DS (Tikomed AB, Viken, Sweden, WO 2016/076780; three concentrations; top concentration 600 µg/ml, 200 µg/ml and 60 µg/ml), 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) for 24 hours at 37° C., 5% CO₂. Following centrifugation, cell culture supernatants were removed and stored at -20° C. awaiting analysis for IL-6 by ELISA. Levels of IL-6 were quantified in the supernatant by ELISA (R&D systems) according to manufacturer’s instructions.

Results

Historical internal data guided selection of the stimulus concentration and the use of sub-maximal concentrations of LPS and peptidoglycan. These also corresponded to the same concentrations of LPS and peptidoglycan used in Example 9 when a human PBMC mix was used as the source of IL-6.

The TLR2 agonist peptidoglycan and the TLR4 agonist LPS evoked release of IL-6 into the cell culture supernatant from the human purified monocytes (FIG. 18).

Average results with monocytes from ten healthy donors demonstrated a concentration dependent statistically significant inhibition of LPS-stimulated IL-6 release into the cell culture supernatant by LMW-DS (FIG. 18A), whereas heparin evoked a concentration dependent statistically significant enhancement of LPS-stimulated IL-6 release into the cell culture supernatant (FIG. 18C). As expected, the glucocorticoid steroid dexamethasone inhibited the LPS-stimulated IL-6 release into the cell culture supernatant in a statistically significant manner (FIG. 18B).

Average results with monocytes from ten healthy donors demonstrated a concentration dependent statistically significant increase of peptidoglycan-stimulated IL-6 release into the cell culture supernatant by LMW-DS (FIG. 18D). This enhancement was mirrored to some extent by heparin although the trend did not reach statistical significance (FIG. 18F). As expected, the presence of dexamethasone resulted in a statistically significant inhibition of peptidoglycan-stimulated IL-6 release into the cell culture supernatant (FIG. 18E).

Monocytes are part of the innate immune system; these myeloid cells can also be used to model responses to other myeloid cells such as macrophages and microglia. In the present studies, the clear and substantial impact of LMW-DS to inhibit the LPS evoked increase in the release of IL-6 from purified monocytes provides strong evidence that these cells represent a target for LMW-DS.

Example 11

Activation of the immune response in diseased, damaged or infected tissues is reflected by changes in the phenotypic balance of peripheral blood mononuclear cells (PBMC). Hence, assessing the impact of investigational drugs upon components of the adaptive and innate immune systems may reveal mechanistic cellular pathways to better understand clinical changes associated with the investigational therapy as well as potentially identifying cellular and/or molecular biomarkers predicting therapeutic efficacy for different pathologies.

Human PBMC can be stimulated in vitro by a variety of agents that will activate directly and indirectly various cell subsets and mimic immune responses associated with compromised tissues. Monitoring cytokine release from PBMC allows the potential impact of drugs to be investigated and to predict drug action in specific patient aetiologies.

Materials & Methods

Example 9 investigated the ability of LMW-DS to modify the secretion of IL-6 from human PBMC arising from the use of various stimuli. The present study performed a broader analysis of the cell culture supernatants arising from Example 9. Thus, peripheral blood mononuclear cells (PBMC) were isolated from healthy donors through Ficoll-Paque PLUS density centrifugation. The PBMC were cultured at 2×10⁵ cells/well in the absence (unstimulated PBS vehicle controls) or presence of stimulation (LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, or Cytostim) in the absence (vehicle) or presence of LMW-DS (Tikomed AB, Viken, Sweden, WO 2016/076780; three concentrations; top concentration 600 µg/ml, 200 µg/ml and 60 µg/ml) for 24 hours at 37° C., 5% CO₂.

