Chemically modified heparin

Abstract

Chemically modified bovine intestinal heparin, as well as pharmaceutical compositions, compositions containing the chemically modified bovine intestinal heparin, and methods for their production and use are provided. In one embodiment, the present disclosure provides a chemically modified bovine intestinal heparin comprising from about 15 to about 90 disaccharide units, wherein about 15% to 50% of the disaccharide units comprise 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide and the anti-Factor IIA activity is less than about 15 IU / mg.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 349,416, filed on June 6, 2022, under 35 U.S.C. § 119(e), the entire disclosure of which is hereby incorporated by reference herein.

[0002] Statement Regarding Government Support This research was supported by $217,928.00 from the Deputy Assistant Secretary of Defense for Health Affairs, under Award Number W81AWH2210093, and approved by the Department of Defense.

Background Art

[0003] Sickle cell disease (SCD) is a devastating disease that affects over 100,000 people in the United States and over 6 million people worldwide. This disease is associated with pain that can be disabling and chronic progressive ischemic damage to nearly every organ in the body, shortening the average life expectancy by more than 20 years. The hallmark of SCD is vaso - occlusive crisis (VOC). VOC is an acute event accompanied by excruciating pain and is a precursor to serious complications such as acute chest syndrome (ACS), which is a type of acute lung injury and a leading cause of death in SCD patients. As a result, the impact on patients is profound, affecting all aspects of life. Some prophylactic drugs, such as anti - P - selectin antibody (crizanlizumab), hydroxyurea, or L - glutamine, have shown promise in at least partially reducing VOC, but disease - specific acute VOC therapies have not been approved to date, and there is a large treatment gap.

Summary of the Invention

[0004] In one embodiment, the present disclosure provides a chemically modified bovine intestinal heparin comprising from about 15 to about 90 disaccharide units, wherein about 15% to 50% of the disaccharide units comprise 1 - (3 - dimethylaminopropyl) - 3 - ethylurea (EDU) - amide and the anti - Factor IIA activity is less than about 15 IU / mg.

[0005] In one embodiment, a chemically modified bovine intestinal heparin and a pharmaceutical composition containing the same are provided, wherein at least a part of the free carboxylic acid moieties on the unmodified bovine intestinal heparin having an anti-factor IIa activity exceeding 90 U / mg is converted to N-acylurea amides, and as a result, the pharmaceutical composition exhibits 1% to about 8% of the anti-factor IIa activity of the unmodified bovine intestinal heparin. The chemically modified bovine intestinal heparin and the composition containing the same disclosed herein have reduced anticoagulant activity and are optimized for selectin and complement inhibition, and when administered to a subject in need thereof, an effective therapeutic effect can be obtained and the risk of harmful bleeding is limited.

[0006] In certain embodiments, a method of treating or alleviating one or more symptoms of sickle cell disease in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of the pharmaceutical composition disclosed herein. In certain embodiments, the subject is in vaso-occlusive crisis. In certain embodiments, the subject is in the early stage of vaso-occlusive crisis, such as the prodromal stage. In certain embodiments, the subject has vaso-occlusive crisis (VOC).

[0007] In certain embodiments, a method of preventing or reversing cell adhesion in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of the pharmaceutical composition disclosed herein.

[0008] In certain embodiments, a method of preventing or reversing complement activation in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of the pharmaceutical composition disclosed herein.

[0009] In certain embodiments, a method of treating a solid tumor in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of the pharmaceutical composition disclosed herein. In certain embodiments, the solid tumor expresses at least one of sLex or sLea (sialyl Lewis x or sialyl Lewis a ). In certain embodiments, the solid tumor is a gastrointestinal tumor, breast tumor, prostate tumor, ovarian tumor, colorectal tumor, liver tumor, lung tumor, cervical tumor, head tumor, neck tumor, esophageal tumor, brain tumor, or pancreatic tumor.

[0010] Also provided is a method of treating a disease or disorder in a subject in need thereof, which is at least partially mediated by inhibition of cell binding to P-selectin and / or inhibition of the complement activation pathway, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified bovine intestinal heparin described herein, or a composition comprising the same, wherein the disease or disorder is cancer, blood cancer, melanoma, leukemia, multiple myeloma, chemotherapy-induced peripheral neuropathy (CIPN), beta thalassemia, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), neurological disease, amyotrophic lateral sclerosis (ALS), sickle cell disease (including but not limited to vaso-occlusive crises), immune response in gene therapy with adeno-associated virus (AAV), acute respiratory distress syndrome (ARDS), cardiovascular disease, ophthalmic disease or disorder, kidney disease, thrombotic microangiopathy (TMA), hereditary angioedema, thrombotic thrombocytopenic purpura (TTP), Shiga toxin-positive HUS, post-infectious HUS, thrombotic microangiopathy, membranoproliferative glomerulonephritis (MPGN), primary MPGN, C3 glomerulopathy (C3G), transplant rejection, delayed kidney transplant rejection, antibody-mediated kidney transplant rejection, kidney transplant reperfusion injury, kidney transplant in CAPS patients, neuromyelitis optica, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, lupus nephritis, IgA nephropathy, rheumatoid arthritis, Crohn's disease, ulcerative colitis, hemolytic anemia, autoimmune hemolytic anemia, pemphigus and pemphigoid, antiphospholipid syndrome, cold agglutinin disease, severe thrombocytopenia, macular degeneration, uveitis, ANCA-associated vasculitis, atherosclerosis, mood disorder, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, sepsis, cerebral malaria, psoriatic arthritis, dermatomyositis, osteoarthritis, dementia, glaucoma, diabetic angiopathy, myocardial infarction, stroke, after bypass surgery, multiple trauma, nerve trauma, antiphospholipid syndrome, pregnancy-induced hypertensive nephropathy, or hemodialysis, but not limited thereto. In certain embodiments, the treatment comprises reducing inflammation, or reducing or inhibiting an inflammatory response as a result of the disease or disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0012] All numerical values, for example, pH, temperature, time, concentration, and molecular weight, are approximate values that vary by (+) or (-) in increments of 0.1 or 10%, including ranges. It should be understood that the term "about" is attached before all numerical values, although it is not always explicitly stated. Also, although not always explicitly stated, the reagents described herein are merely illustrative, and equivalents of such are known in the art.

