Compounds for treating and preventing pathologies mediated by extracellular histones - Patent Application 20070122999

Abstract

To provide a drug for treating and preventing extracellular histone mediated ailments.SOLUTION: A drug comprises a polyanionic heptasulfated cellobioside modified with a small uncharged glycosidically linked substituent at its reducing terminus or a pharmaceutically acceptable salt thereof, the small uncharged glycosidically linked substituent being selected from the group consisting of O-(C1-6)alkyl and S-(C1-6)alkyl, and the extracellular histone mediated ailments being selected from the group consisting of systemic inflammatory response, trauma, burn, acute pancreatitis, acute kidney injury, acute respiratory distress syndrome, multiple sclerosis, systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, ulcerative colitis, Crohn's disease, and rheumatoid arthritis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compounds and methods for inhibiting the pathological activity of extracellular histones in a subject. Specifically, the present invention relates to diseases mediated by extracellular histones (e.g., sepsis). Inhibits various adverse reactions, such as inflammatory bowel disease, systemic immune response syndrome (SIRS), and ischemia-reperfusion injury (IRI) More specifically, the present invention relates to compounds, uses and methods for treating or ameliorating poly Anionic sulfated cellobiosides with small uncharged glycosides at their reducing ends Methods and uses of polyanionic sulfated cellobiosides modified with linking substituents The presence of this substituent is mediated by extracellular histones, resulting in a molecule with high chemical stability. Methods and uses that result in the molecule being effective in treating disease without affecting its ability to do so For example, the present invention relates to a method for treating various diseases mediated by extracellular histones in a subject. β-O-methyl cellobioside sulfate (mCBS) or its pharmaceutical preparations in the treatment of qi The present invention relates to methods and uses of physiologically acceptable salts (e.g., mCBS.Na). [Background technology]

[0002] Histones regulate gene expression by forming nucleosomes in complex with DNA. A small basic protein that functions in the cell nucleus to regulate the nucleosome In addition to its nuclear function, Xu et al. (Nat Med. 2009) .15:1318-21) has demonstrated the cytotoxic activity of histones released in response to inflammatory processes. This extracellular histone has been reported to be involved in endothelial cell dysfunction and organ failure in sepsis. It acts as a mediator of survival and death.

[0003] Histones are currently considered endogenous danger signals or DAMPs during their translocation from the nucleus to the extranuclear space. Histones are also recognized as important regulators of immune cells, cerebellar neurons, and It is often detected on the cell surface or in the cytoplasm of Schwann cells and microglia. , which activates Toll-like receptors and the inflammasome pathway, resulting in systemic inflammatory responses. It has been shown to cause oxidative and toxic reactions. Elevated levels of ATP are associated with multiple pathophysiological mechanisms of disease (e.g., autoimmune diseases, inflammatory diseases, and cancer). Histones are involved in the processes and progression of many human diseases, supporting the role of extracellular histones in many human diseases. There are.

[0004] In recent years, there have been many efforts to find effective new treatments for diseases mediated by extracellular histones. Several attempts have been made, including the development of monoclonal antibodies against key mediators of inflammation. Considerable efforts have been made to generate clonal antibodies, but these have been clinically ineffective. It has been shown to have dangerous side effects, especially in patients with sepsis.

[0005] Antihistone treatment (e.g., neutralizing antibodies, activated protein C, recombinant thrombomodulin and heparin) are used to treat fatal endotoxemia, sepsis, ischemia / reperfusion injury, trauma, pancreatitis, and peritonitis. has been shown to protect mice from stroke, blood clots, and thrombosis, but lack of efficacy or have limited clinical value due to unacceptable side effects. factors (e.g., activation promoters such as recombinant human APC (e.g., Xigris®) Anti-inflammatory cytokines (APCs) have had little clinical impact for several reasons. One reason is the anticoagulant activity of APC, which leads to an increased risk of bleeding. Therefore, APC drugs are not suitable for the treatment of SIRS, which can occur in post-surgical or post-traumatic patients. For similar reasons, APC-based therapies are associated with a high risk of bleeding. It is excluded from use in sepsis that occurs in patients with leukemia. The relatively slow mode of action of APC is a disadvantage, as it can cause severe side effects. As a result, Xigris® was discontinued on October 25, 2011.

[0006] Therefore, despite these efforts, diseases mediated by extracellular histones remain Although some of the most debilitating and deadly diseases present in humans, few treatments are available. Therefore, a significant clinical problem is presented.

[0007] It is applied to the treatment of conditions or diseases mediated by extracellular histones (e.g., sepsis). One class of compounds that has been used is disclosed in U.S. Pat. No. 9,226,939. This U.S. patent relates to a method for inhibiting the cytotoxic activity of extracellular histones in a subject. The invention is directed to a method which involves administering to a subject an effective amount of a polyanion. This US patent publication disclosed a variety of polyanions with very different structures. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention comes against this backdrop of increasing recognition of the role of extracellular histones in multiple diseases. It is being developed for. [Means for solving the problem]

[0009] Extracellular histones released in response to inflammatory challenges contribute to endothelial dysfunction, organ failure, and These are mediators that contribute to cell death, particularly during sepsis. The stable polyanionic compounds interact electrostatically with histones, leading to cellular alterations of these molecules. The discovery that the compound can neutralize the inflammatory, red blood cell damaging, platelet activating and procoagulant properties of erythrocytes. The interaction of such polyanionic molecules with extracellular His in the circulation of living animals is based on Complexation with histones provides a means to at least ameliorate the cytotoxic activity of extracellular histones. do.

[0010] Specifically, the present inventors have demonstrated that specific sulfated disaccharides mediate these pathological effects of histones. It has been confirmed that neutralization is effective. For example, polyanionic sulfated cellobioside Therefore, poly(A) modified at its reducing end with a small, uncharged glycosidic bond substituent. Anionic sulfated cellobiosides are involved in diseases mediated by extracellular histones (e.g., sepsis). It can also provide highly effective treatment for inflammatory bowel disease (SIRS, SIRS, and IRI) and in patients The present invention provides chemically stable polyanions that can at least ameliorate these conditions.

[0011] The present inventors have demonstrated that the compounds of the present invention are useful for the diagnosis and prognosis of diseases mediated by extracellular histones. It also confirms that it provides a method for follow-up and management.

[0012] The present invention relates to a polyanionic sulfated cellobioside having a small reducing end. Polyanionic sulfated cellobiosides modified with uncharged glycosidic bond substituents are protects endothelial cells from the cytotoxicity of extracellular histones in a histone-dependent manner and inhibits the cytotoxicity induced by histones reduce or even reverse damage (e.g., clumping and lysis of red blood cells) that occurs during treatment, and Protects against cell injury and organ dysfunction in subjects with, for example, sepsis, SIRS, and IRI This is also based on the discovery of the present inventors.

[0013] As a result, a chemically stable polyanionic sulfated cellobioside is obtained, and its reducing end The use of sulfated cellobiosides modified at the ends with small uncharged substituents allows the histone The field of treatment of patients suffering from pathologies mediated by HIV and / or patients at risk The present invention provides a novel approach to the prevention of histone-mediated pathologies in humans. The general principles of the rule are presented or provided.

[0014] In a first aspect of the present invention, a method for treating or preventing diseases mediated by extracellular histones is provided. 1. A compound for use in a pharmaceutical composition comprising a polyanionic sulfated cellobioside and its reduced end Polyanionic sulfated cesium modified at the ends with small uncharged glycosidic substituents. A compound comprising lobioside or a pharmaceutically acceptable salt thereof is provided. Small uncharged glycosidic bond present at the reducing end of polyanionic sulfated cellobiosides. Substitutions alter the chemical structure of a polyanion compared to the same polyanion that is sulfated at the reducing end. Improve stability.

[0015] The compounds of the present invention, when present in a therapeutically or pharmaceutically effective amount, inhibit extracellular His The present invention provides a means for ameliorating, treating or preventing diseases transmitted by monocytes.

[0016] In one embodiment of the invention, the modified polyanionic sulfated cellobioside is , the whole structure: [ka] where R1 is a small uncharged glycosidic bond substituent, such as O or S—(C 1~6 )a and R2-R8 are each (i) a small uncharged O-bonded substituent, or (i i) sulfate groups) It has.

[0017] Preferably, R1 is O or S—(C 1~6 ) alkyl. Preferably, R1 is Chemical stability of polyanions compared to the same polyanions bearing sulfate groups at R1 Improve.

[0018] Preferably, R2 to R8 are each (a) an unmodified hydroxyl group, or (b) a sulfonyl group. The aryl group is selected from the group consisting of aryl, ...

[0019] More preferably, R1 is a methoxy group or an ethoxy group, and R2 to R8 are each independently selected from the group consisting of methoxy, ethoxy ... Each is a sulfate group selected from O-sulfate or N-sulfate.

[0020] Desirably, this class of compounds has a high net negative charge, i.e., is polyanionic. do.

[0021] The anomeric configuration of this small uncharged glycosidic substituent (R1) can be either the α- or β-position. Preferably, the small uncharged substituent is in the β configuration.

[0022] In a highly preferred form of the invention, the compound is a sulfated β-O-methyl cellobioside dimer. Illustratively, this compound is mCBS, a β Sodium salt of -O-methyl cellobioside sulfate, i.e., sodium β-O-methyl It is cellobioside sulfate (mCBS.Na).

[0023] mCBS is much more stable than CBS and is well tolerated at high concentrations. BS has minimal anticoagulant effect and suppresses histone-induced plasma coagulation disruption. The anticoagulant activity of mCBS is 110 times lower than that of low molecular weight heparin. 750 times lower than unfractionated heparin.

[0024] In a second aspect of the present invention, a medical condition, condition or disease involving extracellular histones in a subject is detected. A method for treating (therapeutically or prophylactically) a disease comprising administering a polyanionic sulfated cellobioside a polysaccharide modified at its reducing end with a small, uncharged glycosidic bond substituent; A therapeutically effective amount or amount of anionic sulfated cellobioside or a pharmaceutically acceptable salt thereof The method includes administering to a subject a therapeutically effective amount of the polyamine esterase inhibitor. The small uncharged glycosidic bond substituents present at the reducing end of anionic sulfated cellobiosides , the chemical stability of the polyanion compared to the same polyanion that is sulfated at the reducing end. More preferably, the modified sulfated cellobioside is mCBS. or more specifically a pharmaceutically acceptable salt of mCBS (e.g., mCBS.Na). is.

[0025] For example, in certain embodiments of the second aspect of the invention, sepsis, SIRS or Treating or preventing medical conditions or diseases associated with sepsis and / or SIRS 1. A method for preventing or ameliorating atopic dermatitis, the method comprising: Polyanions modified at their reducing ends with small, uncharged glycosidic substituents a therapeutically effective amount or a pharmaceutically effective amount of a soluble sulfated cellobioside or a pharmaceutically acceptable salt thereof; Preferably, the modified The sulfated cellobioside is mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS. An acceptable salt (e.g., mCBS.Na) is

[0026] According to this embodiment of the present invention, this treatment of sepsis / SIRS results in the reduction of sepsis / SIRS. S or septic / SIRS state or related disease is resolved and secondary condition This allows the physician to administer other drugs to treat the condition.

[0027] In another embodiment of the second aspect of the present invention, a method for treating IRI or a medical condition associated with IRI in a subject is provided. A method for treating, preventing, or ameliorating a biological condition or disease, comprising administering to a subject A dianionic sulfated cellobioside with a small uncharged glycosyl group at its reducing end. Polyanionic sulfated cellobiosides modified with hydroxyl-linked substituents or pharmaceutically acceptable salts thereof and administering to a subject a therapeutically or pharmaceutically effective amount of a salt thereof. Preferably, the modified sulfated cellobioside is mCBS or or more specifically a pharmaceutically acceptable salt of mCBS (e.g., mCBS.Na). do.

[0028] In a third aspect of the present invention, there is provided a method for ameliorating extracellular histone accumulation in a subject. and polyanionic sulfated cellobiosides, which have small uncharged groups at their reducing ends. Polyanionic sulfated cellobiosides modified with glycosidic substituents or pharmaceutical compositions thereof a therapeutically or pharmaceutically effective amount of a salt thereof acceptable for Preferably, the polyanionic sulfated cellobioside at the reducing end is The small uncharged glycosidic bond substituents are the same polyanions that are sulfated at the reducing end. The modified polyanion is preferably a polyanion having improved chemical stability compared to the modified polyanion. The sulfated cellobioside is mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS. An acceptable salt (e.g., mCBS.Na) is

[0029] For example, in one embodiment of the third aspect of the invention, the method is used to detect extracellular histidine. Prevent conditions or illnesses associated with cancer-related complications (e.g., sepsis, SIRS, or IRI) do.

[0030] In certain exemplary embodiments according to the second or third aspects of the invention, the specified method comprises anti-inflammatory agents, antibiotic agents, antiviral agents, antifungal agents and / or antiviral agents if the subject is suffering from or has or any other form of pharmaceutical composition that treats one or more conditions that a patient is at risk of suffering from. a therapeutically effective amount or pharmacologically effective amount of one or more second active agents, compounds or compositions selected from and a therapeutically effective amount of the modified sulfated cellobioside, or and administering the compound to a subject concomitantly with a steroid. obtain.

[0031] According to this embodiment, the second active agent, compound or composition is a compound that associates with extracellular histones. complications (e.g., sepsis, SIRS, or IRI) and / or those associated with such complications The present invention provides adjunctive treatment to treatments directed at associated medical conditions or diseases. Alternatively, the second active agent, compound or composition comprises one or more anti-inflammatory agents.

[0032] Preferably, the second active agent is a steroid drug associated with the medical condition being treated by the compounds of the present invention. suggesting a means of medical intervention for a condition from which the patient is suffering that is different from or related to this medical condition; The second active agent provides adjunctive treatment to the patient.

[0033] In a fourth aspect of the present invention, a medical condition or disease associated with extracellular histones in a subject is provided. 2. A method for treating or preventing a disease comprising administering to a subject a polyanionic sulfated cellobioside, the reduction Polyanionic sulfates modified at their original termini with small uncharged glycosidic bond substituents. a therapeutically effective amount or a pharmaceutically effective amount of a hydroxylated cellobioside or a pharmaceutically acceptable salt thereof Preferably, the polyanionic sulfate is The small uncharged glycosidic bond substituents present at the reducing end of oxidized cellobiosides are Improves the chemical stability of the polyanion compared to the same polyanion that is sulfated. More preferably, the modified sulfated cellobioside is mCBS or a similar modified sulfated cellobioside. More specifically, it is a pharmaceutically acceptable salt of mCBS (for example, mCBS.Na).

[0034] In a preferred embodiment of the fourth aspect of the present invention, the method is used to: (i) administer a therapeutic agent to the endothelium of a subject; and / or (ii) extracellular histopathological factors that contribute to endothelial dysfunction in a subject. Additionally or alternatively, this method may be used to treat infection, inflammation, or other conditions in a subject. or extracellular hCG in a subject following hypoxia or any infectious, inflammatory or hypoxic response. The release of, or destruction caused by, or mediated by, sepsis or SIRS state, or IRI, or sepsis, SIRS, or IRI Treat associated diseases.

[0035] In a fifth aspect of the present invention, a therapeutic agent for use in the treatment of extracellular histone-related complications is provided. A composition or pharmaceutical composition comprising a polyanionic sulfated cellobioside, the reduction of which is Polyanionic sulfation modified at the termini with small uncharged glycosidic bond substituents. Therapeutic or pharmaceutical compositions containing at least cellobioside or a pharmaceutically acceptable salt thereof Preferably, the polyanionic sulfated cellobioside is present at the reducing end. The small uncharged glycosidic bond substituents are the same polyaniline that is sulfated at the reducing end. Preferably, the compound improves the chemical stability of the polyanion relative to the therapeutic The composition or pharmaceutical composition is present in a therapeutically or pharmaceutically effective amount. The product may also include a therapeutically acceptable or pharmaceutically acceptable carrier, excipient and / or diluent. The compounds in this treatment or medicament are either in neutral free base form or in salt form. Preferably, the polyanionic sulfated cellobioside compound is mCBS, or More specifically, it is the sodium salt of β-O-methyl cellobioside sulfate.

[0036] In certain exemplary embodiments of the fifth aspect of the invention, the identified composition is an anti-inflammatory antiviral, antifungal, and / or one or more of the following drugs that the subject is suffering from: One or more of any other form of therapeutic or pharmaceutical compound that treats multiple conditions The composition may also include a second active agent, compound or composition selected from:

[0037] In accordance with this embodiment, the second active agent, compound or composition desirably is a compound that inhibits sepsis, S Adjunctive treatment of IRS or IRI or medicine related to sepsis, SIRS or IRI Preferably, the second active agent, compound Alternatively, the composition may include one or more anti-inflammatory agents.

[0038] In a sixth aspect of the present invention, a method for treating a medical condition, disease or disorder involving extracellular histones is provided. A polyanionic sulfated cellobioside, the reducing end of which is used in the manufacture of a drug for Polyanionic sulfated cellulosic acid modified with small uncharged glycosidic substituents in The use of a therapeutically effective amount or a pharmaceutically effective amount of bioside or a pharmaceutically acceptable salt thereof Preferably, the polyanionic sulfated cellobioside is present at the reducing end. Small uncharged glycosidic substituents are sulfated at the reducing end of the same polyanion. More preferably, the modified polyanion has improved chemical stability compared to the modified polyanion. The sulfated cellobioside is mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS. The salt is a salt (e.g., mCBS.Na).

[0039] For example, in one embodiment of the sixth aspect of the invention, a method for treating sepsis, SIRS or other conditions in a subject is provided. or IRI or the treatment of a medical condition or disease associated with sepsis, SIRS, or IRI A polyanionic sulfated cellobioside in the manufacture of a medicament for the treatment or prevention of Polyanions modified at their reducing ends with small, uncharged glycosidic substituents a therapeutically effective amount or a pharmaceutically effective amount of a soluble sulfated cellobioside or a pharmaceutically acceptable salt thereof; Preferably, the modified sulfated cellobioside is mC mCBS, or more specifically a pharmaceutically acceptable salt of mCBS (e.g., mCBS .Na).

[0040] In one embodiment of such a use, the medicament is used to treat sepsis or SIRS in a subject. or for treating a medical condition or disease associated with sepsis or SIRS and the treatment results in the sepsis or SIRS or the combination of sepsis or SIRS. The associated condition or disease is ameliorated or inhibited.

[0041] In another embodiment of such use, the medicament is a drug for treating IRI or a condition associated with IRI in a subject. and for treating a medical condition or disease associated with said IR, said treatment resulting in said IR I or said condition or disease associated with said injury is ameliorated or inhibited.

[0042] In yet another embodiment of such use, the agent is used to (i) induce endothelial or (ii) contributes to endothelial dysfunction in a subject; or i) initiating clotting by activating the subject's platelets, or (iv) It neutralizes extracellular histones that induce red blood cell fragility and consequent anemia in red blood cells.

[0043] In yet another embodiment, the manufactured medicament is a therapeutic agent for a second active agent, compound or composition. In accordance with this embodiment, the second active agent may also include a therapeutically effective amount or a pharmaceutically effective amount. The compound or composition may be used as a supplement for treating a medical condition, disease or disorder involving extracellular histones. Desirably, the second active agent, compound or composition is an agent for treating sepsis, SIR, Adjunctive therapy for the treatment of sepsis, SIRS, or IRI or associated with sepsis, SIRS, or IRI Preferably, the second active agent provides adjunctive treatment for a medical condition or disease associated with the The agent may be an anti-inflammatory agent, an antibiotic agent, an antiviral agent, an antifungal agent, and / or a drug that is effective against one or more of the following conditions: or any other form of therapeutic or pharmaceutical compound that treats multiple conditions. More preferably, the second active agent, compound or composition is selected from one or more contains multiple anti-inflammatory agents.

[0044] In any of the methods of the present invention, a modified sulfated cellobioside compound is used. If so, can the compound be administered to a subject in need thereof in a single dose of the formulation? In certain alternative embodiments, the modified sulfonyl group may be administered or formulated for this administration. The oxidized cellobioside compounds are administered as a multi-dose formulation to a subject in need thereof. or formulated for this administration.

[0045] Additional objects, advantages and novel features are set forth in the following description or may be further illustrated in the drawings and the accompanying drawings. As will become apparent to those skilled in the art upon consideration of the detailed description of several non-limiting embodiments that follow. Deaf.

[0046] The present disclosure provides details in the following description of preferred embodiments with reference to the following drawings. [Brief explanation of the drawings]

[0047] [Figure 1]Figure 1A shows a comparison of the stability of CBS to mCBS when stored at 5±3°C (Figure 1A), 25±2°C / 60% RH (Figure 1B), and 40±2°C / 75% RH (Figure 1C), as measured by HPLC. The figure shows the percent (%) change of mCBS and CBS relative to the starting amount (T=0). [Figure 2] 1 is a graph showing the metabolic stability of mCBS in human liver microsomes, as represented by the measured concentration (μM) of mCBS (mean±SEM) in the presence of human liver microsomes under conditions permissive for phase I metabolism. [Figure 3] 1 is a graph showing the metabolic stability of mCBS in human rat microsomes, as represented by the measured concentration (μM) of mCBS (mean±SEM) in the presence of rat liver microsomes under conditions permissive for phase I metabolism. [Figure 4] 1 is a graph showing the metabolic stability of mCBS in dog liver microsomes, as represented by the measured concentration (μM) of mCBS (mean±SEM) in the presence of dog liver microsomes under conditions permissive for phase I metabolism. [Figure 5]Panels A–D are graphs showing flow cytometry output, and panels E–G are images showing confocal microscopy results demonstrating that mCBS protects human microvascular endothelial cells (HMECs) from histone damage. Cultured HMECs were treated for 60 minutes with (A and E) an equal volume of water, (B and F) 400 μg / mL histones, (C and G) 100 μg / mL mCBS + 400 μg / mL histones, or (D) 25 μg / mL mCBS + 400 μg / mL histones. They were then labeled with the dyes calcein-AM and PI and analyzed for the extent of dye uptake using flow cytometry (A, B, C, and D) or confocal microscopy (E, F, and G). Numbers in quadrants indicate the percentage of cells present in each quadrant. Viable cells take up calcein-AM (green) and exclude PI, whereas damaged and dead cells take up PI (red) and fail to retain calcein-AM. The flow cytometry-based assay used suspensions of HEMCs (panels A-D), whereas the confocal microscopy experiments used HMEC monolayers (panels E-G), which are more sensitive to histone damage. [Figure 6]

[0023] Figure 1 shows that the protective effect of mCBS against histone-induced damage to HMECs is concentration-dependent. Cultured HMECs were exposed to 400 μg / mL histones after the addition of increasing concentrations of mCBS and then analyzed for the uptake of calcein-AM (viable) or PI (dead) using flow cytometry. Dead / viable cells were expressed as % of control untreated cells. Error bars indicate SEM. [Figure 7] Graph showing that mCBS can reverse damage in a portion of HMECs exposed to histones for 1 hour. Cultured HMECs were exposed to 400 μg / ml of histones for 60 minutes, treated with mCBS (100 μg / ml) for 10 minutes, and then analyzed for PI uptake using flow cytometry. Cytotoxicity was expressed as a percentage of PI uptake by cells treated with histones (+ve control) for 60 minutes. Error bars indicate SEM. [Figure 8]Panels A–C show graphs of flow cytometry output, and panels D–F show scanning electron microscopy results demonstrating that histone-induced RBC aggregation is prevented by mCBS. Isolated human RBCs were analyzed by flow cytometry using log FSC vs. log autofluorescence (FL-1 channel) parameters or visualized using scanning electron microscopy for the extent of aggregation after (A and D) no treatment, (B and E) incubation with histones (400 μg / mL) for 60 minutes, and (C and F) treatment with histones (400 μg / mL) for 60 minutes followed by exposure to mCBS (200 μg / mL) for 10 minutes. [Figure 9] Figure 1 shows a graph demonstrating that mCBS inhibits histone-induced RBC aggregation in a dose-dependent manner. Isolated human RBCs were exposed to various concentrations of histones for 60 minutes (Panel A), and the extent of aggregation was measured by the level of autofluorescence (FL-1) as in Figure 8. Similar to (A), except that various concentrations of mCBS were added to the RBCs before the addition of 400 μg / mL of histones (Panel B). Error bars indicate SEM. Asterisks indicate significant differences from the control (no histones present), with a P value of <0001 (****). [Figure 10]Graphs show that mCBS inhibits histone-induced RBC fragility, an effect exacerbated by higher shear flow rates and duration of shear exposure. Isolated human RBCs diluted in 60% saline (normal saline:water at a ratio of 6:4) were incubated with increasing concentrations of histone for 60 minutes and then exposed to increasingly faster flow rates (mm / s) with 40 repetitions in a robotic system (Panel A), or to various repetitions of pipetting at a flow rate of 100 mm / s (Panel B), or treated with various concentrations of mCBS and then exposed to 400 μg / mL histone for 60 minutes at a shear flow rate of 100 mm / s and 40 repetitions of pipetting (Panel C). Supernatants from each sample were then measured for hemoglobin content at A540 nm as an indicator of the extent of RBC lysis. Error bars indicate SEM. Asterisks indicate significant differences from previous treatments, with P values ​​<0.5 (*), <0.01 (***) and <0.001 (****). [Figure 11] Graphs showing that mCBS reverses the susceptibility of RBCs to histone-induced lysis and aggregation. Isolated human RBCs were exposed to 400 μg / mL histone for 55 minutes, followed by various concentrations of mCBS for 5 minutes (Panel A). After applying shear force (100 mm / sec flow rate and 40 pipetting repetitions), the extent of RBC aggregation was analyzed by measuring hemoglobin in the supernatant at A540 nm and the level of autofluorescence at FL1 using flow cytometry (Panel B). Error bars indicate SEM. Asterisks indicate significant differences from the control treatment (histones only, no mCBS); all P values ​​are <0.001 (****). [Figure 12]This shows that a high level of mCBS sulfation is required for mCBS to be an effective inhibitor of histone-mediated pathology. a. Structure of di-, tri-, tetra-, and penta-sulfated mCBS preparations compared to fully sulfated (heptasulfated) mCBS. b. Inhibition curve showing that only pentasulfated mCBS weakly inhibits histone-mediated cytotoxicity on HMEC-1. c. Similar results obtained when investigating the inhibition of histone-induced erythrocyte fragility. Data are expressed as mean ± sem (n = 3). [Figure 13] These graphs show that histones induce platelet aggregation and degranulation, and these effects can be inhibited by mCBS and CBS. (Panel A) Isolated human platelets were incubated with various concentrations of histones for 1 hour, followed by analysis of aggregation by flow cytometry using FSC and SSC to distinguish between single and aggregated platelets. (Panel B) As in (A), but various concentrations of mCBS, CBS, and non-sulfated CB were added before histones (150 μg / mL). (Panel C) Human platelets in whole blood were analyzed for degranulation (ATP release) after the addition of increasing concentrations of histones using chemiluminometry; thrombin was included as a positive control. (Panel D) As in (C), except that increasing concentrations of mCBS, CBS, and non-sulfated CB were added before the addition of histones (400 μg / mL). Error bars indicate SEM. [Figure 14]Histone-mediated cytotoxicity does not require cell surface heparan sulfate. a. HMEC-1 cells in suspension were treated with either bacterial heparinases 1, 2, and 3 (derived from Flavobacterium) or human platelet heparanase. The sensitivity of untreated (histones alone) and heparinized / heparanase-treated HMEC-1 cells to histone-mediated cytotoxicity was then determined. Enzymatic removal of heparan sulfate from HMEC-1 cells did not affect the cells' sensitivity to histone-mediated cytotoxicity, and treatment had no effect on HMEC viability (Hepase alone). b. Suspensions of wild-type CHO-K1 cells and GAG-deficient pgsA-745 CHO-K1 cells were incubated with increasing concentrations of histones at 37°C for 1 hour, and dead cells were detected by flow cytometry. The absence of GAGs slightly but significantly reduced the sensitivity of cells to histone cytotoxicity at high histone concentrations. Data are expressed as mean ± sem (n = 3). *P ≤ 0.05, **P < 0.01 (two-way ANOVA with Sidak's multiple comparison test). [Figure 15] Histones disrupt lipid bilayers and induce intracellular Ca2+ flux, a process blocked by mCBS and CBS. a. Lifetime of artificial lipid bilayers exposed to histone (HIS) (1 μM) alone (n = 47) or in the presence of CBS (n = 52) (10 μM). Control bilayers (n = 125) contained the RμR1 ion channel protein. P values ​​were calculated using the nonparametric Kruskal-Wallis test. b. Representative flow cytometry plots using the Ca2+-sensitive dye Indo-1 showing Ca2+ flux in HMEC-1 cells after histone addition (100 μg ml-1). c. Time course of the effect of mCBS (100 μg ml-1) on histone-induced Ca2+ flux in HMEC-1 cells. [Figure 16]Graphs showing that histones reduce blood clotting. Using the ROTEM whole blood clotting assay (Panel A), the addition of increasing histone concentrations to human whole blood prolonged clotting times in all assays, but particularly in the NATEM and INTEM assays. (Panel B) The same anticoagulant effect of histones on clotting was demonstrated using a plasma-based clotting assay (activated partial thromboplastin time (APTT)). Error bars indicate SEM. [Figure 17] Figure 1 shows a ROTEM analysis of the effects of sulfated saccharides on whole blood clotting. (Panel A) Whole blood was supplemented with mCBS, maltotriose sulfate, or melezitose sulfate (200 μg / mL) immediately before performing the NATEM (non-activation), EXTEM (extrinsic pathway activation), INTEM (intrinsic pathway activation), and FIBTEM (extrinsic pathway activation by neutralized platelets) assays. Data show clotting times expressed as fold increases over the water control. (Panel B) Whole blood supplemented with various concentrations of mCBS, melezitose sulfate, or maltotriose sulfate was analyzed for clotting times using the NATEM assay. (Panel C) Similar to (B), except that data show clot amplitude at 20 minutes. No clotting was detected with the two trisaccharides at 50 and 100 μg / mL. Error bars indicate SEM. [Figure 18] 1 is a graph showing the anticoagulant effect of mCBS compared to heparin and the low molecular weight heparin, enoxaparin. The NATEM assay was used to measure whole blood clotting after the addition of heparin, enoxaparin, the trisaccharide maltotriose sulfate, and mCBS at the concentrations (μg / mL) shown in parentheses. [Figure 19] 1 is a graph showing that mCBS inhibits histone-induced disruption of whole blood coagulation. Using the EXTEM assay, the prolongation of clotting time induced by 400 μg / mL and 800 μg / mL histones could be inhibited by prior addition of 200 μg / mL mCBS. Addition of an equal volume of water had no inhibitory effect. Error bars indicate SEM. [Figure 20] Graphs show that mCBS and CBS protect mice from histone-induced organ damage. Mice were injected intraperitoneally with the indicated concentrations of mCBS, CBS, or nonsulfated CB (or an equivalent volume of PBS) 10 minutes before intravenous injection of 50 mg / kg histone (or an equivalent volume of PBS). Four hours later, blood was collected retroorbitally for analysis of markers of cell injury (LDH - lactate dehydrogenase), liver dysfunction (ALT - alanine aminotransferase), and kidney dysfunction (Crea - creatinine). Error bars indicate SEM. Data are expressed as mean ± SEM. *P≦0.05, **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA with Dunnett's multiple comparison test). [Figure 21] Graphs showing that mCBS attenuates or prevents histone-mediated reductions in circulating white blood cells, platelets, and red blood cells. Mice were injected intraperitoneally with 100 mg / kg mCBS (or an equivalent volume of PBS) 10 minutes before intravenous injection of 50 mg / kg histone (or an equivalent volume of PBS), followed by retro-orbital bleeding 10 minutes later. Whole blood was analyzed for white blood cell, platelet, and red blood cell counts and hemoglobin concentration using an ADVIA 2120 hematology system. Error bars indicate SEM. Asterisks indicate significant differences from the PBS+HIS control, with p values ​​of <0.01 (**) and <0.01 (***) (one-way ANOVA with Dunnett's multiple comparison test). [Figure 22]mCBS protects rats from cell damage induced by moderate sepsis. Figures 1A and 1B show the survival rates of mCBS-treated (mCBS) and untreated (control) rats in a rat model of moderate sepsis (Panel A), and demonstrate that mCBS reduces cell damage (lactate dehydrogenase (LDH)) in an animal model of moderate sepsis (Panel B). Male Wistar rats (n=8 / group) underwent laparotomy and cecal ligation and puncture (CLP) to induce fecal peritonitis and subsequent sepsis. They were then treated with saline (control CLP) or 50 mg / kg mCBS via i.p. injection at 0, 5, and 10 hours postoperatively and monitored for 20 hours. When rats reached severe morbidity, they were humanely euthanized, and deaths were recorded. As seen in panel A, rats treated with mCBS showed a 100% survival rate, and as seen in panel B, mCBS treatment significantly reduced circulating LDH levels compared with rats in the control CLP group (statistical analysis: two-tailed Student's t test, p ≤ 0.05). [Figure 23] CBS protects rats from morbidity and organ damage induced by severe sepsis. a. Survival rate of rats (n=8 / group) subjected to cecal ligation and puncture (CLP) and administered saline (control) or CBS. P values ​​were obtained by the Log rank (Mantel-Cox) test. b. Liver and kidney damage in CLP rats measured by blood levels of ALT and creatinine, respectively. [Figure 24] CBS reduces microvascular obstruction and myocardial necrosis in a cardiac ischemia-reperfusion injury model. The effects of CBS (n=6 / group) on cardiac IRI were measured by ischemic zone (IZ), microvascular obstruction (MVO), and myocardial necrosis (infarct area) in the left ventricle (LV). [Figure 25]mCBS has been shown to improve tissue flap survival after ischemia-reperfusion. Fascia flaps were excised from the abdomen of rats (n = 3-5 per group) with the vascular pedicles intact, and the feeding vessels were clamped for 10 hours and then released. mCBS (50 mg / kg) or saline was administered i.p. 5 minutes before clamp application and 5 minutes after clamp removal. Rats were monitored for a total experimental period of 72 hours, during which additional compound or saline was administered i.p. 24 and 48 hours postoperatively. Valve survival was determined at 72 hours using the extent of valve necrosis (blackened or reddened areas) in the representative photographs shown. [Figure 26] CBS prevents histone-induced deep vein thrombosis. The inferior vena cava (IVC) of mice (n = 8 / group) was ligated to approximately 10% patency. All mice were then intravenously injected with histone (10 mg / kg) or an equivalent volume of saline via the tail vein, followed 5 minutes later by intravenous injection of CBS (50 mg / kg) or saline. Mice were monitored for 48 hours, after which they were re-anesthetized and any thrombi that had developed distal to the IVC stenosis were removed for analysis. Data are presented as mean ± sem. *P ≤ 0.05. (ANOVA with Dunnett's multiple comparison test). [Figure 27] Figure 1 shows the activity of mCBS in a multiple sclerosis model. C57Bl / 6 mice were immunized with MOG35-55 / CFA / PT to induce EAE. mCBS or PBS alone (vehicle) was administered i.p. daily from days 0 to 9. Mice were monitored daily for signs of disease over a period of 17 to 35 days post-immunization. Mean clinical scores for mCBS-treated (n=36) and vehicle-treated (n=33), EAE-induced only (n=14), and untreated controls (n=32) are shown. Data are from six independent pooled experiments. Shown is the mean disease score + / - SEM. Statistical significance was determined using the Sidak-Bonferroni method. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention relates to a method for treating a disease mediated by extracellular histones in a subject (e.g., sepsis, S Modified sulfates with high chemical stability in the treatment or prevention of IRS or IRI The present invention relates to the use of hydroxylated cellobioside compounds in a subject. The cytotoxic effects of exohistones may be ameliorated, inhibited, or prevented.