Accordingly, the treatments were as follows for each PBMC donor:

• i. Vehicle
• ii. Simulation
• iii. Stimulation + LMW-DS (60 µg/ml)
• iv. Stimulation + LMW-DS (200 µg/ml)
• v. Stimulation + LMW-DS (600 µg/ml)

with each assessed in triplicate such that the total numbers of samples were:

• 1. LPS; 165 samples (from 11 donors)
• 2. Peptidoglycan from B. 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. Pokeweed mitogen; 90 samples (from 6 donors)
• 7. 10 µl/ml Cytostim; 90 samples (from 6 donors)
• 8. 30 µl/ml Cytostim; 90 samples (from 6 donors)

Total number of supernatant samples from all stimulations (all cell types) = 795

Following treatment, cell culture supernatants were removed, centrifuged and stored at -20° C. before thawing for multiplex analysis of various cytokines using the 5-plex human magnetic Luminex assay from R&D systems (Cat. No. LXSAHM-05). Luminex analysis was carried out exactly following manufacturer’s protocols.

Results

Historical internal data guided selection of stimulus concentration to use sub-maximal concentrations of LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15 and Cytostim; use of sub-maximal concentrations of stimulus tends to allow both increases and decreases in modulation to be identified when present. With the PBMC mix, all the stimulations increased release of cytokines into the cell culture supernatant although some stimuli were more effective than others (FIGS. 19-23).

LPS

LPS (lipopolysaccharide) is a toll-like receptor (TLR) 4 agonist. In a human PBMC mix, the main cell type activated directly by LPS is monocytes, which express TLR4. Monocytes are part of the innate immune system; these myeloid cells can also be used to model responses to other myeloid cells such as macrophages and microglia. In the present studies, when evaluating the effect of LPS, there was a modest increase in the secretion of IFNy, TNFα, IL-1β and IL-10 but a substantial increase in IL-8 secretion (FIGS. 19-23). LMW-DS was responsible for a concentration dependent but small reduction in IFNy and IL-10 (FIGS. 19, 23), and the presence of the highest concentration of LMW-DS resulted in a modest reduction in the stimulated secretion of IL-1β, IL-8 and TNFα (FIGS. 20-22).

Peptidoglycan

Peptidoglycan is a TLR2 agonist, which in a PBMC mix is expressed predominantly by monocytes and B lymphocytes; the latter are a component of the acquired immune system best known for displaying an integral role in the generation of specific antibodies to antigens. Peptidoglycan evoked an increase in the release of IL-1β, IL- 8 and TNFα into the cell culture supernatant but overall, from examining the results from all the donors, there was little evidence of LMW-DS, even at the highest concentration tested, of causing a general reduction in cytokine release although there were associated increases in IL-1β and TNFα secretion (FIGS. 19-23).

Pha-l

PHA-L (phytohemagglutinin-L) is the L-type subunit lectin from Phaseolus vulgaris (red kidney beans), which crosslinks T lymphocyte surface receptors resulting in their activation. In the PBMC mix, PHA-L induced increases in IFNy, IL-8, IL-10 and TNFα and a modest increase overall in IL-1β (FIGS. 19-23). LMW-DS resulted in small decreases in stimulated release of IFNy, IL-8, TNFα but not IL-1β (FIGS. 19-22). By contrast, LMW-DS resulted in a large, concentration dependent decrease in IL-10 secretion (FIG. 23).

Pokeweed Mitogen

Pokeweed mitogen is a lectin purified from Phytolacca americana. It evokes a T lymphocyte-dependent activation of B lymphocytes. In the present studies, pokeweed mitogen evoked robust increases in the secretion of IFNy, IL-1β, IL-8, IL-10 and TNFα by the PBMC mix (FIGS. 19-23), but this release was not impacted generally by LMW-DS except for a concentration-dependent decrease in IL-10 secretion (FIG. 23).