[0013] "Chemically unmodified" bovine intestinal heparin refers to natural bovine intestinal heparin that has not been modified by chemical means. Exemplary heparins modified by chemical means include, but are not limited to, LMWH, heparan sulfate, biotechnology-derived heparin, synthetic heparin, or other heparin analogs derived from natural heparin. Exemplary chemical modifications include one or more of partial or complete N- or O-desulfation (e.g., 2-O-sulfated heparin, 3-O-sulfated heparin, 2,3-O-desulfated heparin, etc.), oxidation (e.g., periodate-oxidized heparin), reduction (e.g., reduction of heparin carboxyl groups, borohydride-reduced heparin, etc.), N-acetylation (including N-desulfation, O-desulfation, followed by N-resulfation), sulfation, and the like, but are not limited thereto.

[0014] "Chemically modified" bovine intestinal heparin refers to bovine intestinal heparin that has not been chemically modified or natural bovine intestinal heparin modified to include a covalent bond to 1-(3-dimethylaminopropyl)-3-ethylurea.

[0015] As used herein, "1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide" is an amide formed by the reaction of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, EDAC, or EDCI) with a carboxylic acid (such as on heparin). The reaction of EDAC with a carboxyl group generally proceeds by adding a free carboxylate to one of the diimide-based double bonds to form an O-acylurea product. In the presence of a nucleophile, an acyl-nucleophile product is formed, and further, the urea of the carbodiimide is formed. If no nucleophile is added, the O-acylurea rearranges to the more stable N-acylurea isomer shown below by intramolecular acyl transfer,

Chem.

Chem.

[0016] In many cases, the chemically modified bovine intestinal heparin compounds of the present disclosure can form acid and / or base salts due to the presence of sulfoxide and / or carboxyl groups, or groups similar thereto. In certain embodiments, salts, compositions, dosage forms, or other materials useful for the preparation of pharmaceutical compositions suitable for veterinary or human pharmaceutical use are provided.

[0017] A "pharmaceutical composition" is intended to include a combination of an active agent and an inert or active carrier, and to render the composition suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

[0018] A "subject" for diagnosis or treatment is an animal such as a mammal including a human.

[0019] An "effective amount" refers to an amount of a drug sufficient to induce a desired biological and / or therapeutic result. The result can be a reduction in the signs, symptoms, or causes of a disease, or any other desirable change in a biological system.

[0020] As used herein, terms such as "treating," "treatment," etc. are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disorder or its signs or symptoms, and / or may be therapeutic in terms of partially or completely curing a disorder and / or the adverse effects resulting from the disorder.

[0021] "Administration" can be carried out in a single dose continuously or intermittently throughout the course of treatment. Methods for determining the most effective means of administration and dosage are known to those of ordinary skill in the art and vary depending on the pharmaceutical composition used in the treatment, the purpose of the treatment, the target cells being treated, and the subject being treated. Administration can be carried out in single or multiple doses at dosage levels and patterns selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art.

[0022] Chemically modified bovine intestinal heparin Chemically modified bovine intestinal heparin and compositions containing the same are provided. Heparin is a naturally occurring glycosaminoglycan. Glycosaminoglycans (GAGs) or mucopolysaccharides are long linear polysaccharides composed of repeating disaccharide units. Except for keratan, the repeating units consist of an amino sugar and a uronic acid or galactose. The molecular weight of native heparin ranges from 3 to 30 kDa. In this specification, for example, heparins of various molecular weights can be used, from a single disaccharide unit of about 650 - 700 Da to a glycan of about 50 kDa. In some embodiments, the heparin is about 10 - about 20 kDa. In some embodiments, the heparin is about 15 - about 20 kDa. In some embodiments, the heparin is up to about 15, or about 16, or about 17, or about 18, or about 19, or about 20 kDa.

[0023] The main disaccharide units present in heparin include GlcA-GlcNAc, GlcA-GlcNS, IdoA-GlcNS, IdoA(2S)-GlcNS, IdoA-GlcNS(6S), and IdoA(2S)-GlcNS(6S). GlcA represents β-D-glucuronic acid, IdoA represents α-L-iduronic acid, IdoA(2S) represents 2-O-sulfo-α-L-iduronic acid, GlcNAc represents 2-deoxy-2-acetamido-α-D-glucopyranosyl, GlcNS represents 2-deoxy-2-sulfamido-α-D-glucopyranosyl, and GlcNS(6S) represents 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate. The most common disaccharide unit in heparin is composed of 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S).

[0024] The heparin compounds and compositions with reduced anticoagulant activity enable the administration of higher doses of heparin to subjects for whom anticoagulant activity is contraindicated (e.g., subjects taking aspirin, ibuprofen, or other anti-inflammatory drugs (e.g., NSAIDs), or drugs containing these components). The anticoagulant activity of heparin can also be measured with respect to its activity of inhibiting factor Xa (fXa) or factor IIa (thrombin). An example can be found, for example, in Stuart, M, Johnson, L, Hanigan, S, Pipe, SW, Li, S-H. Anti-factor IIa (FIIa) heparin assay for patients on direct factor Xa (FXa) inhibitors. J Thromb Haemost. 2020;00:1-8 (doi.org / 10.1111 / jth.14806) and the examples disclosed herein. The bovine intestinal heparin used herein is derived from bovine intestine.