[0049] For convenience, the following section provides a general overview of the various meanings of terms used herein. After this discussion, general exemplary embodiments illustrating the invention are disclosed, followed by a detailed description of the present invention. Specific examples are disclosed that provide more specific illustrations of the properties of various exemplary embodiments of the invention.

[0050] overview The invention described herein is intended to be within the spirit and scope of the invention described herein. Variations and modifications other than those specifically described are possible without departing from the scope of the present invention. Those skilled in the art will recognize that the present invention includes all such variations and modifications. The present invention also includes all of the compounds, individually or collectively, mentioned or shown in this specification. and any and all combinations of said steps or features. Functionally equivalent products, compositions of matter and processes are not expressly defined. Certainly within the scope of the invention described herein.

[0051] All publications, references cited herein, either above or below, All publications, patents and patent applications are incorporated herein by reference in their entirety, These publications, references, literature, patents and patent applications should be read as part of this text. Any publications, references, or other information cited in this text are also included. The references, publications, patents and patent applications are not repeated in the text solely for the sake of brevity. However, the publications, references, literature, patents and patent applications referred to herein are not to be construed as limiting the scope of the present invention. The protocols reported in these publications and which may be used in connection with the present invention are The terms "col" and "reagent" are used interchangeably herein to describe and disclose the compounds, reagents, and products disclosed herein. neither the present invention nor any third party is entitled to antedate such disclosure by virtue of prior invention or for any other reason. Nothing in this document should be construed as an admission that no right exists. Any statements or representations regarding the contents are based on information available to the applicants and are not intended to be limiting. This document does not constitute any admission as to the accuracy of the dates or contents of the documents.

[0052] Referenced in any document mentioned herein or incorporated by reference herein Any manufacturer's instructions, manuals, product specifications and product sheets for any product covered is incorporated herein by reference and may be used in the practice of the present invention.

[0053] Definitions of selected terms used herein are provided in the Summary of the Invention and the Detailed Description of the Invention. and apply throughout. All other scientific and technical terms used herein are understood to be within the meaning of the appended claims and are within the meaning of the appended claims. The term has the same meaning as understood in the art. In the event of an apparent discrepancy between the stated definition of this term and the The definitions set forth therein shall prevail.

[0054] definition Except in the working examples or where otherwise indicated, the ingredients or All numbers expressing quantities of reaction conditions are modified in all instances by the term "about." For example, the term "about" when used in connection with a percentage should be understood to mean ±10 It can mean %.

[0055] Unless the context otherwise requires or the specification clearly states to the contrary, the singular Any integer, step or element of the invention recited herein as an integer, step or element An element explicitly encompasses both the singular and plural of the listed integers, steps or elements. Throughout this specification, unless otherwise stated or the context otherwise requires, A reference to a step, composition or substance, group of steps or group of compositions of substance includes both one and multiple. number (i.e., one or more) of or these steps, compositions or substances, groups of steps or things Therefore, the present specification and the appended claims are intended to encompass a group of compositions of this type. When used in a range, the singular forms "a," "an," and "the" are Unless the context clearly dictates otherwise, plural references are included. Thus, for example, "sulfated cello Sulfated cellobioside modified with a small uncharged substituent at its reducing end. Reference to "a sulfate or a pharmaceutically acceptable salt thereof" includes multiple such modified sulfates. The present invention includes a compound selected from the group consisting of a hydroxylated cellobioside compound and a plurality of its salts and the like.

[0056] Throughout this specification and claims, unless the context otherwise requires, the word "including" will be used. "comprise" or "comprises" or "including" Variations such as the above may be made by changing the steps, elements, or integers or steps. implies the inclusion of a step, or element, or group of integers, but does not include any other step, or or elements or integers or groups of steps or elements or integers. It will be understood that you do not.

[0057] As used herein, the terms "including" and "included" are used interchangeably. It will be understood that variations such as "there is no limiting value" are also not limiting.

[0058] In this application, the use of "or" means "and / or" unless stated otherwise.

[0059] The invention described herein involves the use of one or more ranges of values ​​(e.g., size, displacement, A range of values ​​may include all values ​​within this range, such as the values ​​that define this range. and results that are identical or substantially identical to the values ​​immediately adjacent to the values ​​that define the boundaries of this range. It will be understood that the range includes adjacent values ​​that lead to the above-mentioned range. One may note that a 10% variation of the upper or lower limits of the range may be entirely appropriate and is encompassed by the present invention. More specifically, the variance at the upper or lower end of the range is 5% or , or whichever is greater as is commonly recognized in the art.

[0060] As used herein, the terms "condition," "illness," or "disease" (used interchangeably) ( ) refers to extracellular histone-related complications mediated by the release of extracellular histones. do.

[0061] As used herein, the phrase "extracellular histone-related complications" is particularly limited to (a) e.g., sepsis (bacterial, viral, fungal) infections such as those caused by surgery, trauma, bleeding, etc. (including sepsis induced by parasites, parasites, and prions) , systemic inflammatory responses to non-infectious triggers, including burns, acute pancreatitis, and acute kidney injury, (b ) for example, after arterial blockage due to atherosclerosis, spontaneous rupture of a blood vessel, or traumatic injury to a blood vessel Hypoxia or drowning at the local tissue level, including cardiac and transplant-related IRI following respiratory arrest due to death, gas exposure or cardiopulmonary arrest, e.g., acute respiratory distress syndrome; (c) hypoxia at the systemic level, including diseases such as chronic obstructive pulmonary disease and drug-mediated tissue injury; Hemostasis or vascular obstruction, such as cardiovascular disease or chronic cardiovascular disease, such as atherosclerosis, clotting and (d) autoimmune and inflammatory disease states, e.g., Multiple sclerosis, hyper-inflammatory disease states, systemic lupus erythematosus, spondyloarthropathy, ankylosing spine inflammation, psoriatic arthritis, reactive arthritis, enteropathic arthritis, ulcerative colitis, Crohn's disease, irritable bowel The disease, rheumatoid arthritis, or juvenile rheumatoid arthritis, is characterized by destruction and inflammation of small blood vessels. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), e.g., ventriculitis with polyangiitis eosinophilic granulomatosis, polyangiitis, and microscopic polyangiitis), Familial Mediterranean Fever, amyotrophic lateral sclerosis, Sjogren's syndrome, early arthritis, viral arthritis, psoriasis, Age-related organ fibrosis, idiopathic pulmonary fibrosis, juvenile diabetes (type 1), diabetes (type 2), anti-inflammatory drugs It refers to phospholipid syndromes and various central nervous system diseases such as Huntington's disease.

[0062] As used herein, the term "sepsis" has the meaning given in standard medical references. Any of the septic diseases or conditions that have been characterized in the literature and / or are known to those skilled in the art. For example, sepsis includes stages of severe sepsis, acute and chronic sepsis, and septicemia. The term "sepsis" as used herein refers to an infection-related illness. The term "SIRS" (systemic immune response syndrome) as used herein refers to: Non-infection-related episodes (e.g., trauma, burns, pancreatitis, organ transplant, surgery, cancer treatment) These include post-radiotherapy tumor lysis, perinatal complications, and prevention of allograft immunosuppression.

[0063] As used herein, the term "medical condition associated with sepsis or SIRS" or "Sepsis or SIRS-related illness" means any illness or condition that is not classified as a sepsis or SIRS-related illness in the context of standard medical practice. Sepsis or SIRS diseases characterized in the literature and / or known to those skilled in the art directly or indirectly related to or resulting from any or all stages of a disease or condition including all signs and symptoms caused by or associated with For example, medical conditions or diseases associated with sepsis or SIRS, with or without infection Any disease or condition of sepsis or SIRS in a subject that may manifest in the subject. or associated with, resulting from, caused by, or in any stage may be accompanied by one or more of the following signs or symptoms: arterial hypotension, metabolic Cirrhosis, decreased systemic vascular resistance, increased heart rate (tachycardia), increased respiratory rate (tachypnea), overall Inflammation or systemic inflammation, elevated white blood cell count or decreased white blood cell count (leukocytosis or leukopenia) minor), increased extracellular histones in the blood, organ dysfunction, e.g., acute organ dysfunction, circulatory System dysfunction, multiple organ dysfunction syndrome, disseminated intravascular coagulation (DIC), one or more organs fibrin deposition in the microvasculature of the kidneys, fever, confusion, pneumonia, cough with pneumonia, kidney infection, kidney Painful urination and / or septic shock with infection.

[0064] As used herein, the terms "reduce," "reduced," "reduction," and "decrease" " or "inhibit" are all used generally to mean a decrease by a statistically significant amount. However, for the avoidance of doubt, "reduced," "reduce," or "decrease" Or "inhibit" means, for example, at least 10% compared to baseline levels in the absence of the agent. means a reduction of, for example, at least about 20%, or at least about 30%, or at least 40%, or at least 50%, or at least about 60%, or at least about 70%, or means a reduction of at least about 80%.

[0065] As used herein, the terms "improve," "increase," "increase" or "Enhance" or "activate" are both generally understood to mean an increase by a statistically significant amount. and for the avoidance of any doubt, the terms "improve", "increase" and "increase" "Enhance" or "enhance" or "activate" refers to, for example, the reduction of a baseline level in the absence of an agent. This means an increase of at least 10% compared to the previous level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%), or or at least about 60%, or at least about 70%, or at least about 80% Increase or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or or at least about a 5-fold, or at least about a 10-fold increase or increase compared to baseline levels It means any increase between 2-fold and 10-fold or more.

[0066] As used herein, the terms "administer," "administered," and "administering" mean and at least partially localizing the composition at the desired site so that the desired effect occurs. The term "administration" refers to the placement of the composition into a subject by any method or route that allows administration of the composition to a subject. The compounds or compositions may be administered by any suitable route known in the art, Examples of routes of administration include, but are not limited to, oral or parenteral routes, such as intravenous administration. , intramuscular administration, subcutaneous administration, transdermal administration, respiratory tract (aerosol) administration, pulmonary administration, nasal administration, rectal administration These include, but are not limited to, topical (e.g., buccal and sublingual) administration and topical (e.g., buccal and sublingual) administration. In certain embodiments, the compound is a polyanionic sulfated cellobioside, the reduction Polyanionic sulfates modified at their original termini with small uncharged glycosidic bond substituents. The polyanionic surfactant is a hydroxylated cellobioside or a pharmaceutically acceptable salt thereof. The small uncharged glycosidic bond substituents present at the reducing end of sulfated cellobiosides are Improved chemical stability of polyanions compared to the same polyanions that are sulfated at the ends. When the compound is present in a composition such as a therapeutic composition, the compound The product may be formulated for parenteral administration or another method that allows delivery to the target site. Some exemplary modes of administration include injection, infusion, infusion, inhalation, or Examples of methods include, but are not limited to, oral ingestion.

[0067] As used herein, the term "injection" includes, but is not limited to: Intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, Tracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal and intrasternal injections and In a preferred embodiment, the composition is administered by intravenous infusion or injection.

[0068] As used herein, any of the conditions or diseases listed herein. Insofar as this is the case, in the context of the present invention, the terms "treat," "treatment," "treating," and the like will be used. The condition or disease is characterized by the progression, worsening, or halting of at least one symptom or complication associated with such condition or disease. aggravation, deterioration, progression, expected Relieve, alleviate, ameliorate, inhibit, or slow the progression or severity of In one embodiment, the symptoms of a condition or disease are , at least 5%, at least 10%, at least 20%, at least 30%, at least are also reduced by 40% or at least 50%.

[0069] As used herein, the phrases "effective amount," "therapeutically effective amount," or "effective dose" ( (used interchangeably herein) in these senses refer to the compounds or compositions of the invention. The compound contains a sufficient, but non-toxic, amount of the compound required to produce the desired effect. The exact amount of the substance or composition will depend on the desired effect, the species to be treated, the age and general condition of the subject, the The efficacy and safety of the drug will vary from subject to subject depending on factors such as the severity of the condition, the drug being administered, the mode of administration, and the like. Therefore, it is not possible to specify an exact "effective amount." For any given case, an appropriate effective amount (dosage) can be determined using only routine experimentation. Generally, a therapeutically effective amount will depend on the subject's medical history, age, condition, sex, and and the severity and type of the subject's medical condition and the administration of other pharmaceutically active agents. obtain.

[0070] As used herein, reference to the use of compounds and compositions in therapeutic or medicinal applications. It is understood that the present invention is applicable to human and non-human (e.g., veterinary) uses alike. Therefore, unless otherwise indicated, the terms "patient," "subject," or "individual" will be used interchangeably. Reference to a "human body" (used interchangeably herein) refers to a human or non-human (e.g., social is understood to mean any individual of any species of national, economic or research importance; Such individuals include, but are not limited to: mammals, birds, lagomorphs, and rhesus. More preferably, the patient is a mammal, such as a rhesus, bovine, equine, porcine, feline, canine, primate, or rodent. The subject or individual is an animal belonging to a mammalian species. The mammalian species is preferably a human. or non-human primates, or companion animals (e.g., domesticated dogs, cat, horse, monkey, mouse, rat, rabbit, sheep, goat, cow or pig) In one particularly preferred example, the patient, subject or individual is a human.

[0071] Definitions of selected terms used herein may be found in the Detailed Description of the Invention. and applies throughout. All other classes used herein unless otherwise defined. Scientific and technical terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It has.

[0072] Exemplary Embodiments of the Invention The following detailed description of the invention is included for the purpose of illustrating the invention only and is not intended to be limiting unless otherwise specified. should in no way be construed as a limitation on the broad description of the invention as set forth herein. stomach.

[0073] 1. Compounds of the Present Invention In a first aspect of the present invention, a method for the treatment of complications mediated by extracellular histones is provided. The compound for the purpose of the present invention is a polyanionic sulfated cellobioside having a reducing end thereof Polyanionic sulfated cellobioside modified with small uncharged glycosidic substituents The present invention provides a compound comprising a substituted or unsubstituted hydroxyl group or a pharmaceutically acceptable salt thereof. Although it is a polyanion, it has a higher molecular weight than polyanions that are sulfated at the reducing end. Preferably, compounds of this class have a high net negative charge. It should be.

[0074] The compounds of the present invention may be used to treat complications mediated by extracellular histones (e.g., sepsis or ischemia). blood reperfusion injury) as a preventative measure (i.e., as a prophylactic pre-treatment for a medical procedure) or to treat this condition or It can be ameliorated both therapeutically and during treatment after the disease has developed.

[0075] In one embodiment of the invention, the modified polyanionic sulfated cellobioside is , the whole structure: [ka] where R1 is a small uncharged glycosidic bond substituent, such as O or S—(C 1~ 6) alkyl, and R2-R8 are each (i) a small uncharged O-bonded substituent, or is selected from (ii) sulfate groups) It has.

[0076] Preferably, R1 is O or S—(C 1~6 ) alkyl. Preferably, R1 is Chemical stability of polyanions compared to the same polyanions bearing sulfate groups at R1 Improve.

[0077] Preferably, R2 to R8 are each (a) an unmodified hydroxyl group, or (b) a sulfonyl group. The aryl group is selected from the group consisting of aryl, ...

[0078] More preferably, R1 is a methoxy group or an ethoxy group, and R2 to R8 are each independently selected from the group consisting of methoxy, ethoxy ... Each is a sulfate group selected from O-sulfate or N-sulfate.

[0079] Desirably, this class of compounds has a high net negative charge, i.e., is polyanionic. do.

[0080] The anomeric configuration of this small uncharged glycosidic substituent (R1) can be either the α- or β-position. Preferably, the small uncharged substituent is in the β configuration.

[0081] In a highly preferred form of the invention, the compound is a sulfated β-O-methyl cellobioside dimer. β-O-methyl cellobioside sulfate, a sugar, or a pharmaceutically acceptable salt thereof For illustrative purposes, this compound is the sodium salt of β-O-methyl cellobioside sulfate. is.

[0082] mCBS is much more stable than CBS and is well tolerated at high doses. BS has minimal anticoagulant effect and disrupts plasma coagulation induced by histones The anticoagulant activity of mCBS is 110 times lower than that of low molecular weight heparin, 750 times lower than fractionated heparin.

[0083] Small uncharged glycosidic bonds present at the reducing end of polyanionic sulfated cellobiosides. The substituents alter the chemistry of the polyanion compared to the same polyanion that is sulfated at the reducing end. Improves stability.

[0084] Chemical stability, as used herein, refers to the resistance of the compounds of the present invention to change, particularly to their natural state. Decomposition in the natural environment or when exposed to air, heat, light, pressure or other natural conditions represents the tendency to resist decomposition due to internal reactions.

[0085] The compounds of the present invention have a shelf life of at least one month under the conditions of expected use or under normal environmental conditions. After storage, there is no significant degradation compared to the same polyanion that is sulfated at the reducing end. In some cases, it is "stable."

[0086] The compounds of the present invention have a shelf life of at least one month under the conditions of expected use or under normal environmental conditions. If three or more sulfate groups are lost after storage, significant degradation may have occurred. Preferably, the compounds of the present invention have at least If both of them lose two sulfate groups after one month of storage, they are significantly degraded. Most preferably, the compounds of the present invention will be resistant to the expected conditions of use or normal environmental conditions. Loss of one sulfate group after storage at room temperature for at least one month indicates significant degradation. It will have been solved.

[0087] Preferably, the compounds of the present invention are stored in a phosphate formulation buffered at pH 7.5. And when stored at 2-8℃, it can be used for at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months Month, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd It is chemically stable for 24 months or more. More preferably, it is buffered to pH 7.5. For compounds stored in phosphate formulations and stored at approximately 2-8°C, the shelf life is 6 months to 2 years. Stability is measured over a period of time.

[0088] As used herein, the phrase "pharmaceutically acceptable salt" means a salt that is pharmaceutically acceptable within the scope of sound medical judgment. and in humans within the limits of their intended use without undue toxicity, irritation, allergic reactions, and the like. Suitable for use in contact with tissues of animals and lower animals, and with a reasonable benefit / risk ratio Contains salt.

[0089] Pharmaceutically acceptable salts are well known in the art. These salts include, for example, salts of inorganic acids. (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., citric acid, acetic acid, oxalic acid, tartaric acid Acid addition salts (formed with free amino groups of proteins) derived from acids, mandelic acid, and the like Similarly, salts formed with the free carboxyl groups of proteins are also , inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine It can be derived from various compounds (e.g., benzocaine, procaine, and the like).

[0090] In a preferred embodiment of the present invention, the modified sulfated cellobiosides of the present invention are used pharmaceutically. It exists as an acceptable salt. Illustratively, this compound is β-O-methyl cellobioside. It is the sodium salt of sulfate, i.e., sodium β-O-methylcellobioside sulfate (mCBS.Na).

[0091] The modified sulfated cellobioside compounds or The pharmaceutically acceptable salts thereof can be prepared by methods known to those skilled in the art. The present invention provides a method for preparing sulfated compounds modified at their reducing ends with uncharged substituents. Methods for preparing the Katrin C Probes are described in detail in the Katrin C Probes references, which are incorporated herein by reference in their entirety. t and Hans Peter Wessel,2001,J.Carbohydr and is generally described in Chemistry, 20(7&8):549-560. .

[0092] 2. Treatment method The compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are useful for the treatment of extracellular histone The present invention provides a method for the treatment of a pulmonary arthritis, which can ameliorate or prevent the pathological activity of a protein of the present invention. In a second embodiment, there is provided a method for treating or preventing an extracellular histone-related complication, comprising administering to a subject a polyaniline-containing compound. ionic sulfated cellobiosides with a small uncharged glycosidic bond at their reducing ends Polyanionic sulfated cellobiosides modified with substituents or pharmaceutically acceptable salts thereof administering to a subject a therapeutically or pharmaceutically effective amount of a salt thereof. Preferably, the small uncharged residues present at the reducing end of the polyanionic sulfated cellobioside are Glycosidic bond substituents are more likely to form polyanions than the same polyanions that are sulfated at the reducing end. Improves the chemical stability of the anion. More preferably, the modified sulfated cellobiose The oside is mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS (e.g., For example, mCBS.Na).

[0093] Furthermore, in a third aspect of the present invention, there is provided a method for ameliorating extracellular histone accumulation in a subject. A method for producing a polyanionic sulfated cellobioside having a small carboxyl group at its reducing end. Polyanionic sulfated cellobiosides or their derivatives modified with uncharged glycosidic bond substituents and administering to a subject a therapeutically effective amount or a pharmaceutically effective amount of a pharmaceutically acceptable salt of Preferably, the reducing end of the polyanionic sulfated cellobioside is The small uncharged glycosidic bond substituents present in the hydroxyl group are the same as those sulfated at the reducing end. This improves the chemical stability of the polyanion compared to the lyanion. The sulfated cellobiosides that are being studied are mCBS or, more specifically, drugs for mCBS. and physiologically acceptable salts (e.g., mCBS.Na).

[0094] The present invention as illustrated by the second and third aspects of the invention provides a method for the release and and the resulting extracellular toxicity caused by various different extracellular histones. According to these aspects of the invention, The method may be used to treat or prevent extracellular histone-related complications in subjects with: (a) For example, sepsis (induced by bacteria, viruses, fungi, parasites, prions) infections such as sepsis (including sepsis caused by HIV infection) or surgery, trauma, bleeding, burns, acute pancreatitis, and acute kidney injury (b) systemic inflammatory responses to non-infectious triggers, such as atherosclerosis, vascular damage, IR following rupture, arterial occlusion due to traumatic injury to the blood vessel, and in relation to the heart and transplant Hypoxia at the local tissue level, including I, or due to, for example, drowning, gas exposure, or cardiopulmonary arrest respiratory arrest and for example, acute respiratory distress syndrome, chronic obstructive pulmonary disease and drug-mediated tissue injury (c) hemostasis or vascular obstruction, e.g., cardiovascular disease; or chronic cardiovascular diseases, such as atherosclerosis, blood clotting and thrombosis (e.g., deep vein thrombosis) (d) autoimmune and inflammatory disease states, e.g., multiple sclerosis, hyper-inflammatory disease states; Systemic lupus erythematosus, spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, intestinal Inflammatory arthritis, ulcerative colitis, Crohn's disease, irritable bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis Antineutrophil cytoplasmic antibody (ANCA)-associated disease characterized by thrombosis, destruction of small blood vessels, and inflammation. Arterial vasculitis (AAV), including granulomatosis with polyangiitis, polyangiitis, and microscopic polyangiitis Eosinophilic granulomatosis with vasculitis, familial Mediterranean fever, amyotrophic lateral sclerosis, Sjögren's disease Syndrome, early arthritis, viral arthritis, psoriasis, age-related organ fibrosis, idiopathic pulmonary fibrosis Diabetes mellitus, juvenile diabetes (type 1), diabetes (type 2), antiphospholipid syndrome and various central nervous system disorders Systemic diseases, such as Huntington's disease.

[0095] In certain embodiments of the second or third aspects of the invention, the respective methods comprise administering to a subject a compound of the invention and administering to a subject suffering from or at risk of suffering from a disease, simultaneously with or in conjunction with the compound. and a second therapeutic agent (e.g., a compound of the present invention) different from the compound of the present invention, which provides adjunctive treatment for a clinical condition. , anti-inflammatory agents, antibiotics, antivirals, antifungals or other forms of medical intervention) The method may further comprise administering to

[0096] Preferably, as an example of these aspects of the invention, each method comprises determining whether or not a patient has an adverse reaction to a drug in a subject. sepsis or SIRS or a medical condition or disease associated with sepsis or SIRS As another example of these aspects of the invention, the respective methods provide a means for treating or preventing The Act provides for the treatment or prevention of an IRI or a medical condition or disease associated with an IRI in a subject. Provide a means to do so.