CpG + IL-15

CpG-ODNs are short single-stranded DNA molecules that activate TLR9, which within a PBMC mix is expressed mainly by monocytes and B cells. IL-15 synergizes with CpG in the stimulation of B lymphocytes. Unlike pokeweed mitogen, CpG + IL-15 activates B lymphocytes directly, i.e., is T lymphocyte independent. In the present studies, there was only robust evidence for this stimulation to increase secretion of IL-8. Overall, LMW-DS displayed little impact on this response in these experiments (FIGS. 19-23).

Cytostim

Cytostim is an antibody-based activator of T lymphocytes. It binds to the T cell receptor (TCR) and cross links this to the major histocompatibility complex (MHC) molecule of an antigen-presenting cell. Thus, Cytostim stimulates both CD4 and CD8 T lymphocytes. Overall, Cytostim evoked an increase in the secretion of IFNy, IL- 1β, IL-8, IL-10 and TNFα (FIGS. 19-23), with small reductions associated with the presence of LMW-DS except for IL-10 secretion where there was a concentration dependent large reduction evident in the presence of LMW-DS (FIG. 23).

Overall, the data support an action of LMW-DS to benefit patients with inflammatory reactions following infection, such as sepsis. Sepsis following infection is considered a dysregulated immune response resulting in organ dysfunction. Sepsis is responsible for major morbidity, mortality and healthcare expenditure. The urgent need for new therapeutics has again been underlined in the current pandemic with most deaths in critically ill COVID-19 patients caused by sepsis.

During sepsis, in response to an infection, excessive production of inflammatory cytokines (cytokine storm) may cause septic shock. Several of the cytokines modulated by LMW-DS are both elevated and considered to contribute to the pathogenesis of sepsis. For example, IFNy, IL-1β, IL-6, IL-8 and TNFα exhibit a persistent increase in non-survivors4. TNFα and IL-1β are considered to play major roles in sepsis and act on cells such as macrophages, where they amplify inflammatory cascades to increase release of other pro-inflammatory cytokines as well as reactive oxygen and nitrogen species, and endothelial cells, where they mediate inflammation-induced activation of coagulation. Additional roles of TNFα include promoting neutrophil extravasation through action on endothelial cells and it has been demonstrated that blockade of TNFα with monoclonal antibodies may also improve survival in patients with severe sepsis. IL-6 can enhance the activation of T cells, B cells and the coagulation system, and levels of IL-6 correlate with the clinical severity of sepsis. Knockout of IL-6 reduces lung damage 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. In addition to pathological roles in sepsis, cytokines may play host protective roles in host defense and immune regulation, and so despite the promise in targeting them described above, the role of cytokines in sepsis remains a ‘double-edged sword’.

One challenge in inflammatory diseases, such is sepsis, is how to target elements of a response without generating prolonged immunosuppression. Whilst benefits of neutralizing TNFα in sepsis have been demonstrated, monoclonal antibodies, with their long half-life, e.g., infliximab, adalimumab and certolizumab have half-life values of around 14 days, raise challenges in the timing and route of administration. Agents that target multiple cytokines, in a specific phase of the disease with a relatively short duration of action, like LMW-DS, could therefore bring potential additional clinical benefit to patients with inflammatory diseases, such as sepsis. In the case of COVID-19 associated sepsis, a further benefit of LMW-DS may arise from a direct effect on the virus; thus, incubation of SARS-CoV-2 with exogenous LMW-DS reduces attachment to human epithelial cell lines and reduced viral replication within ex vivo human lung tissue explants.

Example 12

The objective of this Example was to evaluate the effect of repeated LMW-DS administration on the concentrations of serum metabolites in serum samples from a cohort of Swedish ALS patients who had participated in the trial entitled ‘A single-centre, open single-arm study where the safety, tolerability and efficacy of subcutaneously administered ILB® will be evaluated in patients with Amyotrophic Lateral Sclerosis’. Using a longitudinal design, this study aimed to determine the changes of selected serum metabolites in each ALS patient before and after weekly LMW-DS treatment. Changes in the measured metabolites indicate the biochemical response of the patient to LMW-DS that is underpinning potential disease modification and the mechanisms of action of the drug in this patient population.