[0025] In certain embodiments, chemically modified heparin and pharmaceutical compositions containing the same are provided, wherein at least a portion of the free carboxylic acid moieties on unmodified heparin having an anti-factor IIa activity of greater than 90 U / mg are converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amides, such that the pharmaceutical composition exhibits from 1% to about 8%, 1% to about 7%, 1% to about 6%, 1% to about 5%, 1% to about 4%, or about 3% to 8%, or about 3% to 4%, or about 3% to 5%, or about 4% to 8%, or about 5% to 8%, or about 6% to 8%, or about 6% to 7%, or about 7% to 8% of the anti-factor IIa activity of unmodified heparin.

[0026] In certain embodiments, pharmaceutical compositions containing chemically modified heparin and pharmaceutically acceptable excipients are provided, wherein at least a portion of the free carboxylic acid moieties on unmodified heparin having an anti-factor IIa activity of greater than 90 U / mg are converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amides, such that the pharmaceutical composition exhibits from 1% to about 8%, 1% to about 7%, 1% to about 6%, 1% to about 5%, 1% to about 4%, or about 3% to 8%, or about 3% to 4%, or about 3% to 5%, or about 4% to 8%, or about 5% to 8%, or about 6% to 8%, or about 6% to 7%, or about 7% to 8% of the anti-factor IIa activity of unmodified heparin.

[0027] In certain embodiments, the chemically modified bovine intestinal heparin or pharmaceutical composition exhibits about 3% to 8%, or about 4%, or about 7% of the anti-factor IIa activity of unmodified bovine intestinal heparin.

[0028] In certain embodiments, when comparing herein, "chemically modified" heparin and "unmodified" heparin are from the same source. In certain embodiments, when comparing herein, "chemically modified" heparin and "unmodified" heparin are not from the same source.

[0029] In certain embodiments, unmodified porcine intestinal heparin has an anti - Factor IIa activity greater than 90 U / mg. In certain embodiments, unmodified porcine intestinal heparin has an anti - Factor IIa activity of about 100 U / mg, or about 90 U / mg to about 135 U / mg.

[0030] In certain embodiments, modified porcine intestinal heparin has an anti - Factor IIa activity of less than 135 U / mg.

[0031] In certain embodiments, modified porcine intestinal heparin has an anti - Factor IIa activity of 90 U / mg to 135 U / mg.

[0032] In certain embodiments, modified porcine intestinal heparin exhibits about 3 - 8%, or about 3%, or about 3.5%, or about 4%, or about 4.5%, or about 5%, or about 5.5%, or about 6%, or about 6.5%, or about 7%, or about 7.5%, or about 8% of the anti - Factor IIa activity of unmodified porcine intestinal heparin.

[0033] In certain embodiments, modified porcine intestinal heparin exhibits about 6 - 8%, or about 7% of the anti - Factor IIa activity of unmodified porcine intestinal heparin.

[0034] In certain embodiments, modified porcine intestinal heparin exhibits about 3 - 5%, or about 4% of the anti - Factor IIa activity of unmodified porcine intestinal heparin.

[0035] In certain embodiments, the chemically modified bovine intestinal heparin disclosed herein is composed of heparin reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, EDAC or EDCI) in the absence of a nucleophilic reagent, and thus EDAC is not used to bind heparin to another compound or substance. Further, the degree or level of sulfation of bovine heparin is not reduced or altered by chemical modification (i.e., EDU modification). Further, the molecular weight of bovine heparin is not reduced or altered by chemical modification (i.e., EDU modification).

[0036] In certain embodiments, there is provided a chemically modified bovine intestinal heparin comprising from about 15 to about 90 disaccharide units, wherein from about 15% to about 50% of the disaccharide units comprise 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide.

[0037] In certain embodiments, there is provided a chemically modified bovine intestinal heparin comprising from about 15 to about 90 disaccharide units, wherein from about 15% to 50% of the disaccharide units comprise 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide and the anti-Factor IIA activity is less than about 15 IU / mg.

[0038] In certain embodiments, the anti-Factor IIA activity is less than about 12 IU / mg, or less than about 10 IU / mg, or from 1 to 15 IU / mg, or from 1 to 12 IU / mg, or from 1 to 10 IU / mg.

[0039] In certain embodiments, the anti-Factor IIA activity of the chemically modified bovine intestinal heparin is less than 15 IU / mg, or less than 12 IU / mg, or less than 10 IU / mg, or from 1 to 15 IU / mg, or from 1 to 12 IU / mg, or from 1 to 10 IU / mg.

[0040] In certain embodiments, the pharmaceutical composition increases P-selectin inhibitory activity as compared to unmodified porcine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity of the chemically modified porcine intestinal heparin is about 10% higher than that of unmodified porcine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity of the chemically modified porcine intestinal heparin is about 15%, or about 20%, or about 30%, or about 40%, or about 50%, or about 70%, or more than about 100%, or more than about 150%, or more than about 200%, or more than about 250%, or more than about 300%, or more than about 400% that of unmodified porcine intestinal heparin.

[0041] In certain embodiments, the P-selectin inhibitory activity (IC 50 ) is less than 150% to more than 150% of the inhibitory activity of unmodified porcine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity (IC 50 ) is less than 50% to more than 50% of the inhibitory activity of unmodified porcine intestinal heparin.