[0097] Preferably, this method does not require the physician to administer other drugs to treat secondary conditions. The present invention provides a method for treating a condition or disease state by administering a therapeutically effective amount of the active ingredient to a patient, the method comprising: Polyanionic sulfated cesium ions were used to ameliorate extracellular histone-related complications in patients. Lobioside, modified at its reducing end with a small, uncharged glycosidic bond substituent. Therapeutically effective polyanionic sulfated cellobiosides or pharmaceutically acceptable salts thereof This also includes administering a sufficient amount to a subject.

[0098] In certain exemplary embodiments according to the second or third aspects of the invention, the specified method comprises anti-inflammatory agents, antibiotic agents, antiviral agents, antifungal agents and / or antiviral agents if the subject is suffering from or has or any other form of pharmaceutical composition that treats one or more conditions that a patient is at risk of suffering from. a therapeutically effective amount or pharmacologically effective amount of one or more second active agents, compounds or compositions selected from and a therapeutically effective amount of the modified sulfated cellobioside, or The method may further comprise administering to the subject concomitantly with oside.

[0099] According to this embodiment, the second active agent, compound or composition is an extracellular histone-associated protein. Complications (e.g., sepsis, SIRS, or IRI) and / or any other conditions associated with such complications Preferably, the method provides adjunctive treatment to treatments directed at a specific medical condition or disease. The second active agent, compound or composition comprises one or more anti-inflammatory agents.

[0100] Preferably, the second active agent is a steroid drug associated with the medical condition being treated by the compounds of the present invention. suggesting a means of medical intervention for a condition from which the patient is suffering that is different from or related to this medical condition; The second active agent provides adjunctive treatment to the patient.

[0101] The therapeutic and / or pharmaceutical compositions of the invention disclosed herein may be used for therapeutic or For therapeutic use, the compounds and compositions may be administered to treat extracellular histone-related complications. or to treat patients already suffering from diseases associated with histone-related complications. The compound or composition is administered in an amount sufficient to terminate or at least partially halt the in a single dose or as part of a treatment regime (e.g., a multi-dose treatment regime). The active compound should be provided in an amount sufficient to effectively treat the patient. In use, the compounds and compositions of the present invention can be used to treat diseases associated with extracellular histone-related complications. At least partially arresting the symptoms and / or complications of this disease in subjects at risk of developing it. Administer in an amount sufficient to stop

[0102] In a fourth aspect of the present invention, a method for detecting a histone-mediated pathology in a subject is provided. 1. A method for treating or preventing a medical condition, disease or disorder caused by a polyanionic sulfated Cellobiosides modified at their reducing ends with small, uncharged glycosidic bond substituents Therapeutic use of polyanionic sulfated cellobiosides or pharmaceutically acceptable salts thereof is In one embodiment, a method is provided comprising administering to a subject an effective or pharmaceutically effective amount of a compound of formula (I) or (II) to a subject.

[0103] Preferably, the sulfated cellobioside is β-O-methyl cellobioside sulfate ( mCBS) or a pharmaceutically acceptable salt thereof.

[0104] In one preferred example, the method is used to determine whether a cytotoxic agent (i) is cytotoxic to the endothelium of a subject; or (ii) contribute to endothelial dysfunction in a subject; or (iii) activate platelets in a subject. or (iv) increasing red blood cell fragility and Treating extracellular histones induces the resulting anemia.

[0105] Highly preferred aspects of the present invention according to any aspect, embodiment or example described herein In one exemplary embodiment, the compounds of the present invention are used to treat one or more of the diseases or conditions discussed below. Treat or prevent

[0106] A. Sepsis Sepsis (including septic shock) is a condition characterized by arterial hypotension, metabolic acidosis, and systemic A systemic response to infection characterized by decreased vascular resistance, tachypnea, and organ dysfunction. Sepsis (including septic shock) is caused by cytokine networks, leukocytes, and complement This is associated with and results from the activation of many host defense mechanisms, including the clotting and fibrinolytic systems. It is also a systemic inflammatory response to infection, mediated by the Disseminated intravascular coagulation (DIC), which is the widespread deposition of ATP, may be an early symptom of sepsis. IC is an important mediator in the development of multiple organ dysfunction syndrome and septic shock This contributes to the poor prognosis of patients with glaucoma.

[0107] The immunological response that causes sepsis is a global response that results in widespread activation of inflammatory and coagulation pathways. This is a systemic inflammatory response that can progress to cardiovascular dysfunction, even in the most optimal cases. Even with treatment, it can lead to multiple organ dysfunction syndrome and ultimately death.

[0108] Symptoms of sepsis are often related to the underlying infectious process and, if left untreated, and severe sepsis (sepsis accompanied by acute organ dysfunction) or septic shock (refractory heart disease). may manifest as sepsis with pulsed hypotension. Criteria for systemic inflammatory response syndrome (e.g. For example, generalized inflammation, fever, elevated white blood cell count (leukocytosis), and elevated heart rate (tachycardia). If two or more of the following criteria are met without evidence of infection: They may simply be diagnosed with "SIRS," a septic inflammatory condition that affects the body.

[0109] Many patients with sepsis show rapid decline over 24 to 48 hours. Rapid treatment is essential for treatment. Unfortunately, diagnosing the type of infection can take several days. Microbiological analysis is required to identify the pathogens that may be present. Treatment to eliminate the pathogen (e.g., antibiotic treatment) is crucial without knowing the type and species of the pathogen. The present invention is based on the idea that the virus must be transmitted to humans in a state where there is no way to know the extent of infection. This provides a method for

[0110] Patients with sepsis have elevated levels of extracellular histones in their blood. This protein has been suggested as a key mediator in the pathology of sepsis.

[0111] In one embodiment of the second, third or fourth aspect of the invention, the cytotoxic activity of extracellular histones is and (prophylactically or therapeutically) preventing extracellular histone-associated sepsis in a subject by inhibiting the activity of the extracellular histone-associated sepsis. A method for treating (potentially) a polyanionic sulfated cellobioside, the reduced end thereof Polyanionic sulfated cesium modified at the ends with small uncharged glycosidic substituents. A therapeutically effective amount or a pharmaceutically effective amount of lobioside or a pharmaceutically acceptable salt thereof is administered to the subject. Preferably, the polyanionic sulfated cellulose is The small uncharged glycosidic bond substituent present at the reducing end of lobioside is sulfated at the reducing end. Improves the chemical stability of the polyanion compared to the same polyanion in a modified form. Preferably, the modified sulfated cellobioside is mCBS or more specifically Specifically, it is a pharmaceutically acceptable salt of mCBS (eg, mCBS.Na).

[0112] As demonstrated herein, the compounds of the present invention (particularly mCBS) inhibit the activity of extracellular histopathological These compounds block the toxic effects of steroids and are therefore useful as treatments for sepsis.

[0113] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are Treatment of sepsis, including both pre-treatment (in the case of therapeutic treatment) and treatment after sepsis has developed The present invention provides a means for ameliorating the cytotoxic activity of extracellular histone proteins in tumors.

[0114] B. Non-infectious SIRS Non-infectious systemic inflammatory response syndrome (SIRS) is an inflammatory condition that affects the entire body. Non-infectious SIRS is the body's response to a non-infectious insult. The definition of SIRS is: Although non-infectious SIRS is referred to as an "inflammatory" response, it is actually an inflammation-induced It has anti-inflammatory and anti-inflammatory properties.

[0115] SIRS is a serious condition associated with systemic inflammation, organ dysfunction, and organ failure. IRS is a subset of cytokine storm, characterized by abnormal regulation of various cytokines. SIRS is also closely related to sepsis, in which the patient is infected with S. Meets IRS criteria and has suspected or proven infection. Examples of causes of chronic SIRS include trauma, surgery, traumatic hemorrhage, burns, and acute pancreatitis. These include:

[0116] In one embodiment of the second, third or fourth aspect of the invention, the cytotoxic activity of extracellular histones is and (prevent) extracellular histone-associated non-infectious SIRS in a subject by inhibiting its activity. 1. A method of treating (topical or therapeutic) a cancer cellulosic acid deficiency virus (CA) comprising administering to a patient a polyanionic sulfated cellobioside, Polyanions modified at their reducing ends with small, uncharged glycosidic substituents a therapeutically effective amount or a pharmaceutically effective amount of a soluble sulfated cellobioside or a pharmaceutically acceptable salt thereof; Preferably, the polyanion The small uncharged glycosidic bond substituents present at the reducing end of the cyclic sulfated cellobiosides are Improved chemical stability of polyanions compared to the same polyanions that are terminally sulfated More preferably, the modified sulfated cellobioside is mCBS or or more specifically a pharmaceutically acceptable salt of mCBS (e.g., mCBS.Na). .

[0117] As demonstrated herein, the compounds of the present invention (particularly mCBS) inhibit the activity of extracellular histopathological These compounds block the toxic effects of steroids and are therefore useful as treatments for non-infectious SIRS.

[0118] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are This includes both pre-treatment (in the case of therapeutic treatment) and treatment after non-infectious SIRS has developed. A means to ameliorate the cytotoxic activity of extracellular histone proteins in non-infectious SIRS provide.

[0119] B.1 Trauma Physical trauma is severe, disfiguring bodily injury (e.g., shattered or amputated limbs) is.

[0120] Blunt force trauma is caused by impact or other force applied from or by a blunt object Penetrating trauma is a type of physical injury in which an object penetrates the skin or tissue. Trauma can also be described as both unplanned, such as in an accident, or planned, as in the case of surgery. Both can be characterized by mild to severe tissue damage, bleeding and / or shock, and both can also cause SIRS, but also significantly increase the risk of subsequent infection and sepsis. do.

[0121] Histones are released after trauma or severe cellular stress in the absence of infection. For example, serum histone levels are significantly elevated after severe non-thoracic blunt trauma. Stone levels are positively correlated with severe complications, incidence and poor prognosis. Exogenous histones are involved in the regulation of various cytokines (e.g., TNF-α, IL-6, and IL-1). 0), stimulates the release of myeloperoxidase, and activates immune cells and Increases calcium influx in endothelial cells and endothelial cells, thereby inhibiting histone-induced In vivo, histone administration has been shown to be effective in animal models of trauma. In this context, it also promotes cytokine release, endothelial injury, coagulation activation and lung injury.

[0122] Patients who have undergone trauma may have elevated levels of extracellular histones present in their blood. This protein has been suggested as an important mediator of trauma pathology. .

[0123] In one embodiment of the second, third or fourth aspect of the invention, the cytotoxic activity of extracellular histones is By inhibiting the activity of extracellular histone-associated trauma in a subject (either prophylactically or therapeutically), A method for treating a polyanionic sulfated cellobioside, the reducing end of which is Polyanionic sulfated cellulosic acid modified with small uncharged glycosidic substituents in A therapeutically effective amount or a pharmaceutically effective amount of bioside or a pharmaceutically acceptable salt thereof is administered. Preferably, the polyanionic sulfated cellophosphate is The small uncharged glycosidic bond substituent at the reducing end of the bioside is sulfated at the reducing end. Improve the chemical stability of the polyanion compared to the same polyanion used. Preferably, the modified sulfated cellobioside is mCBS or more specifically Typically, it is a pharmaceutically acceptable salt of mCBS (e.g., mCBS.Na).

[0124] Compounds such as mCBS can block the toxic effects of extracellular histones, thus potentially contributing to the development of inflammatory bowel disease in trauma patients. It is useful as a treatment for

[0125] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are in trauma, including both pre-treatment (in the case of medical treatment) and treatment after the traumatic injury has occurred. The present invention provides a means for ameliorating the cytotoxic activity of extracellular histone proteins in tumors.

[0126] B.2.Surgery Surgery is performed to help improve the function or appearance of the body or sometimes for some other reason. For the purpose of conducting surgical procedures on patients to investigate and / or treat pathological conditions such as disease or injury, The present invention relates to the use of surgical or instrumental techniques resulting from surgery, as further defined below. It can deal with trauma.

[0127] In one embodiment of the second, third or fourth aspect of the invention, the cytotoxic activity of extracellular histones is By inhibiting the activity of extracellular histones, surgical trauma in a subject can be prevented or prevented. A method of treating (therapeutically) a polyanionic sulfated cellobioside, Polyanionic sulfates modified at their original termini with small uncharged glycosidic bond substituents. a therapeutically effective amount or a pharmaceutically effective amount of a hydroxylated cellobioside or a pharmaceutically acceptable salt thereof Preferably, the polyanionic sulfate is The small uncharged glycosidic bond substituents present at the reducing end of oxidized cellobiosides are Improves the chemical stability of the polyanion compared to the same polyanion that is sulfated. More preferably, the modified sulfated cellobioside is mCBS or a similar modified sulfated cellobioside. More specifically, it is a pharmaceutically acceptable salt of mCBS (for example, mCBS.Na).

[0128] Generally, a procedure is considered surgery when it involves removing tissue from the patient or suturing pre-existing wounds. Other procedures that do not necessarily fall into this category (e.g., angioplasty or endoscopy) examination) as well as general surgical procedures or surgical settings (e.g., sterile environment, anesthesia, aseptic conditions, typical (use of surgical instruments and sutures or staples) may be considered surgery. All forms of surgery are considered invasive procedures, but so-called non-invasive surgery is usually ablation that does not penetrate the target structure (e.g., laser ablation of the cornea) or radiosurgery Refers to a treatment (e.g., irradiation of a tumor).

[0129] The compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are useful for the treatment of extracellular histone The present invention is useful for the treatment of cancer, as it can ameliorate the cytotoxic activity of proteins (in the case of medical treatment). For use in surgical trauma, including both pre-treatment (pre-treatment) and treatment after the surgical injury has occurred. Provide treatment.

[0130] B.3. Traumatic bleeding Traumatic hemorrhage accounts for the majority of widespread injuries worldwide and is the cause of most deaths. Despite differences in prehospital care, traumatic injuries Acute management of bleeding is similar worldwide and follows well-accepted published guidelines. Care of the seriously injured patient involves four major phases: resuscitation, surgery, and critical care. Diagnosis and control of bleeding are integral parts of trauma care. This should be a high priority at all stages, especially in patients with hemorrhagic shock. Initial attempts to control bleeding should be made by applying direct pressure, pressure bandages, or tourniquets to prevent bleeding. Direct control of potentially severe bleeding sources; stabilization of long bone and pelvic fractures; and keeping patients warm. The resuscitation phase includes warmed intravenous fluids, hypotensive resuscitation before surgical control of bleeding, and During the surgical phase, bleeding and any other injuries are prevented. Finally, in the critical care phase, postoperative management is performed. support and tissue perfusion are provided).

[0131] The compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are useful for the treatment of extracellular histone The present invention is useful for the treatment of cancer, as it can ameliorate the cytotoxic activity of proteins (in the case of medical treatment). for use in traumatic hemorrhage, including both pre-treatment (pre-treatment) and treatment after traumatic hemorrhage has occurred. Provide treatment.

[0132] B.4. Burns A burn is an injury caused by heat, cold, electricity, chemicals, friction or radiation. First-degree burns are usually limited to redness (erythema), white spots, and mild pain at the site of the injury. These burns usually extend only into the epidermis. Second-degree burns are more likely to be filled with clear fluid. This can cause superficial blisters on the skin and may be more or less painful depending on the level of nerve involvement. Second-degree burns involve the epidermis (papillary dermis) but also the deep dermis (reticular dermis). Third-degree burns involve additional charring of the skin, resulting in a hard, leathery eschar. An eschar is a scab that separates from the unaffected parts of the body. In some cases, purple fluid is also present. This type of burn occurs when nerve endings are destroyed in the burned area. Because the bones are destroyed, they are often painless. If the smoke is too strong, it can cause death and burns to the lungs (e.g., from smoke inhalation). Any symptom is a medical emergency.

[0133] Burns that damage tissues underneath the skin (e.g., muscle or bone) are classified as fourth-degree burns. This burn can be further divided into three stages: 4th degree burns resulting in irreparable muscle loss, 5th degree burns resulting in irreparable muscle loss and bone Sixth degree burns with charring.

[0134] Various types of thermal injury lead to elevated extracellular histone levels, which are consequently associated with toxicity. To the extent that this toxicity is caused, at least in part, by the extracellular action of histones, The present invention aims to reduce this toxicity by using the pharmaceutical compositions of the present invention, thereby reducing patient discomfort. The aim is to reduce or alleviate the symptoms and allow for higher dose treatment.

[0135] Patients suffering from burns have elevated levels of extracellular histones in their blood. This protein has been suggested as an important mediator of burn pathology. do.

[0136] In one embodiment of the second or third aspect of the invention, the A method for ameliorating histone-induced cytotoxicity, comprising administering a polyanionic sulfate A modified cellobioside at its reducing end with a small, uncharged glycosidic bond substituent. Therapeutic use of modified polyanionic sulfated cellobiosides or pharmaceutically acceptable salts thereof Methods are provided that include administering to a subject an effective or pharmaceutically effective amount. Alternatively, the small uncharged glycols present at the reducing end of this polyanionic sulfated cellobioside may be involved. Cosidic bond substituents are more readily available in polyanions than in the same polyanion that is sulfated at the reducing end. More preferably, the modified sulfated cellobiosins are used to improve the chemical stability of the cellobiosins. The compound is mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS (e.g., , mCBS.Na).

[0137] Compounds such as mCBS can block the toxic effects of extracellular histones, thus potentially contributing to the development of cancer in burn patients. It is useful as a treatment for

[0138] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are useful for treating various conditions including: The present invention provides a means for ameliorating the cytotoxic activity of extracellular histone proteins in thermal burns.

[0139] B.5. Acute pancreatitis Acute pancreatitis is a pancreatic inflammation caused by sterile inflammation and acinar cell death (e.g., necrosis and apoptosis). It is characterized by rapid onset of inflammation. In this regard, extracellular Histone Hsp1 expression in activated immune cells is T-mediated HMGB1 release is suppressed in HMGB1 pancreatic conditional knockout mice Loss of HMGB1 in the pancreas causes l-arginine-induced acute pancreatitis in rats. This leads to widespread nuclear damage and increased histone release into the circulation after cell death. Stone recruits macrophages, promoting their activation and further HMBG1 release. Brings out.

[0140] Depending on its severity, acute pancreatitis can have serious complications and can be difficult to treat. In mild cases, conservative treatment or laparoscopy may be effective. Treatment is often successful, but in severe cases, invasive surgery ( Often multiple interventions are required.

[0141] Patients with acute pancreatitis have elevated levels of extracellular histones in the blood. This protein may be an important mediator of the pathogenesis of acute pancreatitis. It has been done.

[0142] In one embodiment of the second, third or fourth aspect of the invention, the cytotoxic activity of extracellular histones is and inhibiting extracellular histone activity to prevent or treat extracellular histone-associated acute pancreatitis in a subject. A method of treating (therapeutically) a polyanionic sulfated cellobioside, the reduction of which comprises Polyanionic sulfation modified at the termini with small uncharged glycosidic bond substituents. A therapeutically effective amount or a pharmaceutically effective amount of cellobioside or a pharmaceutically acceptable salt thereof is administered. Preferably, the polyanionic sulfated The small uncharged glycosidic bond substituents present at the reducing end of cellobiosides are sulfur-containing at the reducing end. Improves the chemical stability of the polyanion compared to the same polyanion that has been oxidized. More preferably, the modified sulfated cellobioside is mCBS or more preferably mCBS. Specifically, it is a pharmaceutically acceptable salt of mCBS (eg, mCBS.Na).

[0143] Compounds such as mCBS may block the toxic effects of extracellular histones, thus preventing acute pancreatitis. It is useful as a treatment in patients.

[0144] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are acute pancreatitis, including both pre-treatment (in the case of therapeutic treatment) and treatment after acute pancreatitis has developed; The present invention provides a means for ameliorating the cytotoxic activity of extracellular histone proteins in bacteria.

[0145] C. Ischemia-reperfusion injury Ischemia-reperfusion injury (e.g., transplant-related ischemia-reperfusion injury) and drug-mediated tissue injury are mediated by microbial This results in sterile inflammation, a process that occurs in the absence of

[0146] Ischemia is a condition in which the blood supply to tissues is restricted and the oxygen necessary for cellular metabolism is deprived. During prolonged ischemia (more than 60 minutes), hypoxanthine is produced as a breakdown product of ATP metabolism. The enzyme xanthine dehydrogenase is formed as a result of the higher availability of oxygen. This oxidation converts molecular oxygen into the more reactive xanthine oxidase Xanthine oxidase is converted into superoxide and hydroxyl radicals, which are more potent than xanthine oxidase. The enzyme also produces uric acid, which acts as a pro-oxidant and a scavenger of reactive species such as peroxynitrite. Excess nitric oxide produced during reperfusion can act as both a scavenger and a scavenger. It reacts with peroxide to produce the powerful reactive species peroxynitrite. Radicals and reactive oxygen species attack cell membrane lipids, proteins, and glycosaminoglycans. These radicals and reactive oxygen species cause further damage through redox signaling. They may also initiate certain biological processes.

[0147] Reperfusion injury refers to damage that is due in part to the inflammatory response of the injured tissue. White blood cells carried to this area by the fluid release a host of inflammatory factors, including interleukins. They also release free radicals in response to tissue damage. The cells are reintroduced into the body, damaging the cells' proteins, DNA, and plasma membranes. Damage to the membrane can lead to the release of additional free radicals. act indirectly through redox signaling to activate apoptosis. It accumulates in small capillaries, blocking them and causing more ischemia.

[0148] Reperfusion injury also participates in the cerebral ischemic cascade involved in stroke and brain trauma. Repeated bouts of vascular and reperfusion injury also contribute to the formation and maintenance of chronic wounds such as pressure ulcers and diabetic foot ulcers. It is believed to be a factor in poor healing. Continuous pressure restricts blood supply and This causes ischemia, which then leads to inflammation during reperfusion. As this process is repeated, The tissue is damaged sufficiently to effectively create a wound.

[0149] Serum histone levels were measured in an animal ischemia-reperfusion model with liver, kidney, lung, and brain injury. was significantly elevated in the IL-14 receptor agonist, indicating an important role for histones in regulating sterile inflammation. Indeed, circulating histones are involved in several liver injury models (e.g., liver injury models). Canavalin A-induced liver injury, acetaminophen-induced hepatotoxicity, liver I / R and acute liver failure In all cases, it is a major mediator of animal mortality.

[0150] Once released, histones bind to Toll-like receptors such as TLR2, TLR4, and TLR927. It selectively binds to TLRs and inhibits the expression of pro-inflammatory cytokines (e.g., TNF-α and I). L-6), which results in enhanced inflammatory responses and tissue injury.

[0151] Extracellular histones contribute to acute kidney injury not only in the liver but also through direct toxic or pro-inflammatory effects. or ischemic stroke. Similarly, signaling mediated by TLR2 and TLR4 signaling pathways (e.g., MyD88, NF-κb, and mitogen-activated protein kinase MAP kinase (MAPK) contributes to acute kidney injury mediated by extracellular histones.

[0152] Histone injection increases cerebral infarct size and worsens stroke outcomes. Serum H3 and H4 levels are significantly elevated in bronchoalveolar lavage fluid from animal models or patients with ALI. is increasing.

[0153] In summary, extracellular histones function as DAMPs, mediating sterile inflammation and organ damage. Inhibition of histone release and activity represents a therapeutic strategy for tissue injury.

[0154] Susceptible to ischemia-reperfusion injury (e.g., transplant-related ischemia-reperfusion injury) and drug-mediated tissue injury Patients with this condition have elevated levels of extracellular histones in their blood, and these proteins Quality of life has been suggested as an important mediator in the pathology of ischemia / reperfusion and drug-mediated tissue injury. There are.

[0155] In one embodiment of the second, third or fourth aspect of the invention, the cytotoxic activity of extracellular histones is inhibiting the activity of extracellular histone-associated IRI and / or drug-mediated IRI in a subject A method for treating (prophylactically or therapeutically) tissue injury, comprising administering to a subject a polyanionic sulfated cellobiose. glycoside modified at its reducing end with a small, uncharged glycosidic bond substituent a therapeutically effective amount of a polyanionic sulfated cellobioside or a pharmaceutically acceptable salt thereof Alternatively, a pharmaceutically effective amount of the compound is administered to a subject. The small uncharged glycosidic bond at the reducing end of this polyanionic sulfated cellobioside The sulphated substituents increase the cation of the polyanion compared to the same polyanion that is sulphated at the reducing end. More preferably, the modified sulfated cellobioside is m mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS (e.g., mCB S.Na).

[0156] Compounds such as mCBS may block the toxic effects of extracellular histones, thus preventing ischemia / relapse. It is useful as a treatment in patients with perfusion and drug-mediated tissue injury.

[0157] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are Pretreatment (in the case of therapeutic treatment) and treatment after ischemia / reperfusion and drug-mediated tissue injury have occurred Extracellular histone proteins in ischemia / reperfusion and drug-mediated tissue injury, including both This provides a means of ameliorating the cytotoxic activity of .

[0158] D. Blood clotting and thrombosis Blood coagulation is the biological process by which blood forms a clot. bleeding (haemorrhage) or obstructive coagulation (thrombosis) The administration of histones in mice has been shown to prevent abnormal blood clotting, which increases the risk of clotting. This increases microvascular thrombosis accompanied by loss of vascular barrier, which in turn affects multiple organ systems. contribute to disability and failure.

[0159] Histones (e.g., H1, H2A, H2B, H3, and H4) are expressed in vivo and in vitro. In vitro, it induces platelet aggregation and subsequent platelet-dependent thrombin formation. Among them, H4 has the strongest effect on platelet activity. It also induces a procoagulant phenotype, which enhances thrombin generation. TLR2 and TLR4 mediate signal transduction pathways (e.g., activation of ATP (e.g., ERK, Akt, p38, and NF-κB), induction of calcium influx, and It is involved in histone-mediated platelet activation through the mobilization of fibrinogen.

[0160] Histone-DNA complexes enhance thrombin generation, a process that is initiated by the APC. Heparin and albumin destroy histones in vitro and in vivo. In addition, histone injections neutralized platelet toxicity and histone-associated platelet activation. Plasma levels of von Willebrand factor are elevated in mice, leading to platelet activation and its In addition to platelets, histones also contribute to the subsequent development of deep vein thrombosis. Impairs the thrombomodulin system. Exogenous histones are It increases plasma thrombin generation in a dose-dependent manner. Recombinant thrombomodulin (rTM), approved for the treatment of patients with histopathic intravascular coagulation, It directly binds to histones and protects mice from lethal thrombosis. The protective effect of rTM on inflammatory bowel disease is mediated by both APC-dependent and APC-independent mechanisms. will be done.

[0161] Patients suffering from blood coagulation and / or thrombosis caused by extracellular histones are Increased levels of extracellular histones are present in the blood, and these proteins are involved in blood clotting and / or has been suggested as an important mediator of the pathology of thrombosis.

[0162] In one embodiment of the second or third aspect of the invention, the cytotoxic activity of extracellular histones is inhibited. 1. A method for treating blood coagulation and thrombosis in a subject by inhibiting a polyanion A soluble sulfated cellobioside with a small uncharged glycosidic bond substitution at its reducing end. A polyanionic sulfated cellobioside modified with a group or a pharmaceutically acceptable salt thereof Methods are provided that include administering to a subject a therapeutically effective amount or a pharmaceutically effective amount of Preferably, the small uncharged amino acid at the reducing end of the polyanionic sulfated cellobioside is The charged glycosidic bond substituents are more soluble in the polyanion than the same polyanion sulfated at the reducing end. The modified sulfated cellophane improves the chemical stability of the cellophane. The bioside is mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS (e.g., For example, mCBS.Na).

[0163] Compounds such as mCBS can block the toxic effects of extracellular histones, thereby preventing blood clotting and Or it is useful as a treatment in patients with thrombosis.

[0164] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are and treatment after clotting or thrombosis has occurred. Extracellular histone proteins in blood coagulation and / or thrombosis This provides a means of ameliorating the cytotoxic activity of agonist proteins.

[0165] E. Autoimmune / inflammatory diseases The present invention relates to the treatment of multiple sclerosis, spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, Enteropathic arthritis, ulcerative colitis, Crohn's disease, irritable bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis Urine, familial Mediterranean fever, amyotrophic lateral sclerosis, Sjogren's syndrome, early arthritis, virus The treatment of various autoimmune and / or inflammatory disease states such as rheumatoid arthritis or psoriasis is contemplated. The diagnosis and treatment of these diseases is well documented in the literature.

[0166] Histones are involved in diseases such as rheumatoid arthritis, systemic lupus erythematosus, small vessel vasculitis, and transfusion-associated diseases. It is involved in several autoimmune and autoinflammatory diseases. In addition to acting as direct autoantigens, extracellular histones also act as histone-DNA complexes. This structure can prevent DNA degradation, thereby enhancing the autoimmune response. Protein arginine deaminases (e.g., PDA4) mediate histone deimination and citrullin synthesis. It mediates histone silencing, resulting in increased immunogenicity of histones.

[0167] Patients suffering from autoimmune and / or inflammatory diseases have been shown to have elevated levels of extracellular histopathological features present in the blood. The levels of ATP are elevated, and this protein is involved in the pathology of autoimmune and / or inflammatory diseases. has been suggested as an important mediator.