Materials & Methods

After an initial screening visit, patients had five weekly dosing of a single LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016/076780) injection of 1.0 mg/kg body weight in saline into the subcutaneous fat of the lower abdomen.

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

After defrost, an aliquot of 500 µl of each serum sample was supplemented with 1 ml of HPLC-grade acetonitrile, vortexed for 60 seconds, centrifuged at the maximum speed in a top-bench centrifuge to precipitate proteins. Supernatants were washed with large volumes of HPLC-grade chloroform to remove organic solvent, centrifuged and the upper aqueous phases were transferred in different tubes, clearly labeled to identify the sample and stored at -80° C. until analyzed to determine different water-soluble compounds.

A second aliquot of about 300 µl of each serum sample was light-protected and then processed to extract fat-soluble antioxidants using a method 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, vigorously vortexed for 60 s and incubated at 37° C. for 1 h in a water bath under agitation, to allow the full extraction of lipid soluble compounds. Samples were then centrifuged at 20,690 × g for 15 min at 4 C to precipitate proteins and the clear supernatants were saved at -80° C. until the HPLC analysis of fat-soluble vitamins and antioxidants.

In deproteinized serum samples, the following water-soluble compounds were separated and quantified by HPLC, according to methods described elsewhere (Tavazzi et al., Simultaneous high performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis of inborn errors of metabolism. Clin Biochem. 2005; 38: 997-1008; 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 Biochem. 2012; 359: 205-216): hypoxhantine, xanthine, uric acid, malondialdehyde (MDA), nitrite, nitrate, N-acetylaspartate (NAA), citrulline (CITR), alanine (ALA), and ornithine (ORN).

The following fat-soluble vitamins and antioxidants in deproteinized serum samples were separated and quantified by HPLC according to a method as previously described (Lazzarino et al., Cerebrospinal fluid ATP metabolites in multiple sclerosis. Mult Scler J. 2010; 16: 549-554): α-tocopherol (vitamin E) and γ-tocopherol. In addition to the abovementioned fat-soluble antioxidants and vitamins, also the concentration of total bilirubin was determined in serum samples.

Statistics

Comparison of the Pre- and Post-treatment subgroups was performed by the two-tailed Student’s t-test for paired samples. The comparison of each subgroup with the group of control healthy subjects was carried out by the two-tailed non-parametric Mann-Whitney U-test for unpaired observations. Differences with p < 0.05 were considered statistically significant.

Results

According to the statistical analysis, the two pre- and post-treatment subgroups of patients had significantly different serum concentrations of NAA (FIG. 26), uric acid (FIG. 27), MDA (FIG. 31), nitrate (NO₃) (FIG. 29), nitrite + nitrate (NO₃ + NO₂) (FIG. 30), sum oxypurines (FIG. 28), ALA (FIG. 32), CITR (FIG. 33), ORN/CITR ratio (FIG. 34), vitamin E (α-tocopherol and γ-tocopherol) (FIGS. 35 and 36) and total bilirubin (FIG. 43).

Data of the aforementioned compounds are illustrated into box plots (reporting minimum, maximum, median, 25% and 75% percentiles) in FIGS. 26-36, in which values of control healthy subjects (age ranging 25-65 years), from historical data sets derived from an Italian cohort of patients, have also been included and compared to both groups (pre- and post-treatment) of the patients. In all the figures, differences respect to the value of controls is indicated by one asterisk (*), whilst difference between the two subgroups of patients is indicated by two asterisks (**).

The ALS patients had higher serum NAA levels than those measured in control healthy subjects, presumably deriving from a decrease in viable neurons (FIG. 26). Significantly lower NAA values were measured post-LMW-DS treatment, thus suggesting a protective effect on cellular survival of the drug.