[0042] In certain embodiments, the P-selectin inhibitory activity is greater than or not substantially different from that of the unmodified parent porcine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity is greater than or substantially the same as the inhibitory activity of unmodified porcine intestinal heparin (±50%, or ±40%, or ±30%, or ±20%, or ±10%). In certain embodiments, the P-selectin inhibitory activity is substantially the same as the inhibitory activity of unmodified porcine intestinal heparin (±50%, or ±40%, or ±30%, or ±20%, or ±10%).

[0043] In certain embodiments, the P-selectin activity of the chemically modified porcine intestinal heparin is not substantially different from that of the unmodified parent porcine intestinal heparin.

[0044] In certain embodiments, the P-selectin IC 50is less than about 20 μg / mL, less than about 15 μg / mL, less than about 10 μg / mL, or less than about 5 μg / mL.

[0045] In certain embodiments, there is provided a chemically modified bovine intestinal heparin of Formula IA or a salt thereof,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0046] In certain embodiments, there is provided a composition comprising the chemically modified bovine intestinal heparin described herein.

[0047] In certain embodiments, there is provided a chemically modified bovine intestinal heparin composed of bovine intestinal heparin, where at least about 15%, or at least about 20% of the carboxylic acid functional groups on the bovine intestinal heparin are covalently bonded to a 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide moiety. In certain embodiments, about 20% to about 40%, or about 20% to about 35%, or about 20% to about 30%, or about 20% to about 25%, or about 20% to about 27%, or about 22% to about 27%, or about 22% to about 26%, or about 23% to about 26%, or about 22% to about 25%, or about 23% to about 24%, or about 23%, or about 24%, or about 25% of the carboxylic acid functional groups on the bovine intestinal heparin are covalently bonded to a 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide moiety.

[0048] In any of the embodiments described herein, the number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups per heparin is an average value, and a particular chemically modified bovine intestinal heparin compound in the composition may have more 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups per heparin, while others may have fewer 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups. Thus, in certain embodiments, the number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups described herein is an average value in the composition of the chemically modified bovine intestinal heparin.

[0049] For example, in certain embodiments, the chemically modified bovine intestinal heparin has an average number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups per heparin (with an average molecular weight of about 16 kDa) of about 6 to 7, or about 6, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, or about 6.7, or about 6.8, or about 6.9, or about 7, or about 7.1, or 6.1 to 7.1. In certain embodiments, the average number of disaccharide units in bovine heparin is 26 to 30, or about 26, or about 27, or about 28, or about 29, or about 30. In certain embodiments, the average number of disaccharide units in bovine heparin is 26 to 30, and the average number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups per heparin is about 6 to 7, or about 6, or about 6.5, or about 7, or more than 6, or more than about 5, or about 5 to 16.

[0050] In certain embodiments, the chemically modified bovine intestinal heparin can be defined by the number of carboxylic acid functional groups converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide, i.e., the degree of substitution (DOS) or the functionalization rate. The degree of substitution (DOS) or the functionalization rate is based on the proportion of disaccharide units in heparin that are functionalized with 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide on the heparin backbone.

[0051] The total number of available disaccharide units present on heparin can be calculated by dividing the molecular weight (or average molecular weight) of a single disaccharide unit (e.g., about 500 - 600 Da, or about 575 Da) by the molecular weight of the glycan (e.g., about 15 - 17 kDa, or about 16 kDa). In certain embodiments, chemically modified bovine intestinal heparin is provided, where the degree of substitution (DOS) or functionalization rate is greater than about 15%, or greater than about 20%, or about 15% - about 50%, or about 15% - about 40%, or about 20% - about 50%, or about 20% - about 40%, or about 20% - about 35%, or about 20% - about 30%, or about 20% - about 25%, or about 20% - about 27%, or about 22% - about 27%, or about 22% - about 26%, or about 23% - about 26%, or about 22% - about 25%, or about 23% - about 24%, or about 23%, or about 24%, or about 25%.

[0052] In certain embodiments, the number of carboxylic acid functional groups converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amides can be described as a "mass-based functionalization rate" based on the number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide units on the heparin backbone. In certain embodiments, chemically modified bovine intestinal heparin is provided, and the mass-based functionalization rate is 5 - 12%, or 5 - 10%, or 5 - 9%, or 5 - 8%, or 5 - 7%, or 6 - 12%, or 6 - 10%, or 6 - 9%, or 6 - 8%, or 6 - 7%, or about 6%, or about 7%, or about 6.5%, or about 6.6%, or about 6.7%, or about 6.8%, or 6.5 - 7%.

[0053] In certain embodiments, there is provided a chemically modified heparin of formula IA or a salt thereof, [Chemical formula] IA wherein, n is from 15 to 90, R 1 is as defined herein, each R2 is independently hydrogen, -S(O)2O - , -S(O)2OH, or -S(O)2OM, and each R 3 is independently hydrogen, -S(O)2O - , -S(O)2OH, or -S(O)2OM, and each R 4 is independently hydrogen, -S(O)2O - , -S(O)2OH, or -S(O)2OM, and each M is independently a cation.

[0054] In certain embodiments, a chemically modified heparin comprising from about 15 to about 90 disaccharide units is provided, wherein from about 15% to 50% of the disaccharide units comprise 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide and the anti-factor IIA activity is less than about 15 IU / mg.

[0055] In certain embodiments, the heparin is 9 - 50 KDa, or about 9 - 35 KDa, or about 9 KDa, or about 12 KDa, or about 16 KDa, or about 20 KDa, or about 35 KDa, or about 50 KDa. In certain embodiments, n is about 15 - 87, or about 20 - 65, or about 25 - 35, or about 25 - 30.