[0168] In one embodiment of the second or third aspect of the invention, the cytotoxic activity of extracellular histones is inhibited. 1. A method of treating an autoimmune and / or inflammatory disease in a subject by inhibiting , a polyanionic sulfated cellobioside containing a small uncharged glycol at its reducing end. Polyanionic sulfated cellobiosides modified with cosidic bond substituents or their pharmaceutical uses A method comprising the step of administering to a subject a therapeutically effective amount or a pharmaceutically effective amount of an acceptable salt. Preferably, the polyanionic sulfated cellobioside is present at the reducing end. The small uncharged glycosidic bond substituents are the same polyaniline that is sulfated at the reducing end. The modified polyanion is preferably a polyanion having improved chemical stability compared to the polyanion. The sulfated cellobioside is mCBS or, more specifically, a pharmaceutical equivalent of mCBS. Acceptable salts (e.g., mCBS.Na).

[0169] Compounds such as mCBS may block the toxic effects of extracellular histones, thus preventing autoimmunity. and / or are useful as treatments in patients with inflammatory diseases.

[0170] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are Pre-treatment (in the case of therapeutic treatment) and treatment after autoimmune and / or inflammatory disease has developed Cytotoxicity of extracellular histone proteins in autoimmune and / or inflammatory diseases, including both It provides a means for ameliorating sexual activity.

[0171] F. Acute respiratory distress syndrome Also known as respiratory distress syndrome (RDS) or adult respiratory distress syndrome (as opposed to IRDS). Acute respiratory distress syndrome (ARDS), as it is commonly known, is a severe response to various forms of lung injury. This is the most important disorder that leads to increased permeability pulmonary edema.

[0172] ARDS is caused by a variety of direct and indirect insults. Inflammatory mediators often cause inflammation, hypoxemia, and multiple organ failure. It is characterized by inflammation of the lung parenchyma leading to impaired gas exchange with systemic release of steroids. It is life-threatening and often fatal, usually requiring mechanical ventilation and admission to an intensive care unit. Less severe forms include acute lung injury (e.g., transfusion-associated acute lung injury (TRALI)). It is called ALI.

[0173] ARDS can develop within 24 to 48 hours of an injury or acute illness. In such cases, patients typically present with shortness of breath, tachypnea, and other symptoms related to the underlying cause (i.e., shock). Long-term illnesses such as malaria can also trigger ARDS. ARDS may then develop, particularly some time after the onset of acute infection.

[0174] Patients with ARDS have elevated levels of extracellular histones in their blood. This protein may be an important mediator of the pathology of ARDS. It has been done.

[0175] In one embodiment of the second, third or fourth aspect of the invention, the cytotoxic activity of extracellular histones is By inhibiting the activity of extracellular histones, the present invention aims to prevent (prevent) extracellular histone-associated acute respiratory distress syndrome in subjects. 1. A method of treating (prophylactically or therapeutically) a cancer cellulosic acid deficiency, comprising administering to a patient a polyanionic sulfated cellobioside, , a polyaniline modified at its reducing end with a small, uncharged glycosidic bond substituent. A therapeutically effective amount or a pharmaceutically effective amount of a carboxylic acid sulfated cellobioside or a pharmaceutically acceptable salt thereof. The method includes administering to a subject an effective amount of the polyanion. The small uncharged glycosidic bond substituents present at the reducing end of cyclic sulfated cellobiosides are Improved chemical stability of polyanions compared to the same polyanions that are sulfated at their original ends. More preferably, the modified sulfated cellobioside is mCBS or or more specifically a pharmaceutically acceptable salt of mCBS (e.g., mCBS.Na). do.

[0176] Compounds such as mCBS may block the toxic effects of extracellular histones, thus preventing ARDS. It is useful as a treatment for patients with

[0177] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are ARDS, including both pre-treatment (in the case of treatment) and treatment after ARDS has developed The present invention provides a means for ameliorating the cytotoxic activity of extracellular histone proteins in bacteria.

[0178] G. Cardiovascular disease Cardiovascular disease refers to a class of diseases that involve the heart or blood vessels (arteries and veins). The term technically refers to any disease affecting the cardiovascular system, but is usually used to refer to atherosclerosis. These conditions are often referred to as those related to arterial disease. The treatment is similar.

[0179] Treatment of cardiovascular disease depends on the specific form of the disease in each patient, but effective treatments include: , always including the preventative lifestyle changes discussed above. Blood pressure lowering medications, aspirin and Medications such as statins may be helpful. In some circumstances, surgery or warrants reopening, repairing, or replacing damaged blood vessels with angioplasty. It may be converted into

[0180] Various forms of cardiovascular disease include: aneurysms, angina, arrhythmias, atherosclerosis Atherosclerosis, cardiomyopathy, cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valvular disease, coronary artery Disease, dilated cardiomyopathy, diastolic dysfunction, endocarditis, high blood pressure ssure) (hypertension), hypertrophic cardiomyopathy, mitral valve prolapse, cardiac Muscle infarction and venous thromboembolism.

[0181] Patients suffering from histone-related cardiovascular diseases have a low level of extracellular histones present in the blood. levels are elevated, and this protein is an important player in the pathology of histone-related cardiovascular diseases. It has been suggested as a dieter.

[0182] In one embodiment of the second, third or fourth aspect of the invention, the cytotoxic activity of extracellular histones is By inhibiting the activity of extracellular histones, cardiovascular disease associated with extracellular histones can be prevented or prevented in a subject. A method of treating (therapeutically) a polyanionic sulfated cellobioside, Polyanionic sulfates modified at their original termini with small uncharged glycosidic bond substituents. a therapeutically effective amount or a pharmaceutically effective amount of a hydroxylated cellobioside or a pharmaceutically acceptable salt thereof Preferably, the polyanionic sulfate is The small uncharged glycosidic bond substituents present at the reducing end of oxidized cellobiosides are Improves the chemical stability of the polyanion compared to the same polyanion that is sulfated. More preferably, the modified sulfated cellobioside is mCBS or a similar modified sulfated cellobioside. More specifically, it is a pharmaceutically acceptable salt of mCBS (for example, mCBS.Na).

[0183] Compounds such as mCBS can block the toxic effects of extracellular histones, thus inhibiting the production of histones. It is useful as a treatment in patients with related cardiovascular diseases.

[0184] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are Both pre-treatment (in the case of therapeutic treatment) and treatment after histone-related cardiovascular disease has developed The cytotoxic activity of extracellular histone proteins in histone-related cardiovascular diseases, including Provide a means of remission.

[0185] H. Retinal detachment Retinal detachment is an eye disorder in which the neural layer of the retina separates from the retinal pigment epithelium. It is usually caused by a retinal rupture or tear. In patients with retinal detachment, normal The vitreous concentration of histones is higher compared to the eye.

[0186] Extracellular histones are toxic and TLR4 / MAPK (ERK1 / 2 and p38) dependent Induces IL-8 production in vivo and in vitro via the vitreous hyaluronic acid pathway. reduces histone-mediated toxicity by inhibiting histone diffusion Therefore, histones released from the dying retina may act as DAMPs and promote inflammation. It is possible that these compounds induce the release of cytokines and mediate cytotoxicity.

[0187] Patients with retinal detachment have elevated levels of extracellular histones in their blood. This protein has been suggested as an important mediator of the pathology of retinal detachment.

[0188] In one embodiment of the second or third aspect of the invention, the cytotoxic activity of extracellular histones is inhibited. 1. A method for treating retinal detachment in a subject by inhibiting the action of a polyanionic sulfate A modified cellobioside at its reducing end with a small, uncharged glycosidic bond substituent. Therapeutic use of modified polyanionic sulfated cellobiosides or pharmaceutically acceptable salts thereof Methods are provided that include administering to a subject an effective or pharmaceutically effective amount. Alternatively, the small uncharged glycols present at the reducing end of this polyanionic sulfated cellobioside may be involved. Cosidic bond substituents are more readily available in polyanions than in the same polyanion that is sulfated at the reducing end. More preferably, the modified sulfated cellobiosins are used to improve the chemical stability of the cellobiosins. The compound is mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS (e.g., , mCBS.Na).

[0189] Compounds such as mCBS may block the toxic effects of extracellular histones, thus preventing retinal detachment. It is useful as a treatment in patients.

[0190] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are Retinal detachment, including both pre-treatment (in the case of therapeutic treatment) and treatment after retinal detachment has occurred The present invention provides a means for ameliorating the cytotoxic activity of extracellular histone proteins in bacteria.

[0191] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are useful for the treatment of burns. A means for ameliorating the cytotoxic activity of extracellular histone proteins in a subject is provided.

[0192] I. Fibrosis Patients with fibrosis have elevated levels of extracellular histones in their blood. This protein has been implicated as a key mediator of fibrotic pathology.

[0193] In certain embodiments of the second or third aspect of the invention, the method comprises administering to a subject a method for treating histone-induced cytotoxicity in a subject. 20. A method for ameliorating fibrosis caused by a polyanionic sulfated cellobiose. glycoside modified at its reducing end with a small, uncharged glycosidic bond substituent a therapeutically effective amount of a polyanionic sulfated cellobioside or a pharmaceutically acceptable salt thereof Alternatively, a pharmaceutically effective amount of the compound is administered to a subject. The small uncharged glycosidic bond at the reducing end of this polyanionic sulfated cellobioside The sulphated substituents increase the cation of the polyanion compared to the same polyanion that is sulphated at the reducing end. More preferably, the modified sulfated cellobioside is m mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS (e.g., mCB S.Na).

[0194] Compounds such as mCBS may block the toxic effects of extracellular histones, thus reducing the risk of fibrosis. It is useful as a treatment for patients with

[0195] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are and treatment of the subject's fibrosis, including both pre-treatment (in the case of therapeutic treatment) and treatment after fibrosis has developed. The present invention provides a means for ameliorating the cytotoxic activity of extracellular histone proteins in rhesus macular degeneration.

[0196] J. Diabetes Patients with diabetes have elevated levels of extracellular histones in their blood. This protein has been suggested as an important mediator of diabetic pathology. are.

[0197] The present invention provides a method for treating histone-related complications in diabetes (e.g., inflammation and delayed wound healing). The present invention provides a method for treating type 1 diabetes, type 1.5 diabetes, or type 2 diabetes. administering to a subject being diagnosed a therapeutically effective amount of at least one compound of the present invention. Includes:

[0198] In one embodiment of the second or third aspect of the invention, there is provided a method for ameliorating diabetes in a subject. A method for producing a polyanionic sulfated cellobioside having a small carboxyl group at its reducing end. Polyanionic sulfated cellobiosides or their derivatives modified with uncharged glycosidic bond substituents administering to a subject a therapeutically effective amount or a pharmaceutically effective amount of a pharmaceutically acceptable salt of Preferably, the method comprises reducing the polyanionic sulfated cellobioside. The small, uncharged glycosidic bond substituents at the terminal end are the same as those sulfated at the reducing end. Improve the chemical stability of the polyanion compared to the polyanion of The modified sulfated cellobioside is mCBS, or more specifically mCBS and a pharmaceutically acceptable salt thereof (e.g., mCBS.Na).

[0199] Compounds such as mCBS may block the toxic effects of extracellular histones, thereby potentially reducing the risk of diabetes It is useful as a treatment for patients with

[0200] In certain embodiments, the diabetic symptom in which histones are involved is inflammation. Progression can be monitored by physical examination and the presence of reduced inflammatory markers.

[0201] In certain embodiments, the method of treating diabetes comprises administering to said patient a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, ... The present invention provides a method for treating a rheumatoid arthritis, comprising administering a therapeutically effective amount of a drug to treat the rheumatoid arthritis and at least one compound of the present invention. The drug used to treat diabetes can be insulin or It may be another drug selected from the following: biguanides, metformin (Glucopharmaceuticals), ge), liquid metformin (Riomet), sustained-release metformin (Glucophage XR, Fortamet, Glumetza), sulfonylureas, glimepiride (Am aryl), glyburide (Diabeta, Micronase), glipizide (Glu cotrol, Glucotrol XL), micronized glyburide (Glynase), Meglitinides, repaglinide (Prandin), D-F Phenylalanine derivatives, nateglinide (Starlix), thiazolidinediones, pyoglycol Ritazon (TZDs), pioglitazone, (Actos), DPP-4 inhibitors, sitagliptin saxagliptin (Onglyza), linagliptin (T radjenta), alpha-glucosidase, acarbose (Precose), mi Glytol (Glyset), bile acid sequestrants, colesevelam (Welchol), pioglitazone Ritazon and metformin (Actoplus Met), glyburide and metformin (Glucovance), glipizide and metformin (Metaglip), Sitag Liptin and metformin (Janumet), saxagliptin and metformin (k ombiglyze), repaglinide and metformin (Prandimet), and Oglitazone and glimepiride (Duetact).

[0202] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are useful for treating various conditions including: provides a means to ameliorate the cytotoxic activity of extracellular histone proteins in diabetes Therefore, the present invention provides a method for pre-treatment (in the case of medical treatment) and treatment after diabetes has developed. The present invention provides a treatment for diabetes in a subject, the treatment comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, ...

[0203] K. Toxicity of Chemotherapy, Radiation Therapy, and Cytokine Therapy Various forms of cancer treatment (e.g., chemotherapy, radiation, and cytokines) have been shown to cause cancer death in cancer patients. This toxicity is at least partly due to the extracellular action of histones. To the extent that toxicity is caused by the steroid hormone, the present invention provides a method for reducing this toxicity using the pharmaceutical compositions of the present invention. , thereby reducing or alleviating patient discomfort and allowing for higher dose treatment. Let's say.

[0204] suffer from the side effects of various forms of cancer treatment (e.g., chemotherapy, radiation, and cytokine therapy); Patients with this condition may have elevated levels of extracellular histones present in their blood. This protein has been suggested as an important mediator of this side effect.

[0205] In one embodiment of the second or third aspect of the invention, the cytotoxic activity of extracellular histones is inhibited. By damaging the immune system, various forms of cancer treatment (e.g., chemotherapy, radiation, and chemotherapy) can be prevented in a subject. A method for ameliorating side effects of cytokine therapy, comprising administering a polyanionic sulfated cellobioside modified at its reducing end with a small, uncharged glycosidic bond substituent a therapeutically effective amount of a polyanionic sulfated cellobioside or a pharmaceutically acceptable salt thereof; A method is provided comprising administering to a subject a pharmaceutically effective amount of Small uncharged glycosidic bond present at the reducing end of polyanionic sulfated cellobiosides. Substitutions alter the chemical structure of a polyanion compared to the same polyanion that is sulfated at the reducing end. More preferably, the modified sulfated cellobioside is mCB S, or more specifically a pharmaceutically acceptable salt of mCBS (e.g., mCBS. Na).

[0206] Compounds such as mCBS can block the toxic effects of extracellular histones, thus inhibiting their proliferation in various forms. It is useful as a treatment for side effects of certain cancer therapies (e.g., chemotherapy, radiation, and cytokine therapy). It is useful.

[0207] In a highly preferred form of the invention, various forms of cancer treatment (e.g., chemotherapy, radiation, and for the treatment of side effects of such therapy in patients receiving steroid therapy (e.g., steroid therapy and cytokine therapy). The compound to be treated is the compound β-O-methyl cellobioside sulfate or a pharmaceutically acceptable salt thereof. For example, the compound used in this method is sodium β-O-methylsulfonyl ether. It is lobioside sulfate.

[0208] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are Pre-treatment (in the case of cancer treatment) and various forms of cancer treatment (e.g., chemotherapy, radiation and The clinical significance of these treatments, including both treatments after the initial administration of cytokine therapy, has been unclear. A means for ameliorating the cytotoxic activity of extracellular histone proteins is provided.

[0209] L. Wound healing Also provided are methods for use in wound healing. In this case, "wound healing" refers to the complex process by which skin (or another organ-tissue) repairs itself after injury. The classical model of wound healing involves three or four successive (and more importantly, The stages are: (1) hemostasis (when a blood clot forms), (2) inflammation, and (3) (4) proliferation, and (5) remodeling. When the skin is injured, a complex series of processes take place to repair the damage. A tightly orchestrated cascade of biochemical events occurs. During the inflammatory phase, leukocytes induce cell proliferation. Bacteria and cell debris are phagocytosed and removed from the wound. The ATP (stored in ATP granules) is released into the wound and induces cell migration and division during the proliferation phase. The proliferative phase involves angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction. New blood vessels are formed, fibroblasts grow, collagen and fibrone The excretion of steroids leads to the formation of a new provisional extracellular matrix (ECM). Occasionally, re-epithelialization of the epidermis occurs, with epithelial cells proliferating and "crawling" over the wound bed, forming new tissue. Generate a cover of

[0210] Patients who suffer from wound healing difficulties have lower levels of extracellular histones in their blood. This protein may be an important mediator of wound healing pathology. It has been suggested that:

[0211] In one embodiment of the second or third aspect of the invention, the histones generated during wound healing in a subject are 1. A method for ameliorating induced cytotoxicity, comprising: Polyanions modified at their reducing ends with small, uncharged glycosidic substituents a therapeutically effective amount or a pharmaceutically effective amount of a soluble sulfated cellobioside or a pharmaceutically acceptable salt thereof; Preferably, the polyanion The small uncharged glycosidic bond substituents present at the reducing end of the cyclic sulfated cellobiosides are Improved chemical stability of polyanions compared to the same polyanions that are terminally sulfated More preferably, the modified sulfated cellobioside is mCBS or or more specifically a pharmaceutically acceptable salt of mCBS (e.g., mCBS.Na). .

[0212] Compounds such as mCBS may block the toxic effects of extracellular histones, thus promoting wound healing. It is useful as a treatment for patients who suffer from difficulty in swallowing.

[0213] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are useful for treating various conditions including: US201301226333A1 - Provides a means of ameliorating the cytotoxic activity of extracellular histone proteins in the treatment of human wounds - Google Patents do.

[0214] M. Histones in Central Nervous System Disease Patients suffering from central nervous system diseases have elevated levels of extracellular histones in their blood. This protein may be an important mediator of the pathology of central nervous system diseases. For example, Huntington's disease is caused by polyglutamine repeat expansions. It is an autosomal dominant neurodegenerative disorder caused by a polyclonal antibody in the Huntington protein. The polyglutamine track is elongated. Evidence suggests that transcriptional deregulation mediated by histone modifications is a key pathogenesis in Huntington's disease. Indicates that this is the pathological mechanism.

[0215] Pharmacological manipulation of histone deacetylase activity has been shown to improve the treatment of central nervous system diseases (e.g., Huntington's disease). It has been shown to be beneficial in various experimental models of neurodegenerative disorders (e.g., Alzheimer's disease, epilepsy, and Alzheimer's disease). Cell death, inflammatory responses, and reactive gliosis are markers of major neurological diseases. Evidence suggests that extracellular histone H1 is a neurotoxic, pro-inflammatory factor and a toxic agent in the central nervous system. These findings suggest that histone modifications and extracellular histone suggest that both contribute to central nervous system disease.

[0216] In one embodiment of the second or third aspect of the invention, a histone-induced A method for ameliorating a central nervous system disorder caused by a polyanionic sulfated cellobioside. and a polysaccharide modified at its reducing end with a small, uncharged glycosidic substituent. A therapeutically effective amount or pharmaceutical composition of an anionic sulfated cellobioside or a pharmaceutically acceptable salt thereof. The method includes administering to a subject a therapeutically effective amount of the polyclonal antibody. Small uncharged glycosidic bond substituents present at the reducing end of anionic sulfated cellobiosides. increases the chemical stability of the polyanion compared to the same polyanion that is sulfated at the reducing end. More preferably, the modified sulfated cellobioside is mCBS. or more specifically, a pharmaceutically acceptable salt of mCBS (e.g., mCBS.Na )

[0217] Compounds such as mCBS can block the toxic effects of extracellular histones, thus potentially contributing to the development of central nervous system These compounds are useful as treatments for systemic diseases.

[0218] Therefore, the compounds of the present invention and therapeutic or pharmaceutical compositions containing said compounds are Treatment, including both pre-treatment (in the case of therapeutic treatment) and treatment after the onset of central nervous system disease, A means to ameliorate the cytotoxic activity of extracellular histone proteins in central nervous system diseases in elephants to provide.

[0219] 3. Therapeutic and pharmaceutical forms In a fifth aspect of the present invention, a therapeutic agent for use in the treatment of extracellular histone-related complications is provided. A composition or pharmaceutical composition comprising a polyanionic sulfated cellobioside, the reduction of which is Polyanionic sulfation modified at the termini with small uncharged glycosidic bond substituents. At least cellobioside or a therapeutically acceptable or pharmaceutically acceptable salt thereof Preferably, the composition is a therapeutically acceptable or containing pharmaceutically acceptable carriers, excipients and / or diluents. The compound in may be in neutral free base form or in salt form. The compound is the sodium salt of β-O-methyl cellobioside sulfate.

[0220] As used herein, the terms "pharmaceutically acceptable" or "therapeutically acceptable" mean , with a reasonable benefit / risk ratio and without excessive toxicity, irritation, allergic reaction or other Contact with human and animal tissue without problems or complications and within the bounds of sound medical judgment refers to compounds, substances, compositions and / or dosage forms suitable for use in

[0221] Methods for preparing administrable compositions will be apparent to those skilled in the art, see, for example, the publications herein, which are incorporated by reference in their entirety. Remington's Pharmaceuticals S.A., incorporated herein by reference. science, 15th ed., Mack Publishing Company, Easton, Pa., as described in more detail.

[0222] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" refers to a "excipient" or "pharmaceutically acceptable diluent" or "therapeutically acceptable carrier" or " A "therapeutically acceptable excipient" or "therapeutically acceptable diluent" is a substance that is capable of being absorbed into an organ or part of the body. a substance, composition, or substance involved in the carrying or transport of a compound of interest from one organ to another organ or part of the body Vehicles (e.g., liquid or solid fillers, diluents, additives, manufacturing aids (e.g., lubricants) Lubricants, magnesium, calcium, or zinc stearate, or stearic acid Each carrier, diluent and excipient is intended to be a component of the formulation. It must be "acceptable" in the sense that it is compatible with the ingredients and not harmful to the patient. This is a substance that is not biologically or otherwise undesirable, i.e. The substance may be used without causing any unacceptable biological effects or the effects of the composition in which it is contained. and providing the active agent to an individual without interacting in a deleterious manner with any one or more of the ingredients. Some of the substances that can function as pharmaceutically acceptable carriers, diluents and excipients are: Examples include, but are not limited to: (1) sugars, such as lactose; (2) starches, such as corn starch and distillers; potato starch; (3) cellulose and its derivatives, such as carboxymethyl cellulose Sodium methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, e.g., stearic acid; magnesium phosphate, sodium lauryl sulfate, and talc; (8) additives such as cocoa Butters and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, (10) glycols, such as propylene glycol; (11) Polyols, such as glycerin, sorbitol, mannitol and polyols; Ethylene glycol (PEG); (12) esters, such as ethyl oleate and laurate; Ethyl phosphate; (13) agar; (14) buffering agents, such as magnesium hydroxide and ammonium hydroxide; Aluminum; (15) Alginic acid; (16) Pyrogen-free water; (17) Isotonic saline Saline solution; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer solution; (21 ) polyesters, polycarbonates and / or polyanhydrides; (22) extenders, e.g. Polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LD L;(22)C2~C 12 Alcohols, such as ethanol; and (23) those used in pharmaceutical preparations. Other non-toxic compatible substances that may be used in the formulation, such as wetting agents, binders, fillers, lubricants, colorants, Disintegrants, release agents, coating agents, sweeteners, flavorings, fragrances, preservatives, water, salt solutions, alcohol Cholesterol, antioxidants, polyethylene glycol, gelatin, lactose, amylose, stearyl alcohol Magnesium distearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose , polyvinylpyrrolidone and the like may also be present. Terms such as "a pharmaceutically acceptable carrier" and "a pharmaceutically acceptable carrier" or the like are used interchangeably herein. It is used.

[0223] Examples of therapeutically acceptable or pharmaceutically acceptable carriers, excipients or diluents are listed below. Demineralized or distilled water; saline solution; vegetable-based oils, such as peanut oil, safflower oil, Leaf oil, cottonseed oil, corn oil, sesame oil, e.g. peanut oil, safflower oil, olive oil , cottonseed oil, corn oil, sesame oil, peanut oil or coconut oil; silicone oils, e.g. polysilane siloxanes, such as methylpolysiloxane, phenylpolysiloxane and methylphenyl Polysiloxanes; volatile silicones; mineral oils, such as liquid paraffin, soft paraffin or sulfur Quaran; cellulose derivatives, such as methylcellulose, ethylcellulose, carboxycellulose Methylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose cellulose; lower alkanols, such as ethanol or isopropanol; lower alkanols Lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, such as polyethylene glycol and polypropylene glycol Coal, ethylene glycol, propylene glycol, 1,3-butylene glycol or Glycerin; fatty acid esters, such as isopropyl palmitate, isopropyl myristate propyl or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; tragacanth Gum or acacia gum and petrolatum. Typically, the carrier comprises from 10% to 99% by weight of the composition. It will form 0.9% by weight.

[0224] The compositions described herein may contain other adjunct ingredients conventionally found in pharmaceutical compositions, such as: It may further comprise art-established use levels of this auxiliary ingredient. The composition may contain additional compatible pharmaceutically active substances (e.g., antipruritics, astringents, local anesthetics, However, such substances, when added, may be included in the It should not unduly interfere with the biological activities of the components of the compositions described herein.

[0225] As described in detail below, the therapeutically acceptable or pharmaceutically acceptable salts described herein may be used in combination with any of the The physiologically acceptable compositions may be specially formulated for administration in solid or liquid form, and are described below. (1) Oral administration, e.g., liquid medicine (aqueous or non-aqueous solution) or suspension), lozenges, dragees, capsules, pills, tablets (e.g., buccal, sublingual) (targeting absorption and systemic absorption), bolus, powder, granules, and pamphlets for application to the tongue. (2) For example, as a sterile solution or suspension or sustained-release preparation, for example, subcutaneous injection (3) Parenteral administration, e.g., by injection, intramuscular injection, intravenous injection, or epidural injection; intraperitoneal, intrathecal, intraventricular, or intrahepatic administration to the organ requiring treatment (4) direct injection; e.g., creams, lotions, gels, ointments, or sprays applied to the skin (5) topical application as a controlled release patch or spray; (6) inhalation (e.g., nasal inhalation or (6) an aerosol form suitable for administration by inhalation (or oral inhalation); (7) for vaginal or rectal use as a cream, suppository, or foam; (8) for sublingual use; (9) for eye drops (9) for ophthalmic use; (10) for transmucosal use; or (11) for nasal use.

[0226] In one embodiment, the compositions of the present invention are administered parenterally (e.g., subcutaneously, intramuscularly, or The drug may be administered by injection, for example, intravenous injection, or by intraparenchymal or intrathecal administration, for example, in the brain. The drug is administered locally to tissues and organs by intravenous, intraventricular, or intrahepatic administration.

[0227] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water-soluble) or dispersions and sterile injectable solutions. Ideally, this composition includes sterile powders for the extemporaneous preparation of solutions or sterile injectable dispersions. The composition must be stable under the conditions of manufacture and storage and must be free from the contaminating action of microorganisms (e.g., bacteria and fungi). A preservative may be included to stabilize the composition against

[0228] The pharmaceutical compositions of the present invention may be prepared in the form of a pharmaceutical composition containing a pharmaceutical composition of the present invention in an amount required for the treatment of a variety of conditions, including the administration of a pharmaceutical composition containing ... Sterile injectable solutions are prepared by incorporating the pharmaceutical compositions, followed by sterile filtration as needed. As an example, a single dose can be prepared by dissolving it in 1 ml of isotonic NaCl solution and adding 1000 ml of liquid. 1 or can be injected at the proposed injection site (e.g., "Reming ton's Pharmaceutical Sciences"15th Editi (See pages 1035-1038 and 1570-1580.)

[0229] For injection solutions, the carrier can be a solvent or dispersion medium, including, for example, water, phosphorus, Gel solution, isotonic saline, phosphate buffered saline, ethanol, polyols (e.g., glucan, Glycerol, propylene glycol (e.g., 1,2 propylene glycol) and liquid poly ethylene glycol and the like), suitable mixtures thereof and vegetable oils.

[0230] For example, a coating such as lecithin can be used to maintain the required particle size in the case of dispersions. The prevention of microbial action can be achieved by various methods. This can be achieved by including suitable antibacterial and / or antifungal agents. Suitable agents are known to those skilled in the art. Examples include parabens, chlorobutanol, phenol, benzyl alcohol, and ascorbic acid. These include benzoic acid, thiomerosal, and the like. In such cases, it may be preferable for the composition to contain isotonic agents, for example sugars, polyhydric alcohols, etc. It may be preferable to include ethanol, such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable composition can be achieved by the use of an agent that delays absorption (e.g., benzodiazepine monostearate). This can be achieved by including in the composition a compound such as aluminum and gelatin.

[0231] In a second embodiment, the compositions of the present invention can be combined with, for example, an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the pharmaceutical composition is combined with an excipient and ingested. Take-out tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and It can be used in the form of hearth and the like.

[0232] Some examples of carriers, diluents, additives and adjuvants suitable for oral use include peanuts Oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, aluminum Sodium phosphate, gum acacia, gum tragacanth, glucose, sucrose, sorbitol In addition, these oral formulations may contain other ingredients such as mannitol, gelatin, and lecithin. Suitable flavoring and coloring agents may be included.

[0233] When used in capsule form, the capsule may contain a compound that retards disintegration (e.g., molybdenum). The granules may be coated with glyceryl monostearate or glyceryl distearate. Similarly, tablets, troches, pills, capsules and the like may contain binders, e.g. gum tragacanth, acacia, corn starch or gelatin; additives such as lysine dicalcium phosphate; additional disintegrants, e.g., corn starch, potato starch, alginate carboxylic acids and the like; lubricants, such as magnesium stearate; and sweeteners, such as Sucrose, lactose or saccharin or flavoring agents such as peppermint, oil of wintergreen or is cherry flavoring.