The ALS patients had higher serum concentrations of uric acid and sum of oxypurines (hypoxanthine + xanthine + uric acid) (FIG. 28) than those measured in controls, presumably in consequence of energy metabolism imbalance leading to the activation of the adenine nucleotide degradation pathway with a consequent increase in circulating purine compounds. LMW-DS treatment decreased both these parameters, thus suggesting amelioration of mitochondrial functions with increase in cell energy state.

The ALS patients had higher serum concentrations of nitrate (FIG. 29), nitrite + nitrate (FIG. 30) and MDA (FIG. 31) than those measured in controls, strongly indicating sustained oxidative/nitrosative stress, causing increasing in the circulating levels of these stable end-products of ROS-mediated lipid peroxidation (MDA) and of nitric oxide metabolism (nitrate and nitrite + nitrate). LMW-DS treatment decreased the levels of these parameters, thus, indicating either a direct scavenging activity of the compound, a positive influence on genes, such as BDNF, regulating the levels of scavenger enzymes, and/or or a positive influence on mitochondrial functions ultimately causing lower levels of ROS production.

The ALS patients had higher serum concentrations of ALA than those measured in controls (FIG. 32), presumably because of a higher rate of muscular protein degradation. LMW-DS treatment normalized circulating values of ALA (equal to those of controls and significantly lower than those of the pre-treatment subgroup), suggesting a positive action of the drug on muscle metabolism and functions.

The ALS patients had higher serum concentrations of CITR (FIG. 33) and lower values of the ORN/CITR ratio (FIG. 34) than those measured in controls, thereby corroborating the presence of sustained nitrosative stress caused by excess of nitric oxide production and subsequent increase in reactive nitrogen species (RNS). LMW-DS treatment ameliorated both parameters, possibly by lowering the expression of inducible nitric oxide synthase, the enzyme responsible for triggering nitrosative stress.

The ALS patients had lower serum concentrations of both α-tocopherol and γ-tocopherol (the two main forms of Vitamin E) than those measured in controls (FIGS. 35 and 36), thereby suggesting significant decrease of the main fat-soluble antioxidant playing a key role as an interrupter of the ROS-mediated peroxidation of fatty acids of membrane phospholipids. LMW-DS treatment ameliorated both parameters.

ALS patients have higher serum concentrations of total bilirubin than those measured in controls, presumably reflecting perturbed liver function. LMW-DS treatment decreased this parameter having, again, a tendency to normalize levels of this serum metabolite, indicating improved liver function.

LMW-DS is capable of improving liver function and thereby inhibit deterioration in liver function, which is seen in several COVID-19 patients. Hence, this effect of LMW-DS treatment is in particular beneficial in the recovery phase (stage IV) and for long-term COVID-19 subjects (FIG. 25).

Example 13

This Example investigated the ability of LMW-DS to impact the interaction between SARS-CoV-2 spike protein and ACE2 assessed using the RayBio® COVID-10 Spike-ACE2 binding assay kit.

COVID-19 arises from infection by the virus SARS-CoV-2. It is now understood that one of the mechanisms by which the virus gains entry to human cells is via a direct interaction between SARS-CoV-2 spike protein and angiotensin I converting enzyme 2 (ACE2); the latter is expressed in the cell membrane of various human cells. This interaction can be assessed using the RayBio® COVID-19 Spike-ACE2 binding assay kit, which is a rapid ELISA based colorimetric assay

Materials & Methods

The RayBio® COVID-19 Spike-ACE2 binding assay kit (cat. number CoV-SACE2) was used as per the manufacturer’s instructions (ver 1.6) to assess the ability of LMW-DS to disrupt the interaction between SARS-CoV-2 spike protein and ACE2.

LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016/076780) in saline was added in concentrations ranging from 3.0 µ/mL to 600 µg/ml.