[0056] In certain embodiments, there is provided a chemically modified bovine intestinal heparin of formula IA or a salt thereof,

Chemical formula

[0057] In certain embodiments, there is provided a chemically modified bovine intestinal heparin of formula IA or a salt thereof: [ka] IA During the ceremony, n is 26 to 30; Each R 1 are independently -OH, [ka] , or [ka] and R 2 is hydrogen, -S(O)2O - , -S(O)2OH, or -S(O)2OM; Each R 3 are independently hydrogen, -S(O)2O - , -S(O)2OH, or -S(O)2OM; Each R 4 are independently hydrogen, -S(O)2O - , -S(O)2OH, or -S(O)2OM; Each M is independently a cation, where: i)R 1 More than about 20%, or about 20% to about 50%, or about 20% to about 40%, or about 20% to about 35%, or about 20% to about 30%, or about 20% to about 25%, or about 20% to about 27%, or about 22% to about 27%, or about 22% to about 26%, or about 23% to about 26%, or about 22% to about 25%, or about 23% to about 24%, or about 23%, or about 24%, or about 25% [Chemistry] and [Chemistry] selected from ii) More than about 5, or about 5 to about 9, or about 5 to about 8.5, or about 5 to about 8, or about 5 to about 7.5, or about 5 to about 7, or about 6 to about 7, or about 5, or about 6, or about 7, or about 8 of the R 1 portions are all [Chemistry] and [Chemistry] selected from iii) About 3n / 4 of the R 1 portion is -OH, or iv) Less than about 80%, or less than about 75%, or about 75% of the R 1 portion is -OH. In certain embodiments of formula IA, about 70 - 80%, or about 73 - 77% of the R 1 portion is -OH.

[0058] In certain embodiments of formula IA, each R 1 is independently -OH, [Chemistry] , or [Chemistry] wherein, R 1More than about 20%, or about 20% to about 50%, about 20% to about 40%, or about 20% to about 35%, or about 20% to about 30%, or about 20% to about 25%, or about 20% to about 27%, or about 22% to about 27%, or about 22% to about 26%, or about 23% to about 26%, or about 22% to about 25%, or about 23% to about 24%, or about 23%, or about 24%, or about 25% of the moiety is

Chem.

Chem.

[0059] In certain embodiments of Formula IA, each R 1 is independently -OH,

Chem.

Chem.

Chem.

Chem.

[0060] In certain embodiments of Formula IA, about 3n / 4 of the R 1 moieties are -OH.

[0061] In certain embodiments of Formula IA, R 1Less than about 80%, or less than about 75%, or about 75% of the moiety is -OH. In certain embodiments of Formula IA, R 1 About 70 - 80%, or about 73 - 77% of the moiety is -OH.

[0062] In certain embodiments, n is 26. In certain embodiments, n is 27. In certain embodiments, n is 28. In certain embodiments, n is 29. In certain embodiments, n is 30.

[0063] In certain embodiments, each R 1 is independently -OH or

Chemical formula

[0064] In certain embodiments, the chemically modified bovine intestinal heparin comprises one or more chemically modified sugar units of Formula I,

Chemical formula

Chemical formula

Chemical formula

[0065] In certain embodiments, R 2 is hydrogen, -S(O)2O - , -S(O)2OH, or -S(O)2ONa.

[0066] In certain embodiments, the chemically modified bovine intestinal heparin comprises one or more chemically modified sugar units of Formula IIA,

Chem.

Chem.

Chem.

[0067] In certain embodiments, the chemically modified bovine intestinal heparin comprises one or more chemically modified sugar units of Formula IIB,

Chem.

Chem.

Chem.

[0068] In certain embodiments, R 1 is

Chem.

[0069] The chemically modified bovine intestinal heparin disclosed herein is contemplated to include the above mixture of EDU-amine isomers.

[0070] In some embodiments, unmodified bovine intestinal heparin has an anti - factor IIa activity of greater than about 135 U / mg, greater than about 130 U / mg, greater than about 125 U / mg, greater than about 120 U / mg, greater than about 115 U / mg, greater than about 110 U / mg, greater than about 105 U / mg, greater than about 100 U / mg, greater than about 90 U / mg, greater than about 85 U / mg, greater than about 80 U / mg, about 135 U / mg, about 130 U / mg, about 125 U / mg, about 120 U / mg, or about 115 U / mg, or about 110 U / mg, or about 105 U / mg, or about 100 U / mg, or about 90 U / mg, or about 85 U / mg, or about 80 U / mg, or from about 80 U / mg to 135 U / mg, or from about 90 U / mg to 135 U / mg, or from about 90 U / mg to 120 U / mg, or from about 90 U / mg to 110 U / mg, or from about 80 U / mg to 120 U / mg, or from about 80 U / mg to 110 U / mg, or from about 100 U / mg to 135 U / mg, or from about 100 U / mg to 130 U / mg, or from about 100 U / mg to 125 U / mg, or from about 100 U / mg to 120 U / mg, or from about 100 U / mg to 130 U / mg, or from about 100 U / mg to 120 U / mg, or from about 100 U / mg to 110 U / mg, or from about 110 U / mg to 140 U / mg, or from about 110 U / mg to 130 U / mg, or from about 110 U / mg to 120 U / mg, or from about 120 U / mg to 140 U / mg, or from about 120 U / mg to 130 U / mg, or from about 130 U / mg to 140 U / mg.

[0071] In certain embodiments, the chemically modified heparin exhibits an anti - factor IIa activity of less than about 15 U / mg, or less than about 14 U / mg, or less than about 13 U / mg, or less than about 12 U / mg, or less than about 11 U / mg, or less than about 10 U / mg, or less than about 9 U / mg, or less than about 8 U / mg, or about 7 U / mg, or from about 7 to 8 U / mg. In certain embodiments, the chemically modified heparin exhibits an anti - factor IIa activity of about 8 U / mg, or about 7 U / mg, or from about 7 to 8 U / mg.