[0234] When the dosage unit form is a capsule, the capsule may contain, in addition to materials of the above type: A variety of other materials may be present as coatings or in other ways. The physical form of the dosage unit may be modified in various ways, for example, as a tablet, pill, or capsule. The capsules may be coated with shellac, sugar or both.

[0235] Liquid forms for oral administration (e.g., syrups or elixirs) may contain, in addition to the above agents, , liquid carrier, sweetener (e.g., sucrose), preservatives (e.g., methylparaben and propylparaben) Contains artificial color and flavoring (e.g., cherry flavor or orange flavor). Suitable liquid carriers include: water, oils such as olive oil, peanut oil, and the like. Nut oil, sesame oil, sunflower oil, safflower oil, peanut oil, coconut oil, liquid paraffin, ethylene glycol Recall, propylene glycol, polyethylene glycol, ethanol, propanol , isopropanol, glycerol, fatty alcohols, triglycerides or mixture.

[0236] Suspensions for oral administration may further comprise dispersing agents and / or suspending agents. Suitable suspending agents As the ingredients, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl Suitable dispersing agents include: lecithin, stearin. Polyoxyethylene esters of fatty acids such as polyoxyethylene sorbitol monooleate ester or dioleate, stearate or laurate, polyoxyethylene sorbitan Tanmono- or dioleate, stearates or ralates and the like Emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include Dispersants such as those exemplified above or natural gums such as guar gum, acacia gum or Examples include gum tragacanth.

[0237] In a third exemplary embodiment, the compositions of the present invention are administered in the form of an aerosol or by inhalation. For use as an aerosol, the pharmaceutical compositions of the present invention are administered directly to the respiratory tract of a subject by administration. A solution or suspension of the composition is dispersed in a suitable propellant (e.g., propane, butane, or ethane). a pressurized aerosol container with a hydrocarbon propellant such as sobutan and a conventional adjuvant Such compositions may be filled into a non-pressurized container (e.g., in a nebulizer or atomizer). It may also be administered in the form of a pharmaceutical preparation.

[0238] Aerosols for delivery to the respiratory tract are known in the art; see, e.g., Adjei ,A.and Garren,J.Pharm.Res.,1:565-569(199 0);Zanen,P.and Lamm,JW.J.Int.J.Pharm.,1 14:111-115(1995);Gonda, I. “Aerosols for d elivery of therapeutic a diagnostic age nts to the respiratory tract,”in Critica l Reviews in Therapeutic Drug Carrier Sy stems,6:273-313(1990);Anderson et al.,Am See Rev. Respir. Dis., 140:1317-1324 (1989). I want to be done that.

[0239] In a fourth exemplary embodiment, the composition may be administered in the form of liposomes. are generally derived from phospholipids or other lipid substances and are monolamellar or It is formed of multilamellar hydrated liquid crystals. It is a non-toxic, physiologically acceptable substance capable of forming liposomes. Any lipid that can be absorbed and metabolized may be used. The composition in liposome form may be free of stabilizers, preservatives, etc. , additives and the like. Preferred lipids are phospholipids and phosphatidylcholine (lecithin), both natural and synthetic. Methods for Forming Liposomes is known in the art and in this regard, see Prescott, Ed., Meth. ds in Cell Biology,Volume XIV,Academic P Ress, New York, NY (1976), p. 33 et seq. (This Specific reference is made to the following:

[0240] In addition, the therapeutic agents of the present invention according to any aspect, embodiment or example described herein Alternatively, the pharmaceutically acceptable compositions may be incorporated into sustained release preparations and formulations. Therapeutic or pharmaceutical compositions should contain appropriate buffers to minimize acid hydrolysis. Suitable buffering agents are known to those skilled in the art and may further include phosphates, citrates, carbonates, etc. and mixtures thereof.

[0241] The compounds of the present invention may be administered in the form of a "prodrug." A prodrug is a compound that acts in vivo A suitable prodrug is an inactive compound that is converted to its active form by Examples of compounds of this type include esters and phosphonate esters.

[0242] In addition, the compositions of the present invention may be implanted into a patient or injected using a drug delivery system. Coated delivery devices may also be useful. ,et al.(1984),Ann.Rev.Pharmacol.Toxicol. 24:199-236;Lewis, ed.“Controlled Release of Pesticides and Pharmaceuticals”(Plenu Press, New York, 1981); U.S. Patent No. 3,773,919 See US Pat. Nos. 6,747,014 and 353,270,960. I want to be done that.

[0243] In certain embodiments, the device or dressing used in the process of treating a wound may be used. The composition is delivered using

[0244] A therapeutically effective amount of the pharmaceutical compositions disclosed herein for any particular subject is The efficacy of the pharmaceutical composition will depend on a variety of factors, including: the therapeutic effect; the severity of the disease; the patient's age, weight, general health, sex and diet; the time of administration; the route of administration; the uptake rate of the composition; the duration of treatment; the efficacy of the treatment, together with other relevant factors known in medicine. Drugs used in combination with or simultaneously with a device.

[0245] Toxicity and therapeutic efficacy were assessed using, for example, LD 50 (lethal dose for 50% of the population) and E D 50cell culture or experimental methods to determine the dose that is therapeutically effective in 50% of the population The dose ratio between toxic and therapeutic effect can be determined by standard pharmaceutical procedures in experimental animals. , is the therapeutic index, and the ratio LD 50 / ED 50 Compositions that exhibit large therapeutic indices can be expressed as preferable.

[0246] The data obtained from the cell culture assays and animal models described herein are used in human studies. The therapeutically effective dosage ranges for use in such compounds may be used to formulate therapeutically effective dosage ranges for use in such patients. The dosage is preferably ED with little or no toxicity. 50 Within a range of circulating concentrations including The dosage may vary within this range depending on the dosage form used and the route of administration utilized. may vary.

[0247] The invention described herein can be combined with a carrier material to produce a dosage form. The amount of compound will generally be that amount of the compound that produces a therapeutic effect. Of the 00%, this amount ranges from about 0.1% to 99% of the compound, preferably from about 5% to It will be in the range of about 70%, and most preferably in the range of 10% to about 30%.

[0248] The dosage can be determined by the physician and is adjusted as needed to suit the observed effects of the treatment. By way of example only, the composition may be prepared as follows: It can be administered in amounts of: 1 μg / kg to 150 mg / kg, 1 μg / kg to 1 00mg / kg, 1μg / kg~50mg / kg, 1μg / kg~20mg / kg, 1μ g / kg~10mg / kg, 1μg / kg~1mg / kg, 100μg / kg~100m g / kg, 100μg / kg~50mg / kg, 100μg / kg~20mg / kg, 1 00μg / kg~10mg / kg, 100μg / kg~1mg / kg, 1mg / kg~1 00mg / kg, 1mg / kg~50mg / kg, 1mg / kg~20mg / kg, 1m g / kg~10mg / kg, 10mg / kg~100mg / kg, 10mg / kg~50 mg / kg or 10mg / kg to 20mg / kg. The ranges shown here include all intermediate ranges. For example, the range 1 mg / kg to 10 mg / kg includes 1 mg / kg~2mg / kg, 1mg / kg~3mg / kg, 1mg / kg~4mg / kg, 1 mg / kg~5mg / kg, 1mg / kg~6mg / kg, 1mg / kg~7mg / kg , 1mg / kg~8mg / kg, 1mg / kg~9mg / kg, 2mg / kg~10mg / kg, 3mg / kg~10mg / kg, 4mg / kg~10mg / kg, 5mg / kg ~10mg / kg, 6mg / kg~10mg / kg, 7mg / kg~10mg / kg, 8 mg / kg~10mg / kg, 9mg / kg~10mg / kg, etc. In the intermediate range (e.g., in the range 1 mg / kg to 10 mg / kg, 2 mg / kg to 8 mg / kg) (dose ranges such as mg / kg, 3mg / kg-7mg / kg, 4mg / kg-6mg / kg, etc.) It should further be understood that any of these are within the scope of the methods and compositions described herein. be.

[0249] When the compound of the present invention is mCBS or mCBS.Na, the dosage is 10 to 800 μg. g / ml. Preferably, this dosage ranges from 50 to 500 μg / ml. More preferably, this dosage is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, , 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790 or 800 μg / ml.

[0250] In a particular embodiment of the present invention, an effective amount of the modified sulfated cellobioside compound is administered in a single dose. In certain instances, this dose is administered repeatedly. That is, the treatment regimen is the severity and type of the condition, the patient's overall health and age, and the treating physician's The duration and frequency of treatment will vary depending on various other conditions. A clinical physician typically monitors the subject to determine when the treatment provides therapeutic benefit. and whether to increase or decrease the dose, increase or decrease the frequency of administration, or discontinue treatment. whether to discontinue treatment, restart treatment, or make other changes to the treatment regimen. Make a decision.

[0251] Therapeutic compositions or compositions of the invention according to any aspect, embodiment or example described herein. The pharmaceutically acceptable composition may be administered in a single bolus or in multiple doses. or treatment, and small amounts of the therapeutic composition are administered continuously over an extended period of time. This can be applied by "continuous" treatment.

[0252] Therapeutic or pharmaceutical compositions when multiple doses are used in treatment (including continuous therapy) The modified substance used in the therapeutic or pharmaceutical composition may be selected based on several clinical factors, such as Once a week to once a week depending on the subject's sensitivity to the sulfated cellobioside compounds being modified The dosage regimen will vary from day to day. The desired dose can be delivered at once as a single dose, or in partial doses (e.g., 2-4 times). divided into subdoses) over a period of time (e.g., at appropriate intervals or other times throughout the day) Such subdoses may be administered as unit dosage forms. .

[0253] In some embodiments, administration is chronic, for example over a period of weeks or months. Dosage schedules include one or more doses per day. Examples of dosing schedules include weekly, biweekly, and 3-weekly. 1, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer The dose is once daily, twice daily, three times daily, or four or more times daily.

[0254] The desired dose is administered using continuous infusion or delivered via a controlled release formulation. In this case, the pharmaceutical composition contained in each partial dose achieves the total daily dose. Therefore, it must be correspondingly small.

[0255] The dosage unit may also be a conventional formulation that provides sustained release of the pharmaceutical composition over a period of, for example, several days. The drug may also be formulated for delivery over several days using sustained release formulations of the drug. Certain compounds known in the art and that may be used, for example, with the agents described herein. In this embodiment, the dosage unit is particularly useful for delivering a drug to a site. Includes corresponding multiples of the dose.

[0256] 4. Combination regime In certain exemplary embodiments of the fifth aspect of the invention, the identified composition is an anti-inflammatory antiviral agents, antifungal agents, and / or one or more of the agents that the subject is suffering from. or any other form of therapeutic or pharmaceutical compound that treats a number of conditions. According to this embodiment, the composition may comprise a second active agent, compound, or composition selected from the group consisting of: The second active agent, compound or composition preferably treats sepsis, SIRS and IRI or ulcers. Providing adjunctive treatment for medical conditions or diseases associated with hypertension, SIRS, and IRI Preferably, the second active agent, compound or composition comprises one or more anti-inflammatory agents. .

[0257] Therapeutic benefits may be realized through combination regimens. The described method, when delivered prophylactically, is to prevent a patient from having or suffering from have or are at risk of having or contracting sepsis, SIRS and IRI or septicemia , which is an adjunctive treatment for medical conditions or diseases associated with SIRS and IRI. The method may further comprise administering two active agents to a subject simultaneously or concomitantly with the treatment of the present invention. do.

[0258] The second active agent may be an anti-inflammatory agent, an antibiotic agent, an antiviral agent, an antifungal agent or a compound of the present invention. Other forms of medical intervention other than chemical compounds may be included, but are not limited to:

[0259] Illustratively, the method or treatment can be used to treat a disease mediated by extracellular histones in a subject. and the like, which are intended to treat or ameliorate septic or non-septic disease states associated with If approved, this method may be useful in treating medical conditions involving pathologies mediated by extracellular histones. A second anti-inflammatory, antibiotic, antiviral, or antifungal agent provides adjunctive treatment for the condition. or administering to the subject simultaneously or concomitantly another form of medical intervention different from the compounds of the present invention. It may also include.

[0260] In one example, the second active agent is a compound that inhibits sepsis, SIRS, or IRI, or sepsis, SIRS, or IRI. or a medical condition or disease associated with IRI (e.g., extracellular histone Sepsis, SIRS, or IRI with pathology mediated by or medical conditions or diseases associated with IRI) provide.

[0261] In another example, the second active agent is an adjunct for a medical condition involving extracellular histone cytotoxicity. provides comprehensive treatment or adjunctive prophylaxis.

[0262] Illustratively, the present treatment methods include treating a subject with a pathology mediated by extracellular histones. Treating septic or non-septic disease states associated with sepsis, SIRS, or IRI When directed to restoring or ameliorating a tumor, the method may be directed to the administration of extracellular histones. and a second anti-inflammatory, antibiotic, agent that provides adjunctive treatment for medical conditions with pathology mediated by steroids. antiviral, antifungal, or other form of medical intervention other than the compounds of the present invention. It may also include administering to the subject simultaneously or concomitantly.

[0263] In some instances, the additional agent administered is an anti-inflammatory agent, e.g., a steroid, Corticosteroids, COX-2 inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), aspirin More specifically, the additional agent administered is: The anti-inflammatory agent may be selected from the group consisting of: alclofenac; propionate; Algestone Acetonide ; alpha amylase; Amcinafal; Amcinafide cinafide); amfenac sodium; amiprilose hydrochloride; anakinra; ani Lorac; Anitrazafen; Apazone; Balsalazide disodium; Bendazac; Benon Benzidamine hydrochloride; Bromelain; Properamol; Budesonide; Capsules cycloprofen; zintazone; criprofen; clobetazoline propionate Clobetasone butyrate; Clopirac; Cloticasone propionate e Propionate); Colmetasone acetate; Cortodoxone; Deflazacort; De Sonid; Desoximetasone; Dexamethasone dipropionate; Diclofenac potassium ;Diclofenac sodium;Diflorasone acetate;Diflumidone sodium;Diflunisa Difluprednate; Diphthalone, dimethyl sulfoxide; Drocinonide; Endoli Son; Enlimomab; Enolicam sodium; Epirizole; Etodolac; Etofene Fenamole; Fenbufen; Fenclofen Enac; Fenclorac; Fendosal; Fenpipalon; Fentiazac; Flazaron ;Fluazacort;Flufenamic acid;Flumizole;Flunisolide acetate;Flunixin; Flunixin meglumine; Flucortine butyl; Fluorometholone acetate; Fluquazone ( Fluquazone; Flurbiprofen; Fluretofen; Flutica Propionate Son; Furaprofen; Flobufen; Halcinonide; Halobetasol propionate; Vinegar Halopredone; Ibufenac; Ibuprofen; Aluminum ibuprofen; Ibuprofen Lofenpiconol; Ironidap; Indomethacin; Indomethacin sodium; In Doprofen; Indoxole; Intrazol e); Isoflupredone acetate; Isoxepac; Isoxicam; Ketoprofen; Hydrochloric acid Femizole; Lomoxicam; Loteprednol etabonate; Methicillin-resistant Staphylococcus aureus Clofenamic acid sodium; Meclofenamic acid; Meclorisone dibutyrate (Mecloris one Dibutyrate); Mefenamic acid; Mesalamine; Meseclazone; Methylpropional Rednisolone suleptanate; Morniflumate; Nabumetone; Naproxen; Naproxen Naproxol; Nimazone; Olsalazine sodium Orgothein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Parahydrochloride Nilin; Pentosan polysulfate sodium; Phenbutazone sodium glycerol Piroxicam; Piroxicam cinnamate; Piroxicam olamine; Pip Lofene; Prednazate; Priferon; Prodric acid; Proquazone; Proxazole; Proxazole citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Salsylate (S alycilate); sanguinarium chloride; seclazone; cermetacin; sudoxicam ;Sulindac;Suprofen;Talmetacin;Talniflumate;Talosalate;Tebuful Tenidap; Tenidap sodium; Tenoxicam; Tesicam; Tesimid; Tet Lidamine; Tiopinac; Tixocortol pivalate; Tolmetin; Tolmetin sodium Triflumidate; Zidometacin; Glucocorticoids; zomepirac sodium; and combinations thereof.

[0264] In some instances, the additional agent administered is an antibiotic agent, such as kanamycin. actinomycin D, doxorubicin, bleomycin, mithramycin, aminoglycoside glycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides amides, lincosamides, macrolides, monobactams, penicillins, polypeptides, quinolones The compounds are benzodiazepines, sulfonamides and / or tetracyclines.

[0265] In some instances, the additional agent administered is an antiviral agent, e.g., a non-nucleoside nucleotide reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors (e.g., nucleoside analogs) nucleotide analogues), protease inhibitors and / or nucleotide analogue reverse transcriptase inhibitors.

[0266] In some instances, the additional agent administered is an antifungal agent, such as an imidazole, The compounds are triazole, thiazole, allylamine and / or echinocandin compounds.

[0267] In some instances, the additional drug administered is an agent for treating diabetes. for the treatment of diabetes and / or for having antihyperglycemic activity. Drugs known in the art include, for example, dipeptidyl peptidases Inhibitors of dipeptidyl peptidase-4 (DPP-4) (e.g., alogliptin, linagliptin, saxagliptin) methadone, sitagliptin, vildagliptin and berberine), biguanides (e.g., methotrexate ... formin, buformin, and phenformin), peroxisome proliferator-activated receptors (P PAR) modulators, such as thiazolidinediones (TZDs) (e.g., pioglitazone , rivoglitazone, rosiglitazone, and troglitazone), a dual PPAR agonist (e.g., aleglitazar, muraglitazar, and tesaglitazar), sulfonylureas (e.g., For example, acetohexamide, carbutamide, chlorpropamide, gliclazide, tolbutamide , tolazamide, glibenclamide (glyburide), glipizide, gliquidone, glycerol pyramide and glimepiride), meglitinides (“glinides”) (e.g., nateglinide, repaglinide and mitiglinide), glucagon-like peptide-1 (GLP-1) and analogs (e.g., exendin-4, exenatide, liraglutide, albiglutide), insulin insulin and insulin analogues (e.g., insulin lispro, insulin aspart, insulin Slingullysine, insulin glargine, insulin detemir, Exubera and NPH insulin), alpha-glucosidase inhibitors (e.g., acarbose, miglitol and voglibose), amylin analogs (e.g., pramlintide), sodium-dependent glutamate SGLT T2 inhibitors (e.g., dapagliflozin, remogliflozin) rosin and serugliflozin) and others (e.g., benfluorex and thor Lestat).

[0268] The composition according to any aspect, embodiment or example described may be used to treat sepsis, SIRS or for the treatment of IRI or a disease or condition associated with sepsis, SIRS or IRI Those skilled in the art will recognize that the compounds may be administered as part of a combination therapeutic approach. The treatment may involve administering the agents simultaneously or sequentially in any order. In such cases, it may be preferable to administer each component by the same route.

[0269] In some instances where two drugs are applied separately, both drugs may be advantageously combined. There was no significant lapse between the times of delivery so that the drug could still exert its intended effect. In such instances, both treatments will generally be approximately 12-2 minutes apart. It is contemplated that they will generally be administered within 4 hours of each other, and more preferably within about 6-12 hours of each other. Although a delay of only about 12 hours is most preferred, in some circumstances a delay of time for treatment may be required. It may be desirable to extend the time between doses significantly, allowing several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) have passed. It is also contemplated that multiple doses may be desirable.

[0270] When the composition of the present invention and the second active agent are administered in separate compositions, the routes of administration may be different. For example, the compositions of the present invention can be prepared by any suitable route known in the art, including, for example, It is not limited to oral or parenteral routes, for example, intravenous, intramuscular, subcutaneous, Transdermal, respiratory (aerosol), pulmonary, nasal, rectal and topical (e.g., buccal and sublingual administration) and a second pharmaceutically active agent is administered by another route (e.g., The drug is administered by a route commonly used in the art for the administration of biologically active agents. In a limited example, the composition of the present invention may be administered by injection and the second active agent may be administered orally. .

[0271] 5. Drug manufacturing In a sixth aspect of the present invention, a method for treating a medical condition, disease or disorder involving extracellular histones is provided. A polyanionic sulfated cellobioside, the reducing end of which is used in the manufacture of a drug for Polyanionic sulfated cellulosic acid modified with small uncharged glycosidic substituents in The use of a therapeutically effective amount or a pharmaceutically effective amount of bioside or a pharmaceutically acceptable salt thereof Preferably, the polyanionic sulfated cellobioside is present at the reducing end. Small uncharged glycosidic substituents are sulfated at the reducing end of the same polyanion. More preferably, the modified polyanion has improved chemical stability compared to the modified polyanion. The sulfated cellobioside is mCBS or, more specifically, a pharmaceutically acceptable salt of mCBS. The salt is a salt (e.g., mCBS.Na).

[0272] For example, in one embodiment of the sixth aspect of the invention, a method for treating sepsis, SIRS or other conditions in a subject is provided. or IRI or the treatment of a medical condition or disease associated with sepsis, SIRS, or IRI A polyanionic sulfated cellobioside in the manufacture of a medicament for the treatment or prevention of Polyanions modified at their reducing ends with small, uncharged glycosidic substituents a therapeutically effective amount or a pharmaceutically effective amount of a soluble sulfated cellobioside or a pharmaceutically acceptable salt thereof; Preferably, the modified sulfated cellobioside is mC mCBS, or more specifically a pharmaceutically acceptable salt of mCBS (e.g., mCBS .Na).

[0273] In one embodiment of such a use, the medicament is used to treat sepsis or SIRS in a subject. or for treating sepsis or SIRS in a subject. and for treating a biological condition or disease, said treatment comprising administering to said subject sepsis or S. ameliorate IRS or the condition or disease associated with said sepsis or SIRS; Or inhibit.

[0274] In another embodiment of such use, the medicament is used to treat IRI in a subject. or treating a medical condition or disease associated with IRI in a subject. wherein said treatment is for treating said condition or disease associated with said IRI or said injury. The treatment ameliorates or inhibits the disease.

[0275] In yet another embodiment of such use, the agent is used to (i) induce endothelial or (ii) contributes to endothelial dysfunction in a subject; or i) initiating clotting by activating the subject's platelets, or (iv) It neutralizes extracellular histones that induce red blood cell fragility and consequent anemia in red blood cells.

[0276] In yet another embodiment, the manufactured medicament is a therapeutic agent for a second active agent, compound or composition. In accordance with this embodiment, the second active agent may also include a therapeutically effective amount or a pharmaceutically effective amount. The compound or composition may be used as a supplement for treating a medical condition, disease or disorder involving extracellular histones. Desirably, the second active agent, compound or composition is an agent for treating sepsis, SIR, S or IRI or a medical condition or disease associated with sepsis, SIRS, or IRI Preferably, the second active agent is an anti-inflammatory agent, an antibiotic, or a combination thereof. the agent, antiviral agent, antifungal agent, and / or one or more conditions from which the subject is suffering or any other form of therapeutic or pharmaceutical compound for treating More preferably, the second active agent, compound or composition contains one or more anti-inflammatory agents. include.

[0277] When a modified sulfated cellobioside compound is used in any of the methods of the present invention, the compound may be administered to a subject in need thereof in a single dose formulation, or It may be formulated for administration to a subject in need thereof. The modified sulfated cellobioside compound is provided as a multi-dose formulation, which is Administered to a subject or formulated for administration to a subject in need thereof.

[0278] Preferably, for administration to a subject, the therapeutic or pharmaceutical composition is pharmaceutically acceptable. In this form, (1) the composition contains one or more drugs. a biologically acceptable carrier (additive), and / or diluent, and / or (2) a modification in the composition, The sulfated cellobioside compounds described herein may be formulated in neutral or salt form. [Example]

[0279] The present invention is further described in the following non-limiting examples, which are provided for illustrative purposes only. These are provided as examples and should not be construed as limiting the generality of the disclosure herein throughout. do not have.

[0280] Example 1: Preparation of mCBS·Na β-O-methyl cellobioside was synthesized as described by Jon K Fairweather et al. As described by [David], 2004, Aust. J. Chem., 57:197-205 Prepare the sea urchin.

[0281] β-O-methyl cellobioside sulfate (mCBS) compound and sodium β-O- The compound methyl cellobioside sulfate (mCBS.Na) was added to Katrin C Pr obst and Hans Peter Wessel,2001,J.Carboh Hydrate Chemistry, 20(7&8):549-560 (this disclosure is (the entirety of which is incorporated herein by reference).

[0282] β-O-methyl cellobioside sulfate (mCBS) was prepared according to the following outline: . [ka]

[0283] Step 1: α-D-cellobiose 1 (116 g, 338 mmol) and glacial acetic acid (1. 6 L), acetyl bromide (300 mL, 500.0 g, 4065 mmol) was added at room temperature. , 12.0 equivalents) was added. The resulting creamy mixture was The mixture was heated at 60°C for 45-55 minutes until it turned into a clear solution indicating completion of the reaction.

[0284] Carefully pour this hot solution into a beaker (10 L) containing broken ice (4 kg). Stir the mixture until a white solid precipitates (approximately 10 minutes). Add another portion of cold water (1 L). Add and continue stirring for 10 minutes.

[0285] The solid was filtered off using a sintered funnel and washed with cold water (700 mL x 3 times). The compound was dissolved in DCM (1 L) and the funnel was washed with DCM (300 mL x 2). The collected DCM layer was washed with brine (1.5 L) and back-extracted with DCM (0.5 L). The final DCM layer was dried over Na2SO4, filtered, and concentrated under reduced pressure at <35 °C within 2 h. Concentrate to obtain the desired bromide 2 (172.5 g, 74.3%) which can be used directly in the next glycosylation. Yield) was obtained.

[0286] Step 2: Per-O-acetylated cellobiosyl bromide 2 (171 g, 250 mm ol), anhydrous DCM (800 mL), anhydrous MeOH (800 mL), activated 3A molecule A mixture of silver carbonate (Ag2CO3, 75g, 275mmol, 1. 1 equivalent) was added. The resulting mixture was stirred in the absence of light for 16 hours. The mixture was purified by passing through a plug of silica and eluting with EtOAc. The collected fractions were concentrated to give The crude product was obtained as a brown solid and used directly in the next step. R f = 0.28 (EtOAc-hexane, 1:1).

[0287] Step 3: A mixture of the crude product obtained in Step 2 and anhydrous MeOH (1 L) was added at room temperature. Na chips (1.72 g, 0.3 eq, 75 mmol) were added at rt. A white solid began to precipitate from the solution. The resulting mixture was stirred overnight to ensure deacetylation. The final suspension was filtered and washed with MeOH (300 mL x 2). The cells were collected and dried under vacuum overnight to give the final cellobioside 4 (72.5 g, 2-step The result was 81.4%.

[0288] [ka] Step 4 Synthesis and Purification Procedure: Step 4: Compound 4 (84.0 g, 236 mmol), SO3.TMA (367.4 g, 2.64 mol, 11.2 eq), anhydrous DMF (3140 mL) and anhydrous DCE (7 The mixture (67 mL) was degassed three times under Ar and heated at 80-90°C for 2 hours. Reaction monitoring: After 10 minutes of heating, the creamy mixture turned into a clear solution. After 30 minutes, the solution became cloudy again. After 50 minutes, a flocculated solid was observed on the surface of the flask. After cooling, the resulting mixture was transferred to a cold room (-5°C) and allowed to settle overnight to remove the solvent. The solid was allowed to fully condense from the solution. Complete conversion of compound 4 to 5 was confirmed by 1H-NMR. The solution was decanted into a drain. The crude solid was filtered off and washed with DCM 2-3 times. The solid was dissolved in deionized water and then passed through an ion exchange column [DOWEX 50W x 8 Na form :Resin(H + Pre-pack 3 kg of 1 M NaOH (-6 L) into a glass gravity column. The collected fractions were regenerated by elution with HCl (200 ml) and neutralized with deionized water (-12 L). The resulting solution was concentrated to give the final sulfated cellobioside 6 (232.1 g, 92.0 mL) as a glassy solid. %) was obtained.

[0289] Example 2: Stability study of mCBS·Na compound Stability studies were conducted at 5±3°C, 25±2°C / 60%RH, and 40±2°C / 75%RH. HPLC revealed that the formulated clinical material (i.e., mCBS in phosphate buffer) In this study, both mCBS and cellobiose sulfate (CBS) levels were monitored. Graph of percent (%) change in mCBS and CBS relative to their starting amounts (T=0) The level of mCBS was found to be very stable over time. The CBS levels dropped very rapidly and were almost gone in about three months. The profiles were compared under accelerated conditions (i.e., 25±2°C / 60% RH and 40±2°C / 75% RH). The elimination rate of CBS appears to be faster. An example of the difference in stability between S and CBS is shown in Figure 1. The data show that CBS is very stable in aqueous solution. However, by adding a methyl group to the reducing end of CBS, it becomes very unstable in aqueous solution. It is shown that a molecule (mCBS) that is stable at 1000 kJ / kg is obtained.

[0290] Additionally, various mCBS formulations were tested in stability studies. It was found that the stability of mCBS does not differ depending on the buffer used. 1 shows the composition of various formulations of mCBS in terms of the buffer used.

[0291] [Table 1]

[0292] Table 2a shows the change in pH over time depending on the buffering capacity of various formulations and The pH of the acid buffered formulation does not remain at approximately pH 7.5 even at approximately 5±3°C. However, this data itself indicates differences in mCBS stability during this period.