Results

There was clear evidence in that LMW-DS was able to reduce the interaction between SARS-CoV-2 spike protein and ACE2 (FIG. 37). In more detail, LMW-DS caused a concentration dependent disruption of the SARS-CoV-2 spike protein - ACE2 interaction. Inhibition of the interaction between SARS-CoV-2 spike protein and ACE2, as caused by LMW-DS, would be predicted to be beneficial to patients by reducing the ability of the SARS-CoV-2 virus to gain entry to human cells.

Example 14

Using a longitudinal design, this study was aimed to determine the changes of selected serum metabolites in ALS patients before dextran sulfate administration and after different times following the beginning of the treatment. Changes in the measured metabolites indicated the biochemical response of the patient to dextran sulfate that is underpinning potential disease modification and the mechanisms of action of the drug in this ALS patient population.

Materials & Methods

Dextran sulfate (ILB®, Tikomed AB, Viken, Sweden, WO 2016/076780) was administered at 2 mg/kg by daily subcutaneous injection once a week for ten weeks to 8 human patients suffering from ALS.

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

A serum aliquot of 500 µl was supplemented with 1 ml of HPLC-grade acetonitrile, vortexed for 60 secs, centrifuged at the maximum speed in a top-bench centrifuge to precipitate proteins. Supernatants were washed with large volumes of HPLC-grade chloroform to remove organic solvent, centrifuged and the upper aqueous phases were transferred to different tubes, clearly labeled to identify the sample and stored at -80° C. until analyzed to determine different water-soluble compounds.

ALSAQ-40 was assessed pre-treatment and weekly thereafter for the 10 treatment weeks and for the visits at the follow-up period.

Results

Lactate is made by muscles and accumulates in the blood during exercise in normal and ALS patients. Raised levels of serum lactate indicate improved muscle function/use.

FIG. 38 illustrates serum lactate levels in ALS patient prior to (Week 0) and after dextran sulfate administration. The data shows that the circulating level of lactate, mainly deriving from metabolism of muscle cells, was raised significantly with increasing time of dextran sulfate administration (*significantly different 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), whilst after 10 weeks of dextran sulfate treatment, serum lactate levels had increased by 70% to 3.02±1.59 µmol/L. Hence, dextran sulfate administration led to increased muscular activity in the ALS patients.

The ALSAQ-40 sub-scores entitled ‘Activities of Daily Living/Independence’ (ADL) and ‘Physical Mobility’ (PM) reflect the patients’ view of their level of physical activity and independence. A falling score reflects improved physical activity. After 10 weeks of dextran sulfate treatment the ADL sub-score declined significantly by 18.6% from 58.9 ± 21.4 to 44.4 ± 24.7 (p < 0.05), see FIG. 39, whilst the PM sub-score declined by 16% from 27.2 ± 22.2 to 22.7 ± 20.2. These results indicated improved physical activity of the patients during treatment.

The improvement in muscle function as induced by dextran sulfate administration is beneficial to COVID-19 patients that, otherwise, may suffer from muscle pain and deteriorated muscle function. Hence, muscle improvement as obtained by dextran sulfate is of benefit during the recovery phase (stage IV) (FIG. 25).

Example 15

Materials & Methods

A clinical trial in the form of a Phase IIa, single-center, open label, single-arm study where the safety, tolerability and possible efficacy of subcutaneously administered dextran sulfate was evaluated in patients with ALS of intermediate progression rate. The clinical trial was conducted at the Sahlgrenska University Hospital, Gothenburg, Sweden and was overseen and approved by the Ethics Committee of the University of Gothenburg and by the Swedish Medical Products Agency.

Dextran sulfate (Tikomed AB, Viken, Sweden, WO 2016/076780) was administered at 1 mg/kg by daily subcutaneous injection once a week for five weeks to 13 human patients suffering from ALS.

Blood samples were drawn at defined study intervals by a venous catheter into vacutainer tubes. Laboratory analyses of blood plasma were performed immediately after collection by the Clinical Chemistry Laboratory at the Sahlgrenska University Hospital.

ALSFRS-R was assessed pre-treatment and weekly thereafter for the 5 treatment weeks and for the visits at the follow-up period.