[0072] In certain embodiments, the chemically modified heparin exhibits anti - Factor IIa activity of about 4 U / mg, or about 3.5 U / mg, or about 3 - 5 U / mg.

[0073] In certain embodiments, a pharmaceutical composition is provided that comprises a chemically modified bovine intestinal heparin and a pharmaceutically acceptable excipient, wherein at least a portion of the free carboxylic acid moieties on the unmodified heparin having an anti - Factor IIa activity greater than 90 U / mg is converted to 1 - (3 - dimethylaminopropyl) - 3 - ethylurea (EDU) - amide, and as a result, the pharmaceutical composition exhibits about 6 - 8% of the anti - Factor IIa activity of the unmodified bovine intestinal heparin.

[0074] In certain embodiments, a pharmaceutical composition is provided that comprises a chemically modified bovine intestinal heparin and a pharmaceutically acceptable excipient, wherein at least a portion of the free carboxylic acid moieties on the unmodified heparin having an anti - Factor IIa activity greater than 90 U / mg is converted to 1 - (3 - dimethylaminopropyl) - 3 - ethylurea (EDU) - amide, and as a result, the pharmaceutical composition exhibits about 3 - 5% of the anti - Factor IIa activity of the unmodified bovine intestinal heparin.

[0075] In certain embodiments, the P - selectin inhibitory activity (IC 50 ) is more potent than that of unmodified bovine intestinal heparin. In certain embodiments, the P - selectin inhibitory activity (IC 50 ) of the chemically modified bovine intestinal heparin is about 5 - fold, or 4.5 - fold, or 4 - fold, or about 3.5 - fold, or about 3 - fold, or about 2.5 - fold, or about 2 - fold, or about 2 - 4 - fold, or about 2 - 3 - fold, or about 3 - 4 - fold more potent than the P - selectin inhibitory activity of the unmodified bovine intestinal heparin.

[0076] In certain embodiments, the P - selectin inhibitory activity (IC 50 ) is less potent than that of unmodified bovine intestinal heparin. In certain embodiments, the P - selectin inhibitory activity (IC50 ) is about one-fifth, or one-fourth point five, or one-fourth, or about one-third point five, or about one-third, or about one-half point five, or about one-half, or about one and a half, or about one to four, or about one to three, or about one to two of the inhibitory activity of unmodified bovine intestinal heparin. In certain embodiments, the P-selectin activity is not substantially different from that of the unmodified parent bovine intestinal heparin.

[0077] In certain embodiments, the pharmaceutical composition reduces P-selectin inhibitory activity as compared to unmodified bovine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity of the chemically modified bovine intestinal heparin is about twice that of the unmodified bovine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity (IC 50 ) of the chemically modified bovine intestinal heparin is about 20 - 500%, or about 500%, or about 450%, or about 400%, or about 350%, or about 300%, or about 250%, or about 200%, or about 150%, or about 140%, or about 130%, or about 120%, or about 110%, or approximately the same, or about 90%, or about 80%, or about 70%, or about 60%, or about 50%, or about 45%, or about 44%, or about 43%, or about 42%, or about 41%, or about 40%, or less than about 500%, or less than about 450%, or less than about 400%, or less than about 350%, or less than about 300%, or less than about 250%, or less than about 200%, or less than about 150%, or less than about 140%, or less than about 130%, or less than about 120%, or less than about 110%, or less than approximately the same, or less than about 90%, or about 80%, or about 70%, or less than about 60%, or less than about 50%, or less than about 45%, or less than about 44%, or less than about 43%, or less than about 42%, or less than about 41%, or less than about 40% of the inhibitory activity of the unmodified bovine intestinal heparin.

[0078] In certain embodiments, when measured in the CH50 assay described herein, the pharmaceutical composition reduces complement inhibitory activity as compared to unchemically modified heparin. In certain embodiments, the complement inhibitory activity (IC 50 concentration) of the chemically modified heparin is about 2 times, or more than 2 times, that of unchemically modified heparin. In certain embodiments, the complement inhibitory activity (IC 50 ) of the chemically modified heparin is about 200%, or about 250%, or about up to 350%, or about 150% - 350%, or about 200% - 350%, or about 200% - 300%, or about 150% - 250% of that of unchemically modified heparin.

[0079] In certain embodiments, the P - selectin inhibitory activity is less than 400% to more than 400% of the inhibitory activity of unchemically modified bovine intestinal heparin. In certain embodiments, the P - selectin inhibitory activity is less than 200% to more than 200% of the inhibitory activity of unchemically modified bovine intestinal heparin.

[0080] In certain embodiments, the P - selectin activity is not substantially different from that of unchemically modified parent bovine intestinal heparin. In certain embodiments, the P - selectin IC 50 is less than about 5 μg / mL.

[0081] In certain embodiments, the anti - Factor IIA activity is less than about 15 IU / mg. In certain embodiments, the anti - Factor IIA activity is less than about 12 IU / mg, or less than 10 IU / mg, or 1 - 15 IU / mg, or 1 - 12 IU / mg, or 1 - 10 IU / mg.

[0082] In certain embodiments, when measured in the CH50 assay, the complement inhibitory activity (IC 50 ) is weaker than that of unchemically modified bovine intestinal heparin. In certain embodiments, the complement inhibitory activity (IC 50) is about one-fourth, or about one-half, or about one-third, or about one-third and a half, or about one-fourth, or about one-fourth and a half, or about one-fifth, or about one-half to one-fifth, or about one-half to one-fourth, or about one-half to one-third of the complement inhibitory activity of unchemically modified bovine intestinal heparin. In certain embodiments, the complement inhibitory activity (IC 50 ) is less than about 1 mg / mL, or less than about 0.7 mg / mL, or less than about 0.5 mg / mL, or less than about 0.2 mg / mL when measured by the CH50 assay described herein.