[0293] [Table 2]

[0294] Table 2b shows the results for a representative batch of bulk powder when stored at -20°C for 24 months. Compare the mCBS level with the CBS level at T, where T is the time in months. = 0 or T0 indicates the analysis performed at the completion of manufacturing. Subsequent analyses shown are The CAD(s) are relative to the first analysis of the powder or drug product (e.g., T1 = 1 month from the date of manufacture). The purity of mCBS and its impurity levels were determined using analytical methods using a charged particle detector. The solubility was measured and expressed as CAD%.

[0295] [Table 3]

[0296] Table 2c shows the clinical efficacy of phosphate buffered pH 7.5 HCl when stored at 2-8°C for over 18 months. The mCBS levels are compared to the CBS levels in a representative batch of test material formulation.

[0297] [Table 4]

[0298] Tables 2b and 2c above show the powder or The levels of mCBS and CBS in either of these preparations are shown. The results show that CBS levels decrease significantly over time. It appears to be stable in powder form when stored at -20°C, as it does not appear to change However, CBS is very unstable in aqueous buffer solutions. The level was 0.03 CAD% (i.e., detection limit) within 3-6 months of storage at 2-8°C. In contrast, levels of mCBS do not appear to fluctuate significantly over time. However, it appears to be highly stable when stored in powder or in solution.

[0299] HPLC analysis of mCBS in various buffer formulations after 28 days (Table 3) was also performed. CBS is suitable for all conditions, even under accelerated conditions of 25±2°C / 60%RH and 40±2°C / 75%RH. However, the fluctuations after 28 days in phosphate buffer or citrate buffer were only slightly below the nominal The variability appears to be about - / + 2%, indicating good stability.

[0300] [Table 5]

[0301] Preclinical evaluation of the toxicity and pharmacokinetic profiles of β-O-methyl cellobioside sulfate compounds floor animal research The following Examples 3 to 10 show the β-O-methyl cellobioside sulfate used in the present invention. The toxicity and pharmacokinetic (PK) profiles of the compound were evaluated in animals by the inventors. Exemplary preclinical studies are outlined.

[0302] Example 3: mCBS·Na Compound Used in Animal Studies This example illustrates the sodium β-O -methyl cellobioside sulfate (mCBS·Na) compound.

[0303] The STX studies described below were carried out with particular reference to β-O-methyl-CBS·Na, such as that used in the present invention. Provides a preliminary understanding of the toxicity and PK profile of chiral cellobioside sulfate compounds This study was performed using the same lot of compound, The characteristics are summarized in Table 4 below.

[0304] [Table 6]

[0305] Example 4: Dosage and concentration of β-O-methyl cellobioside sulfate Generally, in the toxicity and PK animal studies described herein, sodium mCBS However, the bioanalytical measurements outlined herein were performed using mCBS. It should be noted that the free base form of α-tocopherol was detected and reported. To clarify the relationship with the biological analysis results, the dose / concentration is expressed as the free base. The sodium content (based on the ratio of the MW of the free base to the salt form) is also reported. The dose of mCBS free base was obtained by correcting for the purity of the test material. The dosage of either (i.e., sodium salt or free base) is determined taking into account the potency of the compound in the batch. Not yet.

[0306] Example 5: Efficacy of β-O-methyl cellobioside sulfate As mentioned above, the reported doses or concentrations of mCBS administered to animals were determined based on efficacy (i.e., hydrolysis). The study was not corrected for the amount of benzophenone (and other impurities) used during the study. The potency analysis of the sodium salt of mCBS (i.e., mCBS·Na) in the batch used must be determined. Subsequent analyses of the batches used in these studies have since been carried out by sodium The actual efficacy of the ammonium salt used in these examples is approximately 74.5%. The dose was very likely lower than what was prescribed.

[0307] Example 6: Toxicity of mCBS after intravenous administration to Sprague Dawley rats This example demonstrates the efficacy and safety of Sprague Dawley in a 1-week dose-ranging (DRF) study. Evaluation of acute toxicity of mCBS after intravenous administration of a single bolus dose to rats and its 7-day after daily intravenous administration of bolus doses to Sprague Dawley rats over a period of 10 days. Evaluation of the toxicity of mCBS.

[0308] In this study, doses of mCBS·Na and mCBS as the free base were first investigated. From this efficacy evaluation, the maximum tolerated dose (MTD) estimate for the sodium salt of mCBS is The dose was approximately 745 mg / kg, whereas the dose for mCBS free base was approximately 600 mg / kg. High potential.

[0309] to Sprague Dawley rats in a 1-week dose-ranging (DRF) study β-O-methylcellobioside sulfate (mCBS) after a single bolus dose of intravenous administration Evaluation of acute toxicity of In this study, a single IV dose of mCBS in rats at doses up to 1000 mg / kg Acute toxicity from administration was investigated.

[0310] Acute toxicity of β-O-methyl cellobioside sulfate (mCBS) after a single bolus dose This dose-ranging study was evaluated in Sprague Dawley rats after intravenous administration of In this study, the sodium salt form of mCBS test article (mCBS·Na) was administered at 10, 30, and 10 A total of five adult female rats (n=3) were used at doses of 0, 300, and 1000 mg / kg. After correction for purity and sodium content, these dose levels were Equivalent to approximately 8.2, 24.5, 81.5, 244.5 and 815 mg / kg of BS free base The rats were then observed for 7 days before termination without necropsy. Treatment with the test article was well tolerated at all dose levels up to the 1000 mg / kg dose. No findings were considered to be related to treatment.

[0311] Therefore, the maximum tolerated dose (MTD) or acute tolerated dose of mCBS·Na from this study is 1000 mg / kg, which corresponds to 815 mg / kg of mCBS free base. No changes in symptoms or body weight (compared to control animals) were observed.

[0312] Daily intravenous administration of bolus doses to Sprague Dawley rats for 7 days Evaluation of the toxicity of β-O-methyl cellobioside sulfate (mCBS) after This study investigated the efficacy of mCB in rats at doses up to 1000 mg / kg for 7 days. Toxicity from repeated IV administration of S was investigated.

[0313] Acute Tolerance and Toxicity of β-O-Methyl Cellobioside Sulfate Over a 7-Day Period This was evaluated in Sprague Dawley rats after daily intravenous dose administration. Dosage studies have been conducted on the sodium salt form of mCBS (mCBS·Na) at 0, 100, 300 and 100°C. A total of four groups of adult female rats (n=3) were treated with 1000 mg / kg and 1000 mg / kg. Corrections for purity and sodium content resulted in these dose levels being 0.1% of mCBS free base. These rats were then terminated. They were observed for 7 days prior to the start of the study, followed by gross necropsy and terminal hematological and biochemical analyses. An additional group of n=3 male rats was administered up to 1000 mg / kg following the same study design. doses were treated.

[0314] mCBS was well tolerated at all dose levels up to 1000 mg / kg No adverse findings were considered to be related to treatment. Hematological parameters in treated animals Metric and biochemical parameters were unremarkable. The observed effects were not considered to be related to treatment.

[0315] Therefore, the MTD in this study was identified as 1000 mg / kg, which is the maximum daily dose for mCBS. This corresponds to 815 mg / kg of free base.

[0316] Example 7: Pharmacokinetics of mCBS after intravenous administration to Sprague Dawley rats This example demonstrates the efficacy of mCBS drugs administered intravenously to Sprague Dawley rats. The pharmacokinetic (PK) evaluation is shown.

[0317] The following studies report the efficacy of mCBS as both the sodium salt and the free base. The latter indicates the relationship between the measured plasma levels of mCBS (free base) and the administered dose. and terminal PK parameters such as clearance (Cl) and volume of distribution (Vz). The STX-09 study is considered important for determining the data for approximately 25 and 50 m Plasma concentrations of mCBS over a 5-hour continuous infusion in rats at 100 mg / kg / hour were shown. vinegar.

[0318] β-O-methylcellobiosis administered intravenously to Sprague Dawley rats Pharmacokinetic studies of dosulfate This study investigated the efficacy and safety of bolus IV administration of mCBS in Sprague Dawley rats. The PK profile of the compound was investigated after administration (20 mg / kg), and most of the compound was rapidly excreted. More than 90% of the administered dose was removed from the central compartment within the first 4 hours. This indicates that residual mCBS from a given dose is eliminated more slowly over a longer period of time. The large volume of distribution indicates that mCBS compounds are rapidly moving from the central compartment into tissues. This indicates that...

[0319] The pharmacokinetics of β-O-methyl cellobioside sulfate (mCBS) was investigated using heptanatrium The sodium salt of mCBS in the form of its sodium salt (mCBS·Na) was administered at 20 mg / kg. -Last dose (or 16.3 mg / kg free base after adjusting for sodium content and purity) The effects of benzodiazepines on the efficacy and safety of benzodiazepines were evaluated in Sprague Dawley rats after intravenous administration of benzodiazepines (base).

[0320] Considering efficacy, the doses administered to the rats were 100 mg of the sodium salt and 100 mg of the free base, respectively. The estimated probabilities were 14.9 mg / kg and 12.65 mg / kg. The corrected mCBS free base dose was approximately 20% lower, which resulted in a lower calculated dose. Clearance (Cl) and volume of distribution (Vz) values ​​were effectively reduced at similar rates.

[0321] A total of 10 blood samples were taken from three rats at time points ranging from pre-dose to 48 hours post-dose. The blood samples were processed into plasma and then analyzed by LC-MS / MS. The concentration of mCBS (free base) was analyzed using the method of The data were used to calculate pharmacokinetic parameters.

[0322] The mean (±SEM) value of mCBS concentration at time zero (C0) was 73400 (±8560 )ng / mL. The mean (±SEM) values ​​for area under the curve to infinity (AUCinf) were 34,300, respectively. (±2460) ng.hr / mL and 35,000 (±2940) ng.hr / mL. The mean (±SEM) apparent elimination half-life (T1 / 2) was 77.5 (±54.5). The mean residence time (MRT) was 5.58 (±3.99 h), while the mean (±SEM) value of MRT was 5.58 (±3.99 h). ) hours, which was relatively short. The mean (±SEM) value of the volume of distribution (Vz) was 46.9 (±30. 7) L / kg, and the mean (± SEM) value of total body clearance (Cl) was 0.472 (± The blood pressure was low at 0.0377 L / hour / kg. 1 / 2 (122% CV), Vz(113% The high intersubject variability of the CV and MRT (124% CV) was observed at the end of the log-linear concentration versus time curve. This was most likely due to incomplete characterization of the missing end portion.

[0323] Pharmacokinetic study of mCBS administered by intravenous route to Sprague Dawley rats This study was conducted in Sprague Dawley rats using a bolus IV (100 The PK of mCBS was investigated after 24 h administration of 100 mg / kg.

[0324] The pharmacokinetics of mCBS was evaluated by administering a bolus of 100 mg of the sodium salt to groups of six rats. mg / kg dose of mCBS (or 81.5 mg after correction for sodium content and purity) was evaluated in Sprague Dawley rats after intravenous administration of 100 mg / kg of the free base. .

[0325] Considering efficacy, the doses administered to the rats were determined for both the sodium salt and the free base. The corrected mCB concentrations were estimated to be 74.5 mg / kg and 63.25 mg / kg. The dose of the S free base was approximately 20% lower, which resulted in a lower calculated clearance (C The values ​​of l) and volume of distribution (Vz) were effectively reduced at similar rates.

[0326] A total of nine blood samples were taken from each rat at time points ranging from pre-dose to 192 hours post-dose. Urine samples were also collected over the first 48 hours. The urine and plasma samples were then analyzed using an LC-MS / MS-based method. The concentrations of mCBS (free base) were analyzed using the ELISA kit.

[0327] Estimates of plasma pharmacokinetic parameters for mCBS were calculated using pooled mean concentration versus time data. The concentration at time zero (0) was 308,000 ng / mL. The area under the curve from the time of the last measured concentration (AUClast) to infinity (AUC The area under the curve when extrapolated to 1000 ng.hr / mL for both is 135,000 ng.hr / mL. In contrast to the mean residence time (MRT) of 1.43 hours, the apparent terminal elimination half-life was The elimination life (T1 / 2) was 56.1 hours. This compound had a comparatively low T of 49.0 L / kg. It showed a relatively high volume of distribution (Vz), but the terminal systemic clearance (Cl) was 0.605 L / h. The mean mean blood glucose concentration was relatively low at 0.05g / kg.

[0328] Urinary pharmacokinetic mass balance estimates for mCBS free base were calculated based on the excreted urinary volume data. The amount of m excreted at collection intervals of 0-4 hours, 0-24 hours, and 0-48 hours after administration was obtained from the The mean (±SEM) values ​​of the percentage of total CBS dose were 52.0 (±5.0)% and 65.0%, respectively. Urinary excretion was 0.0 (±7.0)% and 69.2 (±10.8)% of the initial dose after administration. It has been identified as the major route for excretion of the compound at 48 hours.

[0329] This study demonstrated that mCBS was rapidly removed from the central compartment immediately after administration and The compound was absorbed into tissues as indicated by its small volume of distribution. The half-life, which is the primary determinant of elimination, was estimated to be 0.65 hours. Multiple terminal sampling improves characterization of terminal elimination half-lives; The terminal elimination half-life was calculated to be approximately 56 hours.

[0330] Drugs in Sprague Dawley rats administered mCBS by intravenous infusion Dynamics research This study examined the efficacy of 5-hour continuous infusion in rats at approximately 25 and 50 mg / kg / hour. The plasma concentration of mCBS was investigated.

[0331] After intravenous administration as a 5-hour infusion to male Sprague Dawley rats The pharmacokinetics (PK) of mCBS was studied. This study demonstrated that the mCBS compound The pharmacokinetics of this compound was investigated. The compound was formulated using Hartmann's solution and 126.5 mg / kg / hour to groups of 3 rats (total dose: 126.5 mg / kg Group 1), and administered at a rate of 50.6 mg / kg / hour to a second group of n=3 rats. (Total dose: 253 mg / kg; Group 2).

[0332] In this study, the nominal use of mCBS sodium salt was measured using a 5-hour continuous infusion. The doses were 40 and 80 mg / kg / hour (or 34 and 68 mg / kg as the free base, respectively). kg / hour).

[0333] A total of six blood PK samples were taken from each rat at time points ranging from pre-dose to 5 hours after the start of the infusion. The blood samples were processed into plasma and then analyzed by LC-MS / MS. The samples were analyzed for mCBS concentration using the method of Phoenix 64 Win Nonlin® software was used to measure concentrations over a 0-5 hour injection interval. Area under the curve (AUC) versus time was estimated. Steady-state concentration (Css) and clearance The value of s (Cl) was also estimated.

[0334] Concentrations of mCBS were below the limit of quantitation of the assay in plasma samples collected before dose administration. Concentrations were quantifiable in all plasma samples taken between 30 minutes and 5 hours after administration. The mean (±SEM) AUC values ​​for the 0-5 hour infusion interval were 260,000 (±30,200) ng.h / mL and 532,000 (±25,50 The mean (±SEM) values ​​of Css and Cl in Group 1 were 58,400 (±6,920) ng / ml and 0.601 (±0.081) l / h The mean (±SEM) values ​​of Css and Cl in Group 2 were 120,000 ( ±5,560) ng / ml and 0.571 (±0.025).

[0335] These results indicate that mCBS reaches steady-state plasma levels (Css) after 2 hours of infusion. Both Css and AUC in rats treated with 50 mg / kg / h Both were 2-fold higher than in rats treated with 25 mg / kg / h, indicating that systemic exposure Coagulation studies indicate that the effects of clotting are proportional to the dose used in this study. Rats treated with BS showed higher APTT scores compared to the reference range, which suggests that 4 shows that the presence of mCBS at a dose of 0.05 mg / mL prolongs clotting time.

[0336] Example 8: Plasma protein binding and metabolism of mCBS in humans, dogs, and rats In vitro studies This example describes in vitro studies performed to investigate the metabolism and plasma protein binding of mCBS. Specifically, this study demonstrated that hydroxybenzoates were expressed in human, dog, and rat liver microsomes. investigated the metabolism of mCBS in cerebrospinal fluids and concluded that no metabolism of mCBS was detected. In this study, the binding of mCBS to human, rat, and dog plasma proteins was investigated using ultrafiltration technology. and concluded that binding to plasma proteins was approximately 20% in all species tested. I did it.

[0337] β-O-methyl cellobioside sulfate in human, dog, and rat liver microsomes In vitro metabolism of In summary, this study is the first step in the determination of mCBS by rat, dog, and human liver microsomes. This was an in vitro study designed to investigate phase I and phase II metabolism. , indicates that no metabolism of mCBS was detected.

[0338] (i) β-O-methylcellobioside analogues in human, rat, and dog liver microsomes Phase I metabolism of phate Add a stock concentration of 25 μM mCBS dissolved in 50% methanol in water to a reaction tube. The reaction mixture (final volume 250 μL) contained the following: 0.1 M LiCl Phosphate buffer (pH 7.4), β-nicotinamide adenine dinucleotide 2'-phosphate NADPH (1 mM) and pooled human, rat, or dog liver microsomes ( After a 5-minute pre-incubation period, NADPH was added. The reaction was initiated by HCl, incubated at 37°C in a shaking water bath, and then added ice-cold acetone. The samples were then vortex mixed and quenched with 500 μL of nitrile. The mixture was centrifuged at 100 rpm for 10 minutes. Half of the sample (375 μl) was transferred to a glass tube. The sample was then transferred to mobile phase A (250 The final concentration of mCBS was 1 μM in the incubation medium. It was.

[0339] The reaction mixture was incubated for 1 hour and then incubated at 0, 15, 30, 45 and 60 minutes. A negative control (NADP) was run in parallel with the study samples. Positive control: midazolam, 25 μM (10 μL) for 1 hour. The final concentration of midazolam was 0.01% throughout the incubation period. It was 1 μM in the medium.

[0340] [Table 7]

[0341] (ii) Bioanalysis: calibration curve of mCBS mCBS (30 μg / ml - as free base) and midazoline in 50% methanol in water A mixed stock solution of ram (10 μg / ml) was prepared using 50% methanol in water. For CBS, the values ​​are 25, 20, 12.5, 6.25, 2.5, 1.25, 0.25 and 0. Diluted to 0.125 μg / ml and 8333, 6667, 4167, 2083, 833, Dilute aliquots of the working standard solution (1 0 μL) was placed in a plastic tube. Then, phosphate buffer (100 mM, pH 7.0) was added to the tube. An aliquot (190 μL) of 0.4 and microsomal phosphate buffer (100 mM, pH 7.0) were added. 7.4) solution (50 μL), followed by a 500 μL aliquot of acetonitrile. The tubes were vortexed and centrifuged at 14,000 rpm for 10 minutes. An aliquot (375 μL) was evaporated under a stream of nitrogen and reconstituted in mobile phase A (250 μL). , and directly injected (10 μL) into the LC-MS / MC system.

[0342] (iii) Phase II metabolism of mCBS in human, rat, and dog liver microsomes React with a 1 mg / ml stock concentration of mCBS dissolved in methanol (50 μL). The tube was evaporated to dryness and resuspended in the reaction mixture at a final concentration of 100 μM. (final volume 250 μl) contained: 0.1 M phosphate buffer (pH 7.4), Mg Cl2 (1 mM), uridine 5'-diphosphoglucuronic acid (UDPGA) (5 mM), liver Microsomes (0.3 mg / ml) and alamethicin (25 μg / mg protein). 5 After a 2 min pre-incubation period, start the reaction mixture by adding UDPGA. After incubating at 37°C in a shaking water bath, ice-cold acetonitrile (50 The samples were then vortexed and stirred at 14,000 rpm. Centrifuge for 5 minutes.

[0343] The reaction mixture was incubated in liver microsomes for 2 hours (n=3). Along with the study samples, a negative control (without UDPGA) and a positive control ( Paracetamol 100 μM) was incubated.

[0344] (iv) Mass spectrometry conditions The LC-MS / MS parameters used for the analysis of mCBS and midazolam are summarized in Table 6. do.

[0345] [Table 8]

[0346] (V) Results In the presence of human, rat, and dog liver microsomes under conditions that allow for phase I metabolism The metabolic stability of mCBS under these conditions is shown in Figures 2-4. Figure 2 shows that there was no significant metabolism of the compound. The measured concentrations (μM) of mCBS in the presence of rat liver microsomes (mean ± SEM) 3 is a graph showing the metabolic stability of mCBS in human liver microsomes. of mCBS in the presence of rat liver microsomes under conditions that allow for phase I metabolism. Measured concentrations (μM) (mean ± SEM) in human rat liver microsomes Figure 4 is a graph showing the metabolic stability of mCBS under conditions that permit phase I metabolism. The measured concentrations (µM) of mCBS in the presence of liver microsomes (mean ± SEM) 1 is a graph showing the metabolic stability of mCBS in dog liver microsomes.

[0347] (vi) Phase II metabolism Compounds were tested with human, rat, and dog liver microsomes under conditions that allow for phase II metabolism. The glucuronic acid metabolite of mCBS detected in the reaction sample after incubation with No matching ions were found. Paracetamol gluconate in the incubation medium The formation of lonide was measured by both neutral loss and MRM scan. I checked it out.

[0348] This in vitro study measured changes in the level of the free base form in the reaction medium. The metabolism of mCBS was evaluated by

[0349] Under conditions that allow for phase I metabolism, a 1-hour incubation with liver microsomes from the three species evaluated was observed. After incubation, the mCBS concentration did not decrease. The compound midazolam was almost completely metabolized in the presence of the three microsomes.

[0350] The concentration of mCBS was less than 0.6 μM at time zero in reactions with human liver microsomes. Note that the concentration of the added ATP was 1 μM. was seen in the negative control sample containing ribosomal but no NADPH, but It was not seen in the negative control sample containing PH but no microsomes. Microsomes are complex tissues containing a mixture of proteins, phospholipids, and fatty acids. Microsomal tissue components affect ionization in mass spectrometers, suppressing the signal. Because the composition of liver microsomes differs between species, ion suppression may be The apparent increase in compound concentration was stronger in the chromatosome and was independent of time and the presence of NADPH. This may result in a decrease in performance.

[0351] In summary, in microsomal reactions using human, rat, and dog microsomes, A second in vitro metabolism study (Phase II) detected no / minimal Phase I metabolism of the test article. ), the glucuronide metabolite also contains uridine 5'-diphosphoglucuronic acid (UDPGA ) was not detected after 2 hours of incubation with liver microsomes of various species in the presence of Ta.

[0352] Without being bound by any theory or particular mode of action, applicants believe that mCBS The lack of extensive in vitro metabolism is due to the presence of seven sulfonates that prevent enzyme access to this molecule. However, this result was not However, the possibility of desulfation in vivo following phase I or phase II metabolism cannot be excluded.

[0353] of mCBS binding to human, rat, and dog plasma proteins using ultrafiltration technology investigation In summary, this study investigated the binding of mCBS to plasma proteins in rats, dogs, and humans. This was an in vitro study designed to investigate the effects of hydroxybenzoates on the hydroxybenzoates of all the compounds tested. This indicates that in this species there was approximately 20% binding to plasma proteins.

[0354] In the work described here, existing methods were used to measure the internal standard deuterated mCBS (m This bioanalysis method was modified to include the use of CBS-d3. The plasma protein binding of mCBS to dog and human plasma was evaluated.

[0355] Centrifree (registered trademark) with a molecular weight cut-off point of 30,000 daltons Protein binding was assessed by ultrafiltration using a TETRA-Trademark ultrafiltration device. Briefly, known concentrations of mCBS (as the free base) were added to human, rat, and dog plasma. The sample was then incubated at 37°C for 20 minutes. The mixture was subjected to ultrafiltration to separate the protein-bound and unbound drug. , the extent of protein binding is defined as the percentage difference between the total and unbound drug concentrations. (including subtraction of nonspecific binding to the ultrafiltration device in the presence of phosphate buffer only).

[0356] The reagents used are detailed in Table 7 below. The LC-MS / MS parameters are summarized in Table 8.

[0357] [Table 9]

[0358] (i) Parameters of the analytical method

[0359] [Table 10]

[0360] (ii) Sample preparation mCBS stock solution (10 μL) was prepared to a final concentration of 200 ng / mL and 2000 ng / mL. of each test matrix (490 μL) listed in Table 9 below so that The solution was spiked.

[0361] [Table 11]

[0362] The spiked samples were then incubated in a water bath for 20 minutes at 37°C. Aliquots (500 μL) of samples from the test groups were transferred to ultrafiltration devices (n=3) and analyzed by 1 The mixture was centrifuged at 1000×g for 20 minutes. After centrifugation, an aliquot (50 μL) of the ultrafiltrate was collected. L) along with duplicate aliquots of pre-ultrafiltration samples from each test group for extraction. Transferred to another test tube.

[0363] (iii) Preparation of a calibration curve Aliquots of mCBS standard solution at 200 μg / mL were diluted as shown below.

[0364] [Table 12]

[0365] Aliquots (10 μL) of the resulting standard solutions were then added to the relevant matrices (human, rat, etc.). 490 μL of each of mouse and dog plasma and phosphate buffer (2.5 mM, pH 7.4) was added. Spike and mix well.

[0366] (iv) Extraction Aliquots (50 μL) of spiked standards and samples (incubation and limiting) After ultrafiltration) was mixed with 50 μL of 1 μg / mL internal standard solution. 150 μL) was added and the sample was immediately vortexed. The supernatant solution was transferred to a glass tube. The dried sample was then evaporated under airflow at 37°C. The dried sample was then diluted with mobile phase A' for 15 min. Reconstituted in 0 μL.

[0367] For quantification of samples before ultrafiltration, samples were compared with freshly prepared standards in plasma. The protein-poor samples taken after ultrafiltration were analyzed against the curve. Quantification was performed against a freshly prepared calibration curve in 100 M phosphate buffer, pH 7.4. For estimation of specific binding, samples both before and after ultrafiltration were subjected to a calibration curve in phosphate buffer. was quantified.

[0368] (v) Result: non-specific binding Nonspecific binding to the ultrafiltration device was 0.5% at 200 and 2000 ng / mL, respectively. and -1.97% (Table 10 below). Therefore, the binding of the compound to the ultrafiltration device was was thought to be negligible.

[0369] [Table 13]

[0370] (vi) plasma protein binding Comparison of the concentrations of mCBS spiked into human, rat, and dog plasma before and after filtration Plasma protein binding was calculated using the same assay. The extent of binding ranged from 16 to 23% for all three species. The plasma proteins of mCBS were similar (see Tables 11-13 below). Protein binding was similar at both concentrations tested.

[0371] [Table 14]

[0372] [Table 15]

[0373] [Table 16]

[0374] In this study, mCBS binding to human, rat, and dog plasma proteins was approximately 20%. The degree of binding was similar at 200 and 2000 ng / mL, and the binding to the ultrafiltration device was Non-specific binding was negligible.

[0375] Example 9: Dosage of mCBS after chronic continuous IV infusion for 7 consecutive days in rats and in vivo studies of toxicity This example demonstrates continuous (24 hour / day) Sprague-Dawley steroid therapy over a 7 day consecutive period. Dose range and toxicity study of mCBS when administered by IV infusion to Awley rats. This example outlines an in vivo study of this concentrated dose of mCBS in rats. When administered to rats under the same dosing regimen, the dose was 1394 mg / kg / day (free base; potency and There were no observable side effects of mCBS at doses (after adjusting for salt content). Indicates that.

[0376] The purpose of this study was to administer external steroids to Sprague-Dawley rats for 7 consecutive days. The drug was administered by continuous (24 h / day) intravenous infusion via a surgically placed catheter. The objective of this study was to determine the toxicity of the test article, mCBS, in this case.

[0377] Test and control dose formulations of mCBS (heptasodium salt mCBS 100 mg / kg of 1000 mg ... Groups of rats were administered the drug by intravenous infusion on day 1 of each study.

[0378] [Table 17]

[0379] Parameters monitored during this study included mortality, clinical observations, body weight and food intake. In addition, hematological, coagulation and clinical chemistry parameters were measured on day 9. The meter was evaluated relative to the start of the infusion to analyze the test article concentration in plasma. Blood samples were taken from the animals at time points. At termination (day 9), all animals were euthanized. Organ weights were measured for selected organs and gross lesions were noted. A selected list of such tissues was kept and prepared for microscopic evaluation.

[0380] Sprague-Dawley rats were given surgically placed catheters for 7 consecutive days. Administration of mCBS·Na by continuous (24 hours / day) intravenous infusion via a catheter , 5575 mg / kg / day mCBS (free base) after adjustment for potency and salt level One female died.

[0381] Plasma concentration versus time data showed that steady-state concentrations of mCBS persisted from 5 hours to 96 hours (or 1 hour). The results show that the AUC5-96 hours and Css were maintained over the infusion interval (68 hours). Mean (±SEM) values ​​increased linearly with dose.

[0382] In animals of both sexes treated with mCBS (free base) at 5575 mg / kg / day At doses ≥ 3484 mg / kg, a decrease in body weight gain was observed, correlating with a decrease in food intake. Increases in leukocyte lineages were observed in animals of both sexes at 5575 mg / kg / day. In males at 1 / day, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume and mean red blood cell volume were measured. A decrease in blood hemoglobin concentration and an increase in reticulocytes were observed. / kg / day in males and at doses ≥ 3484 mg / kg / day in females In males administered 5575 mg / kg / day, an increase in thromboplastin time was observed. , liver enzymes, alanine aminotransferase and aspartate aminotransferase In addition, the serum cholesterol and triglycerides increased at 5575 mg / kg In animals of both sexes at 1 / day, urea increased and total protein and albumin It decreased.

[0383] In the kidney, in animals treated with ≥ 2178 mg / kg / day of mCBS (free base), Vacuolation / coarsening of the proximal tubules was observed bilaterally. The pulmonary fibrosis was not associated with any other pathological changes. This correlated with increased kidney weight.