Results

Myoglobin is a protein typically found in heart and skeletal muscle tissues. A raised level of myoglobin is found in the bloodstream when injury/disease has damaged muscle. Reduced levels of serum myoglobin indicate reduced muscle degeneration.

Serum myoglobin data from the patients revealed a statistically significant 30% reduction in myoglobin levels from 133.92 ± 126.28 to 103.69 ± 72.16 µg/L (p = 0.021 vs Day 1) after 4 weeks of dextran sulfate treatment indicating a drug-related reduction in the rate of muscle tissue degeneration and muscle atrophy of the patients during treatment, see FIG. 40.

The appearance in blood of the muscle enzyme creatine kinase is generally considered to be a biomarker of muscle damage, and to be particularly useful for the diagnosis of medical conditions involving muscle atrophy, including ALS. Raised creatine kinase levels are a common characteristic of ALS patients. Reduced levels of serum creatine kinase indicate relief from the disease-related myopathy.

Serum creatine kinase data from the patients revealed a statistically significant 13.3% reduction in levels from 7.15 ± 5.74 to 6.2 ± 5.08 µkat/L (p < 0.05, Wilcoxon signed-rank test) after 4 weeks of dextran sulfate treatment, indicating a drug-related reduction in muscle atrophy of the patients during treatment, see FIG. 41.

Hepatocyte growth factor (HGF) is a naturally occurring growth factor that acts as a potent neuroprotective and myogenic agent and has been shown to be useful against degenerative disease progression in numerous animal models, including in ALS models. Interestingly, Hauerslev S et al. (2014, Plos One 9:e100594) demonstrated an 18% increase in muscle mass after 2 weeks of recombinant HGF treatment in a mouse model of muscle atrophy. The observation that HGF treatment is able to induce such rapid regenerative responses in skeletal muscle in this animal model of muscle atrophy is of relevance to the rapid muscle responses observed in response to dextran sulfate in the ALS patients.

Pharmacokinetic data from the ALS patients revealed a statistically significant (p < 0.001) increase to pharmacologically relevant levels of circulating HGF from 820 ± 581 to a peak at 2.5 hours of 37863 ± 14235 µg/L after dextran sulfate injection, see FIG. 42. This indicates the potential for direct myogenic, as well as indirect neurotrophic, HGF-mediated effects on muscle atrophy after dextran sulfate administration.

The biochemical evidence of reduced muscle degeneration supports the clinical observations of improved muscle function. For example, in the ALSFRS-R, functions mediated by cervical, trunk, lumbosacral, and respiratory muscles are each assessed by 3 items and scores in these categories show close agreement with objective measures of muscle strength. Of note, two patients with severe bulbar paresis experienced almost complete resolution of this symptom during the five-week treatment period.

The improvement in muscle function as induced by dextran sulfate administration is beneficial to COVID-19 patients that, otherwise, may suffer from muscle pain and deteriorated muscle function. Hence, muscle improvement as obtained by dextran sulfate is of benefit during the recovery phase (stage IV) (FIG. 25). In addition, increased levels of HGF activated tissue repair and wound healing in the COVID-19 patients, which is of relevance during the pulmonary phase (stage II) (FIG. 25).

The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible.

Claims

1-21. (canceled)

22. A method for preventing, inhibiting or treating a coronavirus infection or infectious disease in a subject, the method comprises administering dextran sulfate, or a pharmaceutically acceptable salt thereof, having an average molecular weight equal to or below 10,000 Da to the subject to prevent, inhibit or treat a coronavirus infection or infectious disease.

23. The method according to claim 22, wherein the coronavirus infectious disease is selected from the group consisting of Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and Coronavirus disease 2019 (COVID-19).