[0083] In certain embodiments, the pharmaceutical composition reduces the anticoagulant activity by more than 90% (or more than 92%) as compared to unchemically modified bovine intestinal heparin, increases or decreases the P-selectin inhibitory activity by up to 250%, and decreases the complement inhibitory activity by up to 500%.

[0084] In certain embodiments, the pharmaceutical composition reduces the anticoagulant activity by more than 90% (or more than 92%) as compared to unchemically modified bovine intestinal heparin, increases the P-selectin inhibitory activity by up to 250%, and decreases the complement inhibitory activity by up to 500%.

[0085] In certain embodiments, the pharmaceutical composition reduces the anticoagulant activity by more than 90% (or more than 92%) as compared to unchemically modified bovine intestinal heparin, decreases the P-selectin inhibitory activity by up to 250%, and decreases the complement inhibitory activity by up to 500%.

[0086] In certain embodiments, the pharmaceutical composition exhibits an anticoagulant activity of less than about 15 U / mg, or less than about 14 U / mg, or less than about 13 U / mg, or less than about 12 U / mg, or less than about 11 U / mg, or less than about 10 U / mg.

[0087] In certain embodiments, the pharmaceutical composition reduces the anticoagulant activity by more than 90% (or more than 92%) as compared to unchemically modified bovine intestinal heparin and decreases the complement inhibitory activity by up to 500%.

[0088] In certain embodiments, the pharmaceutical composition reduces the anticoagulant activity by more than 90% (or more than 92%) and increases or decreases the P-selectin inhibitory activity by up to 250% compared to unchemically modified bovine intestinal heparin. In certain embodiments, the pharmaceutical composition reduces the anticoagulant activity by more than 90% (or more than 92%) and increases the P-selectin inhibitory activity by up to 250% compared to unchemically modified bovine intestinal heparin. In certain embodiments, the pharmaceutical composition reduces the anticoagulant activity by more than 90% (or more than 92%) and decreases the P-selectin inhibitory activity by up to 250% compared to unchemically modified bovine intestinal heparin.

[0089] In certain embodiments, the pharmaceutical composition increases or decreases the P-selectin inhibitory activity by up to 250% and decreases the complement inhibitory activity by up to 500% compared to unchemically modified bovine intestinal heparin. In certain embodiments, the pharmaceutical composition increases the P-selectin inhibitory activity by up to 250% and decreases the complement inhibitory activity by up to 500% compared to unchemically modified bovine intestinal heparin. In certain embodiments, the pharmaceutical composition decreases the P-selectin inhibitory activity by up to 250% and decreases the complement inhibitory activity by up to 500% compared to unchemically modified bovine intestinal heparin.

[0090] In certain embodiments, the bovine intestinal heparin is unfractionated bovine intestinal heparin.

[0091] In certain embodiments, the chemically modified bovine intestinal heparin is unfractionated bovine intestinal heparin.

[0092] Method Heparin, as well as various other chemically or biochemically modified heparins, have proven useful in many diseases and disorders. The chemically modified heparins described herein, and compositions comprising the chemically modified heparins described herein, may be useful in many diseases and disorders for which heparin is administered, as well as in diseases and disorders for which heparin compositions with some or significant anti-factor IIa activity are contraindicated, because they have reduced or no antithrombin activity.

[0093] In certain embodiments, a method of reducing inflammation in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chemically modified heparin as described herein.

[0094] TNF-α is a characteristic cytokine of inflammation and an important therapeutic target (see, e.g., Esposito, et al. Current medicinal chemistry 16.24 (2009): 3152-3167). The chemically modified bovine intestinal heparin described herein is believed to be able to reduce the production of TNF-α. Thus, in certain embodiments, a method of inhibiting the production of TNF-α in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chemically modified heparin as described herein.

[0095] In certain embodiments, a method of reducing anti-inflammatory properties in a subject in need thereof, such as reducing the recruitment of leukocytes (such as neutrophils) to the endothelium by P-selectin and L-selectin blockade, attenuating cytokine production by NF-kB inhibition, inhibiting complement activation, or modulating neutrophil extracellular traps (NETs), is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chemically modified heparin as described herein.

[0096] In certain embodiments, a method of treating or reducing one or more symptoms of acute respiratory distress syndrome (ARDS) in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chemically modified heparin as described herein. In certain embodiments, acute respiratory distress syndrome (ARDS) is one or more of sepsis, SARS-CoV-2 infection, aspiration vomiting, drowning episode, severe pneumonia, physical injury to the lung, physical injury to the part of the brain that controls breathing, pancreatitis (inflammation of the pancreas), massive transfusion, burns, or inhalation of smoke or chemical fumes.

[0097] In certain embodiments, a method of treating sepsis in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified heparin described herein.

[0098] In one embodiment, the subject is hospitalized. In one embodiment, the subject is being treated with a ventilator. In one embodiment, the subject is suffering from cytokine release syndrome.

[0099] P-selectin is an important therapeutic target for multiple diseases including cancer, cardiovascular disease, and sickle cell disease (SCD) (see, e.g., Ataga, et al. New England Journal of Medicine 376.5 (2017): 429-439; Yeini, et al. Nature communications 12.1 (2021): 1-22; Borsig, Glycobiology 28.9 (2018): 648-655; Ludwig, et al. Expert opinion on therapeutic targets 11.8 (2007): 1103-1117; and Merle, et al. Proceedings of the National Academy of Sciences 116.13 (2019): 6280-6285). In certain embodiments, the compounds disclosed herein inhibit P-selectin-mediated inflammatory responses (e.g., to LPS) (see, e.g., Mayadas, et al. Cell 74.3 (1993): 541-554).