[0384] In the spleen, in animals treated with ≥ 1394 mg / kg / day of mCBS (free base), , observed an accumulation of foamy macrophages associated with an increase in apoptotic cells in the red pulp. These splenic changes include hyperplasia of interstitial cells, cellularity / size of the white pulp (germinal centers), and Microscopic findings of the spleen included an increase in spleen weight, sometimes accompanied by increased blood volume, capsular fibrosis, and increased hematopoiesis. was correlated with

[0385] In the liver, animals treated with ≥ 1394 mg / kg / day of mCBS (free base) Increased sinusoidal juxtaposition (at ≥1394 mg / kg / day), increased sinusoidal juxtaposition (at 5575 mg / kg / day) Accumulation of foamy Kupffer cells, often associated with extramedullary hematopoiesis, was observed. Single-cell necrosis was observed in animals treated with mCBS (free base) at doses ≥ 2178 mg / kg / day. In animals treated with 2178 and 5575 mg / kg / day, focal or multifocal lesions were observed. In the 5575 mg / kg / day group, liver changes were more pronounced in males than in females. and / or frequent, both male animals adequately exposed to mCBS showed minimal to moderate showed liver changes.

[0386] In various lymph nodes (bronchial, mandibular, mediastinal, mesenteric, pancreatic, hepatic and / or auricular), ≥ In many animals treated with 1394 mg / kg / day of mCBS (free base), foamy Accumulation of macrophages was observed.

[0387] Accumulation of foamy macrophages (in the spleen and lymph nodes), foamy Kupf (in the liver) The findings of fer cell accumulation and proximal tubular vacuolization / roughening (in the kidney) were observed with the test article mCB. S and / or its degradation products are captured by the mononuclear phagocyte system (in the spleen, liver, and lymph nodes) This suggests that the drug was most likely to be absorbed into the renal tubular epithelium and then transferred to the renal tubular epithelium. Without being bound by any particular theory or particular mode of action, applicants believe that these However, the mononuclear phagocyte system acts as a result of the phagocytosis and elimination of the test substance and / or its degradation products. We reasoned that this most likely represents adaptive changes in the spleen and kidney. [Increase in apoptotic cells, hyperplasia of interstitial cells, cellularity of the white pulp (germinal center) / cytoplasm] increased hematopoiesis and capsular fibrosis] and other findings in the liver (increased sinusoidal juxtaposition cells and Applicants reasoned that the increased hematopoiesis (hemopoiesis and extramedullary hematopoiesis) is also an adaptive response to an activated phagocyte system.

[0388] However, no significant adverse events were observed at 2178, 3484, and 5575 mg / kg / day (free base). Single-cell necrosis in the liver was considered potentially harmful. At 75 mg / kg / day, focal or multiple focal necrosis of the liver was observed. , which was not seen in animals treated with 3484 mg / kg / day and may occur naturally, Considering the fact that this finding was observed only in animals treated with mCBS, the relationship between the test article and The relevance cannot be ruled out.

[0389] As a result, histopathology observed in the liver at ≥ 2178 mg / kg / day (free base) Due to biological changes, the no-observed-adverse-effect level (NOAEL) of mCBS was 1394 mg / kg / day in this study. The dose was determined to be mg / kg / day (free base).

[0390] Example 10: Prolonged administration of mCBS following continuous IV infusion for up to 14 consecutive days in dogs In vivo studies of dosage and toxicity This example demonstrates the efficacy of steroids when administered by continuous IV infusion to beagle dogs over a 14 day period. This example outlines an in vivo study investigating the dose range and toxicity of mCBS. Under this intensive dosing regime of mCBS in dogs, 2788 mg over 14 days Continuous intravenous infusion (24 hours / day for 48 hours) of mCBS at 1 / kg / day was well tolerated. The data showed good tolerance to the disease, and mortality, clinical observations, body weight, food intake, clinical pathology (hematology, coagulation and clinical signs) were analyzed. (chemical), indicating that there was no effect on organ weights or macroscopic assessment.

[0391] Continuous infusion of mCBS in dogs In summary, this study demonstrates the maximum efficacy of mCBS for use in a 14-day continuous infusion dog study. A dose range study of mCBS in dogs by continuous infusion was conducted to select the appropriate dose. Similarly, the dose selected was determined to be at least equal to the MTD or at least 300 μg / ml of mCBS (immune system). The results showed that the hydroxybenzoates produced 100% hydroxybenzoates (i.e., approximately three times the target human plasma level) in the 200% hydroxybenzoates range.

[0392] Specifically, Beagle dogs were administered up to four dose levels for 48 hours per dose level. Administered by continuous (24 h / day) intravenous infusion via a surgically placed catheter This study was also conducted to determine the maximum tolerated dose (MTD) of the test article mCBS in Ta.

[0393] Phase 1: Dose Escalation The mCBS dosage formulations were administered at up to four dose levels as described in Table 15 below. Continuous (24 hours / day) intravenous infusion for 48 hours per dose level It was administered to guru dogs.

[0394] [Table 18]

[0395] Parameters monitored during this phase of the study included mortality, clinical observations, and physical The following time points were taken from each animal to determine the mCBS plasma levels: Serial blood samples were taken from: 24 and 48 hours after the start of infusion for each dose level. On Day 1, before administration of the new dose level on Days 3, 5, and 7, and on Day 9, clinical Blood samples were taken from each animal for bed pathology evaluation (haematology, coagulation and clinical chemistry).

[0396] No dose-limiting toxicity (adverse clinical symptoms) was observed after escalating doses up to the highest dose level. Therefore, the maximum tolerated dose (MTD) was determined by continuous (24 hours / day) intravenous infusion over 48 hours. The steady-state plasma concentration of mCBS was 2788 mg / kg / day. (Css) was higher than 300 μg / mL, which is three times higher than the target plasma concentration in humans. Therefore, this dose was not escalated further.

[0397] Quantifiable levels of mCB were detected in plasma samples collected from all animals at each time point. S was detected, indicating that the animals were properly administered mCBS. Average concentration in plasma (C ss ) ranged from 53.8 to 358 μg / mL, and mCBS was 222 (Cl) was removed at a rate of ~450 mL / hr / kg.

[0398] Continuous infusion of mCBS in dogs In summary, this study demonstrated that the maximum tolerated dose (MTD) identified in the STX-102 study described above was was used to investigate continuous infusion of mCBS in dogs.

[0399] After confirmation of the MTD (2788 mg / kg / day), the dogs were transferred to Phase 2 of this study. Dosing resumed at the MTD for an additional 6 days of continuous infusion, as shown in Table 16 below.

[0400] [Table 19]

[0401] Parameters monitored during this phase of the study included mortality, clinical observations, and physical These included changes in body weight, food intake, clinical pathology (haematology, coagulation and clinical chemistry) and organ weights. Serial blood samples for toxicokinetic analysis were taken from each animal at the following time points: 24 and 48 hours after initiation, immediately before the end of the infusion, 15, 30 minutes and 1, 1.5 minutes after the end of the infusion. 2, 3, 4, 6, 24 hours.

[0402] On Day 8, after completion of an additional 6 days of continuous infusion and collection of the final toxicokinetic blood sample. All animals were euthanized and subjected to necropsy examination. Histology was performed on the liver and kidneys of all animals. A clinical examination was performed.

[0403] Continuous intravenous infusion of mCBS at 2788 mg / kg / day for 6 days (over 144 hours) ( The study (24 hours / day for 48 hours) was well tolerated and no significant changes in mortality, clinical observations, body weight, food intake, or other parameters were observed. There were no effects on dose, clinical pathology (haematology, coagulation and clinical chemistry), organ weights or macroscopic assessments.

[0404] Microscopic findings include vacuolization / ocularization of the proximal renal tubules in the kidney and foamy Kupf's lesions in the liver. Accumulation of fer cells was noted but was considered non-harmful or adaptive.

[0405] Toxicokinetic analysis was performed using steady-state plasma concentrations (C ss ) is 223-246μg / mL range and AUC 0~144 (AUC 0~168 ) is 43800 (44100) ~ 48 300(48800) hours * After the end of the infusion, mCB S plasma concentrations were estimated at approximately 1 hour for both animals. 1 / 2 The value dropped rapidly. The BS was eliminated at a rate of 472-522 mL / h / kg (Cl). The volume of distribution (Vz ) ranged from 740 to 741 mL / kg, which indicates that mCBS was widely distributed among tissues. There were no notable differences between the sexes.

[0406] Examples 11-22: Comparison of cellobiose sulfate and mCBS The following study compares cellobiose sulfate (CBS) and mCBS. BS is much more chemically stable than CBS and therefore represents a better drug candidate. The methodology used in Examples 11 to 22 is described below.

[0407] Methods and Materials for Examples (11-22) Below Human subjects. All human-related research is conducted under the supervision of ANU Health Human Research Approved by the University of California Ethics Committee for in vitro studies. Healthy adult donors were used as a source of red blood cells and platelets.

[0408] Animals. All animal experiments were carried out under the supervision of Australian National Universities. ity Animal Experimentation Ethics Commit Pathogen-free male and female C57BL / 6 mice (6-8 weeks old) were used. Female BALB / c mice (5-6 weeks old) and male Wistar rats (250-300 kg body weight) were used. 350g) at the Australian National University obtained from the Stratian Phenomics Facility.

[0409] Cell line and cell culture conditions. Human microvascular endothelial cells-1 (HMEC-1) (blood type O) (which does not react with anti-blood group antibodies in human serum) was supplied by ATCC and 0% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 IU ml - 1 of penicillin and 100 μg ml -1 MCDB supplemented with streptomycin 1 Human umbilical vein endothelial cells (HUVECs) were cultured in medium 31 as previously described. Sea urchin (Jaffe, EA) Biology of endothelial cell s.(Martinus Nijhoff Publishers; Distribu tors for the United States and Canada,Kl (Boston, 1984), established from primary cultures and containing 20% ​​FCS, 2 mM of L-glutamine, 100 IUml -1 of penicillin, 100 μg ml -1 Strep Mycin, 130 μg ml -1 of heparin and 1.2mg ml -1 Endothelial cell growth supplement The cells were cultured in Medium 199 supplemented with Sigma-Aldrich Chinese hamster ovary (CHO)-K1 cells and cells lacking HS and GAG were used. Xylotransferase-1-deficient CHO K1 cells (pgsA-745 cells) were supplied by ATCC and supplemented with 5% FCS and antibiotics. All cell lines were grown in RPMI-1640 medium supplemented with 5% CO₂ at 37°C. Incubated under CO2 and ambient O2, and then incubated with MycoAlert Assay Kit (L onza) were repeatedly tested for mycoplasma.

[0410] Assay for histone-mediated cytotoxicity. Calf thymus histones (Sigma-Aldrich) were used. To determine the cytotoxicity of 100-800 μL of histones, various concentrations of histones (100-800 μL) were added. gml -1 ) in a 96-well plate containing HMEC-1 or HUVEC (1 × 10 6 ml -1 ) and incubated at 37°C for 1 hour. , propidium iodide (PI; 2.5 μg ml ) to detect dead cells. -1 )(The and C to detect viable cells. alcein-AM(0.04μM)(ThermoFisher Scientifi c) for 5 min at 37°C, placed on ice, and then incubated with HCl for 5 min at 37°C for 5 min. Dead and viable cells were identified by flow cytometry using a gating strategy developed The percentage of cells that were activated was determined. In the inhibition assay, HMEC-1 cells were incubated with various concentrations of compounds (12. 5 to 400 μg ml -1 ) for 1 hour at 37°C in the presence of histones (400μL) gml -1 After incubation with PI and calcein-AM, The HMEC-1 cytotoxicity at each compound concentration was calculated using the formula:

number

[2017] , Flavobacterium Actinium heparinase (HPNSE) I, II, and III (Sigma-Aldrich) drich) or human platelet heparanase (HPSE) (Freeman, C. & Par ish,CRHuman platelet heparanase:purifi cation, characterization and catalytic ac tivity.Biochem J 330(Pt 3),1341-1350(199 8)) to deplete cell surface HS, and then perform the same procedure as described above for HMEC-1. As described, sensitivity to histone-mediated cytotoxicity was examined. Suspension of live CHO-K1 and HS / GAG-deficient pgsA-745 CHO-K1 cells The suspensions were compared for susceptibility to histone-mediated cytotoxicity.

[0411] Lipid bilayer assay. As previously described (Rebbeck, RT et al.). l.The beta(1a)subunit of the skeletal DH PR binds to skeletal RyR1 and activates the channel via its 35-residue C-termina l tail.Biophys J 100,922-930,doi:10.1016 / j.bpj.2011.01.022(2011).) and prepared at 150 mM or 250 mM. This is an artificial lipid bilayer symmetrically separated in a solution of 100 mM KCl (pH approximately 5.5). Histone (1 μM, 15.2 μg ml -1 ) was added alone or at 10 μM CBS (3.5 μg ml -1 ) or 10 μM MTS (5.1 μg ml -1 ) and one After the initial incubation for 0.5–3 hours, the bilayer was incubated with histones. Current was recorded continuously until the line broke or the experiment was terminated.

[0412] Calcium flux studies in endothelial cells. HMEC-1(2 x10 7 ml -1 ) for 60 min at 37 °C with Indo-1 AM (5 μM) (Ther (Tellam, RL & Paris) h,CRThe effect of sulfated polysacchar ides on the free intracellular calcium i on concentration of lymphocytes.Biochim Biophys Acta 930,55-64(1987)&Weston,SA ,Tellam,RL&Parish,CRDextran sulfate induces changes in the free intracellular r calcium ion concentration of a subpopu lation of immature thymocytes.Immunol Ce ll Biol 69(Pt 6),369-376,doi:10.1038 / icb .1991.53(1991)). In RPMI-1640 medium supplemented with 5% FCS. After three washes with HCl, the cells were resuspended in ice-cold HEPES-buffered saline supplemented with 10 mM HEPES. Saline (NaCl 8g -1 , KCl 0.4gl -1 , CaCl2 0.2gl -1 , MgCl2.6H2O 0.2gl -1 , D-glucose 1.8gl -1 , pH 7. 4) 4 x 10 6 ml -1 The cell suspension was kept on ice and incubated for no more than 3 hours. Flow cytometry was used to measure intracellular Ca 2+ The flow was monitored The cells were pre-equilibrated and placed in an external sheath connected to a heated water bath. The cells were maintained at 37°C during analysis. After removal of debris and clumped cells, 2 min was allowed to stand before adding histones in the presence / absence of novel compounds. Basal Ca 2+ The levels were monitored at a constant flow rate (approximately 300 events / sec). Ca at 1, 4, and 10 min after histone addition 2+ The levels were measured. 2+ Ca flux 2 + Ca for unbound Indo-1 2+ Geometric mean fluorescence intensity (GMFI) of bound Indo-1 ) was determined as an increase in the ratio of

[0413] In vitro red blood cell microscopy, aggregation, fragility and deformability assays. Human red blood cells The inventors have investigated histone-mediated aggregation of β-glucan and its inhibition by various compounds. As already reported by some of the authors (Kordbacheh, F., O'Mear a,CH,Coupland,LA,Lelliott,PM&Paris h,CRExtracellular histones induce eryt hrocyte fragility and anemia.Blood 130,2 884-2888,doi:10.1182 / blood-2017-06-79051 9 (2017)), forward and side scatter parameters or red blood cell autofluorescence. Flow cytometry based on either ELISA or ELISA as previously described (Yabas, M .et al.Mice deficient in the putative ph ospholipid flippase ATP11C exhibit alter ed erythrocyte shape, anemia, and reduced erythrocyte life span.J Biol Chem 289,19 531-19537,doi:10.1074 / jbc.C114.570267(20 14)) Scanning electron microscopy was used to detect the presence or absence of inhibitors. The fragility of red blood cells induced by stone has already been reported by some of the present inventors. (Kordbacheh, F., O'Meara, C.H., Coupland ,LA,Lelliott,PM&Parish,CRExtracell ular histones induce erythrocyte fragili ty and anemia.Blood 130,2884-2888,doi:10 (See .1182 / blood-2017-06-790519(2017)) Finally, the transformation of erythrocytes in the presence of histones was quantified using a shear stress assay. The reduction in erythrocyte function and the effect of inhibitors on this process were assessed by measuring the passage of red blood cells through an artificial human spleen. The sensitivity was evaluated by measuring the sensitivity (Deplaine, G. et al., The Sensin g of poorly deformable red blood cells b y the human spleen can be mimicked in vi tro.Blood 117,e88-95,doi:10.1182 / blood-2 (See 010-10-312801(2011)).

[0414] In vitro platelet aggregation and degranulation assay. For aggregation studies, a two-stage incubation at room temperature was performed. Centrifugation (200 × g for 20 min, followed by 80 × g for 15 min for platelet-rich plasma extraction) Platelets were isolated from human whole blood collected in a sodium citrate vacutainer at 100 × g. The platelet pellet was resuspended in Hanks' balanced salt solution containing calcium and magnesium. Histones were added and the ink was analyzed in the presence or absence of compounds at the indicated concentrations. Flow using the characteristic log FSC vs. log SSC discrimination of platelets Cytometric analysis of platelets was performed to assess the extent of platelet aggregation after 15 minutes of exposure to histones. The platelet aggregation is assessed and an increase in the geometric mean of log FSC indicates platelet aggregation.

[0415] For the platelet activation assay, whole blood collected in a sodium citrate vacutainer was analyzed by Chr Chrono-Lo using o-Lume reagent (Chrono-Log Corp) g Platelet degranulation was monitored using the luminescence mode of the Model 700. Pre-warmed blood (420 μl) was added to saline (3 μL) in situ using a stir bar. 100 μl) was added. Then, Chromo-Lume reagent (100 μl) was added. After a 2-minute incubation, histones ± compounds diluted in water were added at the indicated concentrations. The results were expressed as a percentage of the histone + saline control. The data were expressed as ATP release calculated from the ATP concentration.

[0416] In vivo histone toxicity assay. BALB / c female mice (5-6 weeks old) (C57B At this young age, compared with L / 6 mice, they are more susceptible to histone-induced anemia. and easy to inject IV), phosphate phosphate was measured 10 minutes after ip injection of the test compound at the indicated concentrations. Histones (50 mg kg ) in buffered saline -1 ) was injected intravenously. After 10 minutes, retro-orbital bleeding was performed and collected. Blood was added to acid citrate dextrose (ACD) and this 10-minute blood sample was analyzed by A Platelets and The blood was analyzed for red blood cell content. Ten minutes after the histone injection, the spleen was also removed and the hepatocytes were analyzed. Splenic hemoglobin was measured using a hemoglobin assay kit (Sigma-Aldrich). The content was quantified. For the 4-hour blood samples, male C57 / BL / 6 mice (6-8 weeks old) were used. ) were injected with test compounds and histones as described above, and blood was collected for subsequent biochemical testing. Plasma was isolated and cryopreserved, and markers of liver injury (alanine aminotransferase, A LT), markers of kidney damage (creatinine, Crea) and markers of general tissue damage ( Lactate dehydrogenase (LDH) was detected by the Department of Pathology. , measured by The Canberra Hospital.

[0417] Murine deep vein thrombosis (DVT) model. The procedures used have been largely described previously. (Brill, A. et al. Neutrophil extracellular matrix ular traps promote deep vein thrombosis in mice.J Thromb Haemost 10,136-144,doi: See 10.1111 / j.1538-7836.2011.04544.x(2012) Briefly, 8-week-old male C57BL / 6 mice were anesthetized and subjected to laparotomy. The intestine was then removed from the body, and after gentle separation from the abdominal aorta, the intestine was inserted into the inferior aorta just below the renal vein. The vein (IVC) was ligated to approximately 10% patency, and all involved IVC tributaries were ligated. The peritoneum and skin were closed, and then all mice were administered histones (10 mg kg ) via the tail vein. - 1 ) or an equal volume of saline was injected intravenously, followed 5 minutes later by the test compound (50 mg kg -1 Mice were monitored for 48 hours and were then given an intravenous injection of either thrombus or saline. The patient was then re-anesthetized and reopened, and any thrombus that had developed distal to the IVC stenosis was removed for analysis. Sham-operated control animals underwent laparotomy and 90% IVC ligation. However, this ligature was removed immediately after IVC occlusion.

[0418] Rat cecal ligation and puncture (CLP) assay for sepsis. As described (Hubbard, WJ et al. Cecal ligat ion and puncture.Shock 24 Suppl 1,52-57( 2005) was performed in male Wistar rats. Test compound (50 mg kg -1 ) or an equal volume of saline alone (control cohort). 5 min before CLP and 5, 10, and 15 h after surgery until the end of the experiment at 20 h. Sham CLP rats underwent the same procedure, but the cecum was not ligated or punctured. The rats were administered saline at the same time as above. 20 hours) or if pathological conditions ethically required euthanasia, rats were anesthetized and subsequently artment of Pathology,The Canberra Hospit Cardiac puncture for analysis of liver (ALT) and kidney (creatinine) function by al Blood samples from saline-treated control CLP animals were collected in EDTA. Because of the tendency for clots to form in the tubing (despite the presence of EDTA), Analysis of plasma samples from the specimens was unsuccessful.

[0419] A rat cardiac IRI model. The method used is based on a combination of previously published procedures. Takada, Y., Hashimoto, M., Kasahara, J., Ai hara, K. & Fukunaga, K. Cytoprotective effect of sodium orthovanadate on ischemia / rep erfusion-induced injury in the rat heart involves Akt activation and inhibition of fodrin breakdown and apoptosis.J Phar macol Exp Ther 311,1249-1255,doi:10.1124 / jpet.104.070839(2004)&Hale,SL,Dae,MW .&Kloner, RAHypothermia during reperfus ion limits 'no-reflow' injury in a rabbit t model of acute myocardial infarction.C Cardiovasc Res 59, 715-722 (2003). Wistar rats were anesthetized with isoflurane, intubated via tracheotomy, and administered 1 ml 15 0g -1 tidal volume and 65 breaths per minute -1 Artificial respiration was administered at a respiratory rate of . Oxygen supplementation was administered. A left hemithoracotomy was performed to allow visualization of the left ventricle. The coronary artery plexus (LCA) was atraumatically snare-snapped for 30 minutes before reperfusion for 30 minutes. The ischemia was confirmed by myocardial hyperemia. -1 ) or An equal volume (200 μl) of saline was added 5 min before the release of the snare for the reperfusion phase. The injection was into the lumen of the left ventricle (confirmed by aspiration).

[0420] At the end of reperfusion (30 min), thioflavin S (1 ml 200 g body weight) was administered into the left ventricular lumen. -1 ) was gently injected to define the area of ​​microvascular occlusion (MVO) within the ischemic zone (IZ). This IZ region was defined as the area where the non-invasive snare reocclusion and CD-6800 (Unisoni) cs) Blue microparticles dispersed in a solution into the left ventricle by sonication using an ultrasonicator. The concentration of β-glucan was determined by injection of particles (Unisperse Blue, BASF). The liver was removed from the chest, rinsed with isotonic saline, and then noninvasively placed at right angles to the interventricular line. A 2 mm section was cut distal to the snare. This method was performed by weighing and measuring the area of ​​the MVO. ) and bright light (IZ region) (Sony Handycam®, Four myocardial sections were prepared using a Zeiss 60x optical zoom, and then tetrazolium chloride (T The area of ​​necrotic myocardium was determined by incubation in 1000 μL of PBS. The areas of IZ, MVO, and necrosis were quantified using Image J (Freeware). .

[0421] A rat ischemia-reperfusion tissue flap model was used. The procedure used was largely based on previously described methods. (Askar, I., Oktay, MF, Gurlek, A. & Bac, B. Pr. otective effects of some antiplastic agents on ischemia-reperfusion injury in epigastric island skin flaps.Microsurge ry 26,193-199,doi:10.1002 / micr.20193(200 6) Briefly, male Wistar rats were anesthetized, locally depilated, and 3 cm A 6 cm × 6 cm fascial flap was removed, leaving the vascular pedicle intact. The inferior epigastric artery was clamped and the inferior epigastric artery was A fine rubber sheet was placed under the valve to prevent oxygen diffusion from the tissues. The valve was re-sutured into place. To allow blood flow to return to the valve, 10 hours after application, The clamp was removed at 100°C. -1 ) or saline solution, The rats were administered i.p. 5 min before surgery and 5 min after the removal of the clamp. The rats were given additional compound or saline ip at 48 hours and 72 hours after administration. The rats were monitored for the total experimental period. , had a tissue flap placed under a rubber band before being extracted and re-suturing, but the vessels were not clamped. At the end of the experimental period, the survival rate of the valve was evaluated. The ratio of black necrotic or reddened areas to pink viable areas was determined. Despite the application of acetaminophen and the use of analgesia as a precipitating agent, few rats were found to have autosomal recessive valvular sympathetic syndromes. He had to be euthanized early when he repeatedly started eating.

[0422] EAE model of multiple sclerosis. On day 1, mice were treated with complete Freund's adjuvant (Sigma) 115 μg / mouse myelin oligodendrocyte glycoprotein (MOG35-5 5 genscript) was administered subcutaneously to 8-12 week old C57Bl / 6 mice. EAE was induced in mice. On days 0 and 2, 100 mg of 300 ng / mouse in PBS was administered. Cough toxin (List Biological Laboratories) was administered intraperitoneally (i .p.) injected. 50 mg / kg mCBS in PBS or PBS alone (vehicle) The mice were administered i.p. daily on days 0-9. Mice were monitored daily for signs of disease. The mice were scored on a scale of 0 to 5 based on the physical symptoms of the disease. Scored: 0, clinically normal; 1, tail flaccidity and / or ataxia; 2, hind limb weakness; 3 , hind limb paralysis; 4, hind and forelimb paralysis; and 5, moribund.

[0423] Statistical analysis was performed using Prism software (Graphpad Software). Statistical tests were performed and graphs were generated using the data, and details of the tests used are included in the figure legends.

[0424] In vitro evidence of the biological effects of mCBS Examples 11-14 below demonstrate the biological efficacy of mCBS in neutralizing free extracellular histones. We provide in vitro evidence of this effect.

[0425] Example 11: mCBS protects endothelial cells from histone toxicity This example demonstrates that mCBS protects human endothelial cells from histone damage and The protective effect of mCBS against histone-induced damage to the nuclei was concentration-dependent. This example demonstrates that mCBS inhibits damage in a subset of endothelial cells exposed to histones. We also show that it is possible to invert

[0426] Endothelial cells line the lumen of blood vessels and are essential for the integrity of the vascular system, transporting blood cells. It generates many signals between the underlying tissues to allow blood to pass through, acting as an anticoagulant surface for blood to flow through. Histones damage the cell membrane of endothelial cells, inducing their death, and thus There is a loss of microvasculature integrity and anticoagulant properties of the endothelium, which leads to widespread clot formation. This impairs the delivery of oxygen- and nutrient-containing fluids to vital organs, which in turn damages these organs. This causes the condition to become insufficient.

[0427] To investigate whether mCBS protects human microvascular endothelial cells (HMECs) from histone damage, To evaluate this, two fluorescent dyes, calcein-AM and propidium iodide (PI), were used. Endothelial cell health was determined in vitro using calcein AM. Healthy viable cells were identified using calcein AM. In damaged or dead cells, the opposite was true. The uptake / exclusion of these dyes was measured using flow cytometry, as shown in Figure 5. The cellular concentrations were measured and visualized using a confocal microscope.

[0428] With reference to FIG. 5, cultured HMECs were treated with histone HCl in an equal volume of water (panels A and E of FIG. 5). At 400 μg / mL of mCBS (Panels B and F of Figure 5), 400 μg / mL of mCBS (Panels C and G of Figure 5) or mCBS 25 μg / mL and hCG The cells were treated with 400 μg / mL of estradiol (Panel D of Figure 5) for 60 minutes, followed by dye calcification. Labeling with thiazolinone-AM and PI followed by flow cytometry (Panels A, B, C, and D) The extent of dye uptake was analyzed using confocal microscopy (Panels E, F, and G). As shown in Figure 5, cultured untreated human microvascular endothelial cells (HMECs) were largely viable. The cells contained 76% calcein-AM and 19% PI (Fig. 5A). However, when exposed to histones (400 μg / mL), 37% remained viable. However, 56% of HMECs incorporated PI (Figure 5B). When CBS (100 μg / mL) was added, 74% remained viable and were able to uptake PI. 20% of the cells died based on their accumulation, so mCBS is not mediated by histones. This protective effect was concentration-dependent, and A lower dose of mCBS (25 μg / mL) resulted in a reduced level of protection (Figure 5D). The effects of histones and mCBS on ECs were compared with healthy untreated endothelial cells (Figure 5E) and histone mCBS-treated endothelial cells exposed to α-glucan accumulate green fluorescent calcein-AM dye (Fig. 5G), whereas untreated histone-exposed endothelial cells take up the red fluorescent dye PI (Figure 5F). It can also be seen using a spot microscope.

[0429] Cultured HMECs were incubated with 400 μg / mL of histones after the addition of increasing concentrations of mCBS. and then analyzed using flow cytometry for calcein-AM (viability) or PI ( The effects of histone damage on HMECs were analyzed by analyzing the uptake of mC. The dose-dependent protective effect of mCBS is shown in Figure 6, which shows that increasing concentrations of mCBS significantly reduced the number of surviving cells. The increase in cells (calcein-AM uptake) and the decrease in dead cells (PI uptake) were observed. Therefore, these results suggest that HMECs respond to histone-induced damage. These results show that the protective effect of mCBS on IL-1 is concentration-dependent.