24. The method according to claim 23, wherein the coronavirus infectious disease is COVID-19.

25. The method according to claim 22, wherein the coronavirus infection is selected from the group consisting of a coronavirus infection caused by a coronavirus selected from the group consisting of Middle East respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

26. The method according to claim 25, wherein the coronavirus infection is caused by SARS-CoV-2.

27. The method according to claim 22, wherein the average molecular weight is within a range of from 2000 to 10,000 Da.

28. The method according to claim 27, wherein the average molecular weight is within a range of from 3000 to 10,000 Da.

29. The method according to claim 28, wherein the average molecular weight is within a range of from 3500 to 9500 Da.

30. The method according to claim 29, wherein the average molecular weight is within a range of from 4500 to 7500 Da.

31. The method according to claim 30, wherein the average molecular weight is within a range of from 4500 to 5500 Da.

32. The method according to claim 22, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average sulfur content in a range of from 15 to 20%.

33. The method according to claim 22, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a number average molecular weight (Mn) as measured by nuclear magnetic resonance (NMR) spectroscopy within a range of from 1850 to 3500 Da.

34. The method according to claim 33, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a Mn as measured by NMR spectroscopy within a range of from 1850 to 2500 Da.

35. The method according to claim 34, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a Mn as measured by NMR spectroscopy within a range of from 1850 to 2300 Da.

36. The method according to claim 35, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has a Mn as measured by NMR spectroscopy within a range of from 1850 to 2000 Da.

37. The method according to claim 22, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average sulfate number per glucose unit within a range of from 2.5 to 3.0.

38. The method according to claim 37, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average sulfate number per glucose unit within a range of from 2.5 to 2.8.

39. The method according to claim 38, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has an average sulfate number per glucose unit within a range of from 2.6 to 2.7.

40. The method according to claim 22, wherein the dextran sulfate, or the pharmaceutically acceptable salt thereof, has on average 5.1 glucose units and an average sulfate number per glucose unit of 2.6 to 2.7.

41. The method according to claim 22, wherein administering dextran sulfate, or the pharmaceutically acceptable salt thereof, comprises systemically administering dextran sulfate, or the pharmaceutically acceptable salt thereof, to the subject.

42. The method according to claim 41, wherein systemically administering dextran sulfate, or the pharmaceutically acceptable salt thereof, comprises subcutaneously administering dextran sulfate, or the pharmaceutically acceptable salt thereof, to the subject.

43. The method according to claim 41, wherein systemically administering dextran sulfate, or the pharmaceutically acceptable salt thereof, comprises intravenously administering dextran sulfate, or the pharmaceutically acceptable salt thereof, to the subject.

44. The method according to claim 22, wherein administering dextran sulfate, or the pharmaceutically acceptable salt thereof, comprises administering an aqueous injection solution comprising the dextran sulfate, or the pharmaceutically acceptable salt thereof, to the subject.

45. The method according to claim 22, wherein the pharmaceutically acceptable salt thereof is a sodium salt of dextran sulfate.

46. A method for preventing, inhibiting or treating an inflammatory disease in a subject, the method comprises administering dextran sulfate, or a pharmaceutically acceptable salt thereof, having an average molecular weight equal to or below 10,000 Da to the subject to prevent, inhibit or treat the inflammatory disease, wherein the inflammatory disease is selected from the group consisting of acute respiratory distress syndrome (ARDS) and systemic inflammatory response syndrome (SIRS).

47. A method for preventing, inhibiting or treating an infection or infectious disease in a subject, the method comprises administering dextran sulfate, or a pharmaceutically acceptable salt thereof, having an average molecular weight equal to or below 10,000 Da to the subject to prevent, inhibit or treat an infection or infectious disease caused by a pathogen capable of binding to cell surface heparan sulfate proteoglycans (HSPG).

48. The method according to claim 47, wherein the pathogen is capable of binding to HSPG to facilitate initial cellular attachment and/or subsequent cellular entry.

49. The method according to claim 47, wherein the pathogen is capable of binding to the heparan sulfate part of HSPG.