[0430] We evaluated whether mCBS could reverse damage in endothelial cells following exposure to histones. To evaluate the effect of histone depletion on HMECs, cultured HMECs were exposed to 400 μg / ml of histone for 60 min. Then, the cells were treated with mCBS (100 μg / ml) for 10 minutes, and then with HCl for the final 5 minutes. Calcein-AM and PI were added to the cells. The cells were then analyzed by flow cytometry. The results are shown in Figure 7. The results are important, especially for clinical In a clinical setting, mCBS could reverse the damaging effects of histones in a subpopulation of HMECs. In this situation (histones were added to cultured HMECs, and then 60 minutes later, 10 minutes later), mCBS was added for 5 min, and calcein-AM and PI were added for the last 5 min. In this study, approximately 30% of HMECs were found to undergo a PI uptake and subsequent PI exclusion and calcification after mCBS treatment. Sein-AM uptake was reversed.

[0431] Example 12: mCBS and CBS inhibit histone-induced red blood cell aggregation and lysis Prevent, reduce, and even reverse This example demonstrates that mCBS prevents histone-induced RBC aggregation and Furthermore, this study demonstrates that the compound inhibits RBC aggregation induced by acetaminophen in a dose-dependent manner. An example is mCBS, an effect that is exacerbated by higher shear flow rates and duration of shear exposure. We show that it inhibits RBC fragility induced by certain histones. The examples show that mCBS inhibits histone-induced RBC sensitivity to lysis and aggregation. We show that the passivity can be almost completely reversed.

[0432] Red blood cells (RBCs) are involved in oxygen transport to tissues and act as scaffolding for clot formation. Contributes to clot formation by providing membrane proteins. The soft structure allows RBCs to squeeze through narrow capillaries and experience rapid flow in the bloodstream. RBCs lack a nucleus, allowing them to withstand shear forces. or if RBCs are unable to respond to injury through apoptosis and instead undergo deformity However, damaged RBCs are removed by macrophages in the spleen. Before reaching the In cases of sepsis, etc., histone levels increase, making the blood vessels more susceptible to endothelial dissolution due to exposure to shear forces. Anemia is often observed in diseases that increase blood pressure.

[0433] The damaging effect of histones on RBCs was studied along with the ability of mCBS to reverse this effect. In an early study of isolated human RBCs incubated with histones, flow cytometry was performed. Using fluoroscopy and electron microscopy, significant aggregation was demonstrated. The RBC agglutination effect of stone could be prevented by treatment with mCBS (CBS In this case, isolated human RBCs were analyzed after no treatment (Panels A and D). After 60 min of incubation with histones (400 μg / mL) (Panel B and and E) and mCBS (200 μg / mL), followed by histone (40 The log FSC for the extent of aggregation immediately after exposure to 0 μg / mL (Panels C and F) Flow cytometry was used using the log autofluorescence (FL-1 channel) parameter. The cells were analyzed using a scanning electron microscope (Figure 8, panels A-C) and visualized using a scanning electron microscope (Figure 8, As can be clearly seen from the results in Figure 8, the histone-induced RBC clumping is prevented by mCBS.

[0434] As shown in Figure 9, histones induced RBC aggregation in a dose-dependent manner, and mC It is further confirmed that BS and CBS can significantly reduce this aggregation again in a dose-dependent manner. Subsequent experiments were performed. Specifically, in this case, isolated human RBCs were incubated with various Concentrations of histones (0, 1.25, 25, 50, 100, 200, 400 and 800 μg / mL), and the level of autofluorescence (FL-1) indicated RBC aggregation, as shown in Figure 9A. The degree of cytotoxicity was then measured. Various concentrations of mCB were then added before the addition of 400 μg / mL of histones. S and CBS (0, 12.5, 25, 50, 100, and 200 μg / mL) were added to RBCs. This was repeated except that 100% fluorescein was added. Again, autofluorescence (FL- The degree of RBC aggregation was measured by the level of 1). The results showed that both mCBS and CBS , showing that it inhibits histone-induced RBC aggregation in a dose-dependent manner.

[0435] In addition, the increased activity of the histones when incubated with increasing concentrations for 60 min Under applied shear force (pipetting speed) and shear exposure (repetition of pipetting) The susceptibility of RBCs to lysis was determined using a robotic pipetting system. The results are shown in Figure 10. Specifically, 60% saline (normal saline at a ratio of 6:4) Isolated human RBCs diluted in saline:water were treated with increasing concentrations of histones ( 0, 1.25, 25, 50, 100, 200, 400 and 800 μg / mL) The cells were incubated and then transferred to the robotic system at increasingly faster flow rates (mm 100 mm / s) (Figure 10A) or various repetitive pipetting at a flow rate of 100 mm / s. (Panel B) or at various concentrations (0, 12.5, 25, 50, 100, and 2 100 μg / mL) of mCBS (Figure 10C), followed by 100 μg / mL of HCl for 60 min and 100 μg / mL of HCl. Exposure to 400 μg / mL histones at a shear rate of 100 m / s and 40 pipetting repetitions The supernatant from each sample was then analyzed by A540nm as an indicator of the extent of RBC lysis. The hemoglobin content was measured at 1000 kcal / kg.

[0436] These experiments were performed in physiological saline at 60% tonicity to induce baseline stress in the RBCs. The hemoglobin level in the supernatant was measured at 540 nm. These results suggest that increasing histone concentration leads to increased shear force and shear resistance. The results show that RBC susceptibility to lysis increases dramatically under shear exposure (Figures 10A and B). Treatment of RBCs with mCBS (and CBS, not shown) prior to histone exposure significantly reduced the amount of RBCs under shear stress. In this study, it was found that α-glucan could inhibit histone-induced lysis in a dose-dependent manner (Fig. 10C). Therefore, the results indicate that the histone effect is exacerbated by higher shear flow rates and duration of shear exposure. Even when activated, mCBS (and CBS) inhibit histone-induced RBC fragility. This indicates that the activity of the ATP-dependent agonist was inhibited.

[0437] To more closely mimic the clinical scenario, mC after 55 min of exposure to histones Treatment of RBCs with BS for 5 min significantly reduced their susceptibility to lysis and aggregation under shear forces. We demonstrated that the antibody produced almost complete inhibition of the IL-12 receptor agonist activity (Figure 11). The cells were exposed to 400 μg / mL histone for 55 min, followed by 5 min of various concentrations of histone. After exposure to mCBS (Fig. 11A), the shear stress (flow rate of 100 mm / s and 40 pulses) Repeated pipetting was applied, and hemoglobin in the supernatant was measured at A540 nm. The degree of RBC aggregation was measured by the level of autofluorescence in FL1 using cytometry. The results showed that mCBS significantly inhibited the histone-induced RBC proliferation. It is shown that the susceptibility to lysis and aggregation could be reversed.

[0438] Example 13: Sulfation of mCBS As mentioned above, mCBS is chemically much more stable than CBS, and as a result Therefore, we have demonstrated that CBS requires sulfur for activity. The oxidation was tested in the HMEC-1 cytotoxicity assay (Fig. 12b) and the RBC fragility assay. (Fig. 12c) were used to test these various sulfation states of CBS compounds (Fig. 12a). In doing so, the inventors determined that highly sulfated CBS is required for antihistone activity. , because even if five of the seven O-sulfation sites are occupied, undersulfated mCB S had minimal histone inhibitory activity.

[0439] Example 14: mCBS and CBS inhibit histone-induced platelet aggregation and deconjugation Reduce grain size This example demonstrates that mCBS and CBS inhibit histone-induced platelet aggregation and Shown to inhibit degranulation.

[0440] Platelets play a key role in blood clot formation and act as plasma coagulation proteins after vascular injury. Platelets also interact with immune cells, forming an effective plug. Activation and migration to the infected area are supported by the

[0441] Therefore, the present inventors have investigated the effects of histones on platelets and the role of mCB in inhibiting these effects. Specifically, the ability of S and CBS to induce the erythrocyte proliferation and erythrocyte motility was investigated. Incubate with various concentrations of histones (e.g., 0–1000 μg / mL) over a period of time. Afterwards, FSC and SSC were used to distinguish between single and aggregated platelets. Aggregation was analyzed by flow cytometry (the results are shown in Figure 13A). A range of concentrations of mCBS and CBS were added before the addition of histones (at 50 μg / mL). This was repeated except that human platelets in whole blood were incubated for 1 hour at 4°C for 1 hour, and then ATP was released from the platelets. Use chemiluminometry to detect decondylation after the addition of increasing concentrations of histones. The histones were analyzed for granules and thrombin was included as a positive control (Figure 13C). Before the addition of mCBS (400 μg / mL), increasing concentrations of mCBS and CBS were added. This last step was repeated except that (shown in Figure 13D)

[0442] The results showed that isolated platelets, when exposed to increasing concentrations of histones, exhibited a rapid response to flow cytometry. As measured by platelet luminescence (Figure 13A), platelets showed an increased tendency to aggregate. The tendency to degranulate, as measured using ATP release by ATP analgesia (Fig. 13C), However, the platelet preparations were pretreated with mCBS and CBS. When non-sulfurized erythrocytes were treated with erythrocyte stimulatory agents, aggregation (Fig. 13B) and degranulation (Fig. 13D) were significantly reduced. Oxidized cellobiose (CB) had no inhibitory activity.

[0443] Therefore, these results indicate that histones induce platelet aggregation and degranulation and that these This indicates that the effect of is inhibited by mCBS and CBS.

[0444] Example 15: mCBS prevents lipid bilayer disruption by histones Next, we investigated how histones mediate their cytotoxicity and the resulting We investigated whether CBS and mCBS protect cells from histone-mediated damage. Histones bind to GAGs (especially HS) that are ubiquitously expressed on the cell surface. Histones bind to cell surface HS, which mediates the cytotoxic effect of this histone. It seemed possible that it might start functioning.

[0445] To test this idea, a mixture of three bacterial heparinases or human platelet heparanase was used. Exposure to histones after incubation with either When cell surface HS is depleted and HS removal is monitored by flow cytometry , 86% and 97%, respectively, for these two enzyme treatments.

[0446] The present inventors have demonstrated that HMEC-1 is resistant to either bacterial or human HS degrading enzymes. Pretreatment of cells did not affect their susceptibility to histone-mediated cytotoxicity. Furthermore, pretreatment with these two enzymes did not affect the viability of HMECs (Fig. 14). a) To confirm this finding, the present inventors investigated the mechanism by which GAG chain biosynthesis is initiated. A CHO cell line ( pgsA-745) was used. Compared to the parental CHO-K1 cell line, The deletion had little effect on the cytotoxicity of histones, and at the highest histone concentrations tested, Cytotoxicity was slightly but significantly reduced (Fig. 14b). G is not essential for histone-mediated cytotoxicity.

[0447] Histones have been shown to interact with and damage lipid bilayers, and they have been shown to play a role in cell permeability. It has also been shown that His acts as a transient protein. We investigated whether Tn mediates cytotoxicity by directly disrupting the lipid bilayer.

[0448] To investigate this possibility, we prepared an artificial lipid bilayer and measured the change in current across this bilayer. detected the susceptibility of this artificial lipid bilayer to histone disruption.

[0449] Lipid bilayers have a finite lifetime, typically on the order of 30 to 120 minutes. In this experiment, a control containing the ryanodine receptor 1 (RyR1) ion channel protein was used. The average lifetime of the molten lipid bilayer was 46±4 minutes, and the addition of histones (1 μM) significantly reduced this lifetime. The lifetime of the 47 bilayers was significantly reduced to 5.7 ± 1.2 min (Fig. 15a). Thirteen (28%) of the 125 fragments were disrupted within 0.3–0.5 min of histone addition. Only 2 (1.6%) of the control bilayers ruptured in the same period, resulting in higher histone At concentrations (≥50 μM), most bilayers rapidly ruptured (not shown). In the presence of CBS, the bilayer is less susceptible to disruption by histones, and the average bilayer lifetime is longer than that of CBS. In the case of histone alone (2), the time increased significantly to 18±4 minutes and 36±5 minutes (Fig. 15a). In the case of CBS, rapid bilayer rupture occurred in 3 of 52 bilayers (5.8%) compared with 8%. 0.8%).

[0450] Previous studies have shown that histones act as a non-selective Ca receptor in cells. 2+ Channels and plasma membrane desorption It has also been demonstrated that histones can induce polarization. This finding supports the idea that histones interact with cell surface phospholipids. Further supporting the concept of direct interactions to disrupt membrane integrity.

[0451] mCBS induces histone-induced Ca 2+ Whether to protect cells against flow To investigate this, HMEC-1 cells were cultured in a Ca 2+Loaded with the sensitive dye Indo-1 and mCBS challenged with histones in the presence or absence of α-glucan and analyzed by flow cytometry. a 2+ Histone uptake was measured (Fig. 15b). 2+ Showing the level It induces an approximately six-fold increase in cell population, and this response occurs 4-10 minutes after histone addition. The presence of mCBS substantially inhibited this response (Fig. 15c). The inventors' discovery that histones damage cell membranes by directly disrupting the lipid bilayer of cells , indicating that mCBS neutralizes this undesirable property of histones.

[0452] Example 16: mCBS has minimal intrinsic anticoagulant activity and is induced by histones Reduces plasma coagulation disruptions This example demonstrates that histones reduce blood clotting and that mCBS has minimal anticoagulant effect. These results indicate that the compound has the effect of reducing plasma coagulation disruption induced by histones.

[0453] Although it has been found that histones can promote platelet aggregation and degranulation, The authors demonstrated that histones specifically inhibit plasma coagulation via factors involved in the intrinsic pathway. They also found that this reduced the level of whole blood coagulation.

[0454] This was compared with rotational thromboelastometry (ROTEM) (Figure 16A) and traditional plasma bioassays. This was demonstrated using an activated partial thrombin time (APTT) assay (Figure 16B). .

[0455] Specifically, we used ROTEM (Figure 16A) to measure increasing histone concentrations (0 Addition of 1000 μg / mL of thiazolinone prolonged clotting times (measured in seconds) in all assays. The time to clotting was longer, especially in the NATEM and INTEM assays. The same anticoagulant effect of acetaminophen was demonstrated using the plasma-based clotting assay APTT ( Figure 16B).

[0456] Since mCBS is a sulfated disaccharide, the inventors believe that mCBS is an unfractionated, low molecular weight They reasoned that ROI could be considered a much smaller relative of the anticoagulant heparin. The clotting properties of mCBS (200 μg / mL) were studied using TEM. Two sulfated trisaccharides, melezitose and maltotriose, were included (Figure 17). These include the NATEM (non-activation) assay, the EXTEM (extrinsic pathway activation) assay, and the I NTEM (intrinsic pathway activation) assay and FIBTEM (exogenous pathway activation with neutralized platelets) Immediately before performing the pathway activation assay, whole blood was treated with mCBS, maltotriose, or melamine. Supplemented with chytose (200 μg / mL). Data are presented as fold increase over water control. The clotting times of various concentrations (0-100 μg / mL) of mC are shown in Figure 17A. Whole blood supplemented with BS, melezitose, or maltotriose was analyzed for clotting time using NATE. The data was then analyzed using the M assay and the results are shown in Figure 17B. The same was repeated (panel B) except that the amplitude was shown. The results are shown in Figure 17C. We noticed that no clots were detected with the two trisaccharides at 50 and 100 μg / mL. Ta.

[0457] The results shown in Figure 17 indicate that mCBS has minimal or no effect on whole blood coagulation, but The sulfated trisaccharide compounds were analyzed by NATEM (non-activated thromboelastometry (TEM)) assay. Significant anticoagulant activity was detected by the intrinsic pathway activation TEM (INTTEM) assay. The NATEM assay demonstrated that these compounds induce Since the sulphated compounds were most sensitive to changes in the blood glucose level, lower concentrations of the three sulphated compounds were added to whole blood. This analysis further defined their efficacy as anticoagulants. The clotting time of the control was doubled in g / mL but 100 μg / mL to achieve the same results. 17B).

[0458] Furthermore, the anticoagulant effect of mCBS was compared with that of heparin and low-molecular-weight heparin, enoxaparin. Specifically, the NATEM assay was used to compare the Addition of heparin (at a concentration of 1 μg / mL or 10 μg / mL) The addition of enoxaparin (at a concentration of 100 μg / mL), the trisaccharide maltotriose (at a concentration of 25 μg / mL) Whole blood clotting was observed after the addition of sucrose or mCBS (at a concentration of 25 μg / mL). The results are shown in Figure 18. The results of the measurement of mCBS, unfractionated heparin, and low molecular weight heparin (L MWH) compared with LMWH (enoxaparin), which showed 1.0 times the anticoagulant activity of mCBS compared with LMWH (enoxaparin). It showed a 10-fold reduction and a >750-fold reduction compared to unfractionated heparin.

[0459] The EXTEM assay showed that histones increased clotting time, but mCBS did not affect the same parameters, so using the NATEM assay, The ability of mCBS to inhibit histone-induced disruption of coagulation was also tested. The results are shown in Figure 1. As demonstrated in Figure 9, the addition of 200 μg / mL of mCBS resulted in a 400 and In comparison, the same amount of water was able to inhibit the anticoagulant effect of both 800 μg / mL and 800 μg / mL of histone. had no effect (Fig. 19). Therefore, this result suggests that mCBS is not induced by histones. The results show that the disruption of whole blood coagulation caused by thrombin inhibits the disruption of whole blood coagulation caused by thrombin.

[0460] In vivo evidence of the biological effects of mCBS and CBS Example 17: mCBS and CBS protect organs from histone-mediated injury R This example demonstrates that mCBS and CBS protect mice from histone-induced organ damage. This indicates that the service can be protected.

[0461] Intravenous injection of histones into mice resulted in the formation of microthrombi in organs, leading to cell injury and It has been demonstrated that organ dysfunction occurs (Xu et al., Extra cellular histones are major mediators of death in sepsis.Nat Med.2009 Nov;15(11) :1318-21.2009). The same septic mice as Xu et al., 2009 Using the model, we demonstrate that mCBS and CBS are induced by histones. Mice were given 50 mg / kg of histochemicals to examine whether they could protect against organ damage. 10 minutes before intravenous injection of 6.25, 25 and 100 mg / kg of ethanol (or an equivalent volume of PBS). The mice were intraperitoneally injected with mCBS or CBS at a concentration of 0.01% or an equal volume of PBS. After 4 hours, markers of cell injury (lactate dehydrogenase, LDH) and liver dysfunction were detected. markers of kidney dysfunction (alanine aminotransferase, ALT) Blood was collected retroorbitally for analysis of creatinine (Creat). The results are shown in Figure 20. Specifically, using these results, the inventors have demonstrated that mCBS and CBS are It dose-dependently protected animals from stone-mediated injury and significantly improved liver and kidney function. Although significant conservation was demonstrated (Fig. 20), it could be shown that non-sulfated CB was inactive. .

[0462] Example 18: mCBS protects cells in the bloodstream from histone-mediated injury This example demonstrates that mCBS inhibits histidine dehydrogenase activity in circulating white blood cells, platelets, and red blood cells in mice. These results demonstrate that the agonist-mediated reduction of ATP-mediated ATP synthesis is attenuated and / or prevented.

[0463] Injection of histones into mice has also been shown to induce severe thrombocytopenia. Therefore, in this example, the present inventors investigated the effect of histones on cells in the bloodstream after intravenous injection. The protective effect of mCBS was investigated. Using the same mouse model as in the previous example, mice were 10 min before intravenous injection of 50 mg / kg histone (or equivalent volume of PBS) kg of mCBS (or an equivalent volume of PBS) was injected intraperitoneally, followed by retro-orbital bleeding 10 minutes later. Using the ADVIA 2120 hematology system, whole blood was analyzed for white blood cell count, platelet count, and red blood cell count. The blood count and hemoglobin concentration were analyzed. The results are shown in Figure 21. Not only did the number of infected platelets decrease significantly within minutes of histone injection, but the number of infected white blood cells, red blood cells, and Erythrocytes (red blood cells) and plasma The serum hemoglobin level also significantly decreased. These histone-mediated effects were not completely abolished when injected with The activity was also significantly inhibited by the α-glucanase inhibitor (Fig. 21).

[0464] Therefore, these results suggest that mCBS protects cells in the bloodstream from histone-mediated injury. It has been shown to protect cells.

[0465] Example 19: CBS and mCBS inhibit sepsis We next investigated the effects of cecal ligation and puncture (CLP) on the rat cecal ligation and puncture model of moderate and severe sepsis. The efficacy of mCBS and CBS was investigated in rats with rhesus monkeys. Only a small number of rats died (Figure 22A). In cases of moderate sepsis where a SIRS response is induced, mCBS treatment The results demonstrated a significant decrease in circulating LDH levels compared with the CLP group (0.6 ±0.1 and 1.1±0.2U / L×10 3 , p=0.03; Figure 22B). No difference in serum or creatinine levels was observed, supporting the induction of mild sepsis. (data not shown).

[0466] In cases of severe sepsis, where morbidity was much more evident, mortality in rodents was significantly higher than in PBS controls. The CBS-treated animals had significantly lower levels of β-glucan compared to the control group. There was zero mortality in the 2000-mg mice (Fig. 23A). Importantly, extensive liver and kidney damage was observed. The high ALT and creatinine levels detected in the untreated group, indicative of injury, were This was not seen in treated animals (Fig. 23B).

[0467] Taken together, these results suggest that CBS and the more stable mCBS may be effective in preventing sepsis and SIRS. This may limit the histone-mediated effects of ATP, thereby limiting tissue damage and reducing the risk of death from peripheral organs. This indicates that the function of the vessel is preserved.

[0468] Example 20: CBS and mCBS inhibit IRI To investigate the ability of CBS and mCBS to inhibit IRI, rat cardiac IRI (cI The ischemic area was equal between groups (Fig. 24A). CBS treatment reduced microvascular A significant 50% reduction in the area of ​​ductal obstruction (Fig. 24B) and myocardial necrosis in the ischemic area (Fig. 24C) Furthermore, in a rat skin flap IRI model, mCBS significantly improved the survival area of ​​the skin flap. The results were consistent and significant (Figure 25).

[0469] Example 21: CBS inhibits venous thrombosis To investigate whether CBS regulates the local vascular effects of histones, we investigated histone We established a model of deep vein thrombosis (DVT) mediated by CBS, which was almost completely induced by CBS. It was shown that the ATP-dependent agonist activity was inhibited (Fig. 26).

[0470] This data suggests that both systemic and local vascular pathology mediated by free histones is associated with CB This is consistent with its suitability for inhibition by S / mCBS.

[0471] Example 22: mCBS inhibits autoimmunity The present inventors then investigated the pathogenesis of experimental autoimmune encephalomyelitis (EEE), which is similar to multiple sclerosis in humans. The researchers evaluated the ability of mCBS to inhibit an animal model of autoimmunity called AE. The data, shown in Figure 27, demonstrate that mCBS, when administered daily, provides a 35-day window of action. It was found that the treatment effectively protected mice from developing EAE over a long period of time.

[0472] In the above examples, the inventors have demonstrated that many pathological processes mediated by free histones Highly effective in vivo treatment of erythrocytes (e.g., cytotoxicity, red blood cell fragility / deformability, and platelet activation) The development of small polyanionic molecules as thoracic inhibitors is described.

[0473] These data suggest that CBS / mCBS may be a potential therapeutic option for histone-mediated diseases, e.g. It also provides primary data evidence that vasodilators can inhibit vasodilators (sepsis, IRI, thrombosis and autoimmunity).

[0474] In humans and animals, mCBS is highly stable and well tolerated at high doses. The only dose-limiting property is anticoagulant activity, which is 1.5 times greater than that of LMW-heparin. 10-fold lower than unfractionated heparin and 750-fold lower than unfractionated heparin. Therefore, mCBS has considerable clinical benefits. This represents a new class of therapeutics with promising potential.

Claims

1. A drug for treating or preventing a disease mediated by extracellular histones, the drug comprising a polyanionic heptasulfated cellobioside modified at its reducing end with a small, uncharged glycosidic bond substituent, or a pharmaceutically acceptable salt thereof, wherein the small, uncharged glycosidic bond substituent is O—(C １～６ ) alkyl and S—(C １～６ ) alkyl; The disease mediated by extracellular histones is selected from the group consisting of systemic inflammatory response, acute kidney injury, acute liver injury, acute respiratory distress syndrome, and multiple sclerosis.

2. the systemic inflammatory response is a systemic inflammatory response to an infectious or non-infectious trigger; the infection comprises a bacterial, viral, fungal, or parasitic infection; or The drug of claim 1, wherein the non-infectious trigger comprises surgery, trauma, bleeding, burns, acute pancreatitis, pre-eclampsia or acute kidney injury.

3. (a) the extracellular histone-mediated disease is caused by and / or mediated by and / or involves and / or is associated with an extracellular histone-mediated pathology in the subject; and / or (b) the extracellular histone-mediated condition is caused by and / or mediated by and / or involves and / or is associated with release of extracellular histones in the subject; and / or (c) the extracellular histone-mediated condition is caused by and / or mediated by and / or involves and / or is associated with inflammation or release of extracellular histones following an inflammatory response in the subject; and / or (d) the extracellular histone (i) is cytotoxic to the endothelium of the subject, or (ii) contributes to endothelial dysfunction in the subject, or (iii) initiates blood clotting by activating platelets in the subject, or (iv) induces red blood cell fragility and resulting anemia in the subject, or (v) induces sterile inflammation in the subject, or (vi) induces organ damage in the subject; and / or (e) the agent reduces, minimizes, or inhibits pathology mediated by extracellular histones in the subject; and / or (f) the medicament reduces, minimizes, or inhibits extracellular histone-mediated pathology to the endothelial or organ dysfunction in the subject; and / or (g) the drug is formulated for single dose administration or multiple dose administration to the subject; and / or (h) The drug of claim 1, wherein the drug is formulated for simultaneous or concomitant administration to the subject of a second active agent selected from the group consisting of an anti-inflammatory agent, an antibiotic agent, an antiviral agent, an antifungal agent, or an adjunctive treatment.

4. The polyanionic heptasulfated cellobioside or a pharmaceutically acceptable salt thereof modified with a small uncharged substituent at the reducing end is 【Chemistry 1】 (Wherein, R1 is O—(C １～６ ) alkyl and S—(C １～６ ) alkyl, and R2-R8 are each sulfate groups. The drug according to any one of claims 1 to 3, having an overall structure:

5. (a) R1 is a methoxy group or an ethoxy group, and / or (b) The drug of claim 4, wherein R2 to R8 are each selected from O-sulfate or N-sulfate.

6. The drug according to any one of claims 1 to 5, wherein the polyanionic heptasulfated cellobioside or a pharmaceutically acceptable salt thereof is a sulfated β-O-methyl cellobioside disaccharide or a pharmaceutically acceptable salt thereof, or the polyanionic heptasulfated cellobioside is sodium β-O-methyl cellobioside sulfate.

7. 1. Use of a compound comprising a polyanionic heptasulfated cellobioside, the polyanionic heptasulfated cellobioside being modified at its reducing end with small, uncharged glycosidic bond substituents, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament suitable for the treatment or prevention of a disease mediated by extracellular histones, The small uncharged glycosidic bond substituent is O—(C １～６ ) alkyl and S—(C １～６ ) alkyl; The use, wherein the disease mediated by extracellular histones is selected from the group consisting of systemic inflammatory response, acute kidney injury, acute liver injury, acute respiratory distress syndrome, and multiple sclerosis.

8. the systemic inflammatory response is a systemic inflammatory response to an infectious or non-infectious inducer; the infection comprises a bacterial, viral, fungal, or parasitic infection; or 8. The use of claim 7, wherein the non-infectious trigger comprises surgery, trauma, bleeding, burns, acute pancreatitis, pre-eclampsia or acute kidney injury.

9. (a) the extracellular histone-mediated disease is caused by and / or mediated by and / or involves and / or is associated with an extracellular histone-mediated pathology in the subject; and / or (b) the extracellular histone-mediated condition is caused by and / or mediated by and / or involves and / or is associated with release of extracellular histones in the subject; and / or (c) the extracellular histone-mediated condition is caused by and / or mediated by and / or involves and / or is associated with inflammation or release of extracellular histones following an inflammatory response in the subject; and / or (d) the extracellular histone (i) is cytotoxic to the endothelium of the subject, or (ii) contributes to endothelial dysfunction in the subject, or (iii) initiates blood clotting by activating platelets in the subject, or (iv) induces red blood cell fragility and resulting anemia in the subject, or (v) induces sterile inflammation in the subject, or (vi) induces organ damage in the subject; and / or (e) the agent reduces, minimizes, or inhibits pathology mediated by extracellular histones in the subject; and / or (f) the medicament reduces, minimizes, or inhibits extracellular histone-mediated pathology to the endothelial or organ dysfunction in the subject; and / or (g) the drug is formulated for single dose administration or multiple dose administration to the subject; and / or (h) The use of claim 7, wherein the medicament is formulated for simultaneous or concomitant administration to the subject of a second active agent selected from the group consisting of an anti-inflammatory agent, an antibiotic agent, an antiviral agent, an antifungal agent, or an adjunctive treatment.

10. The polyanionic heptasulfated cellobioside or a pharmaceutically acceptable salt thereof modified with a small uncharged substituent at the reducing end is 【Chemistry 2】 (Wherein, R1 is O—(C １～６ ) alkyl and S—(C １～６ ) alkyl, and R2-R8 are each sulfate groups. The use according to any one of claims 7 to 9, having the overall structure:

11. (a) R1 is a methoxy group or an ethoxy group, and / or (b) The use according to claim 9, wherein R2 to R8 are each selected from O-sulfate or N-sulfate.

12. The use according to any one of claims 7 to 11, wherein the polyanionic heptasulfated cellobioside or a pharmaceutically acceptable salt thereof is a sulfated β-O-methyl cellobioside disaccharide or a pharmaceutically acceptable salt thereof, or the polyanionic heptasulfated cellobioside is sodium β-O-methyl cellobioside sulfate.

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