[0100] In certain embodiments, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified heparin described herein. Exemplary cancers include, but are not limited to, leukemia, multiple myeloma, gastrointestinal cancer, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, liver cancer, lung cancer, cervical cancer, head cancer, neck cancer, melanoma, or pancreatic cancer.

[0101] In certain embodiments, a method of treating a solid tumor in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified heparin described herein. Exemplary solid tumors include, but are not limited to, glioblastoma, metastatic gastrointestinal tumors, breast tumors, prostate tumors, ovarian tumors, colorectal tumors, liver tumors, lung tumors, cervical tumors, head tumors, neck tumors, esophageal tumors, brain tumors, or pancreatic tumors.

[0102] In certain embodiments, the cancer is a cancer associated with the expression of sialyl Lewis, sialyl Lewis X, or sialyl Lewis A (also known as CA19-9), which are high selectin ligands. In certain embodiments, a method of treating cancer in a subject in need thereof is provided, where the subject exhibits the expression or overexpression of sialyl Lewis X or sialyl Lewis A. The overexpression of sialyl Lewis X or sialyl Lewis A can be detected by measuring various biomarkers such as antibodies.

[0103] In certain embodiments, the solid tumor is a CNS cancer such as, but not limited to, glioblastoma.

[0104] In certain embodiments, the tumor is a colorectal tumor, liver tumor, pancreatic tumor, lung tumor, breast tumor, prostate tumor, ovarian tumor, head and neck tumor, esophageal tumor, or other tumor type.

[0105] In certain embodiments, a method of inhibiting the metastasis of cancer cells in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified heparin described herein. In certain embodiments, the cancer cells are melanoma cells. Melanoma, the most malignant type of skin cancer, often becomes incurable once it metastasizes. It has been shown that melanoma can metastasize via the blood or lymphatic system. In certain embodiments, a method of inhibiting the metastasis of cancer cells to the mesentery is provided. In certain embodiments, a method of inhibiting the metastasis of cancer cells to the lung is provided.

[0106] In certain embodiments, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified heparin described herein. Exemplary cancers include, but are not limited to, blood cancers such as leukemia, multiple myeloma, and the like.

[0107] In certain embodiments, a method of treating a disease or disorder in a subject in need thereof that is at least partially mediated by inhibition of cell binding to P-selectin is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified heparin described herein. In certain embodiments, treatment comprises reducing inflammation or reducing or inhibiting an inflammatory response as a result of the disease or disorder. In certain embodiments, treatment comprises reducing P-selectin-mediated sickle cell vaso-occlusive crisis.

[0108] In certain embodiments, the disease or disorder is cancer (e.g., blood cancers such as leukemia, multiple myeloma, or metastatic cancers such as melanoma), chemotherapy-induced peripheral neuropathy (CIPN), beta-thalassemia, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), neurological diseases such as amyotrophic lateral sclerosis (ALS), sickle cell disease (including, but not limited to, vaso-occlusive crisis), immune responses in gene therapy with adeno-associated virus (AAV), acute respiratory distress syndrome (ARDS), cardiovascular diseases (e.g., after myocardial infarction or interventional procedures), ophthalmic diseases or disorders, kidney diseases, or thrombotic microangiopathy (TMA).

[0109] In certain embodiments, a method of treating vaso-occlusive crisis (VOC) in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified heparin described herein. In certain embodiments, the subject is in an early stage of VOC, such as a prodromal stage. In certain embodiments, the administration is by subcutaneous administration at home or at a pharmacy, etc., during the early stage of VOC. The early stage of VOC often precedes established VOC by one or more days, such as 1 day, 2 days, 3 days, 4 days, 5 days, 1 - 5 days, 1 - 4 days, 2 - 5 days, 2 - 4 days, 1 - 3 days, or 2 - 3 days. In certain embodiments, the administration is performed during VOC, or at the time of initial symptoms of VOC, or at the established stage of VOC. Administration may relieve or prevent one or more symptoms of VOC, including but not limited to pain episodes, tissue damage, or hospitalization.

[0110] In certain embodiments, a method of treating or alleviating one or more symptoms of sickle cell disease in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chemically modified heparin described herein. In certain embodiments, the subject has vaso-occlusive crisis. In certain embodiments, the subject in need of treatment is in the prodromal or early stage of vaso-occlusive crisis (VOC).

[0111] In certain embodiments, the term administration includes self-administration, including self-administration outside of a medical setting, such as in a home environment.

[0112] In certain embodiments, provided is a method of treating a disease or disorder in a subject in need of treatment, which is at least partially mediated by inhibition of the complement activation pathway, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chemically modified heparin as described herein, or a composition comprising the same. Exemplary diseases or disorders can be found, for example, in Oberkersch, et al. Thrombosis research 125.5 (2010): e240-e245, and Morgan, et al. Nature reviews Drug discovery 14.12 (2015): 857-877.

[0113] Exemplary diseases or disorders include, but are not limited to, hereditary angioedema, paroxysmal nocturnal hemoglobinuria (PNH), chemotherapy-induced peripheral neuropathy (CIPN), beta thalassemia, atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), Shiga toxin-positive HUS, post-infectious HUS, thrombotic microangiopathy, membranoproliferative glomerulonephritis (MPGN) such as primary MPGN, C3 glomerulopathy (C3G), transplant rejection, delayed renal transplant rejection, antibody-mediated renal transplant rejection, renal transplant reperfusion injury, renal transplantation in CAPS patients, neuromyelitis optica, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, lupus nephritis, IgA nephropathy, rheumatoid arthritis, Crohn's disease, ulcerative colitis, hemolytic anemia, autoimmune hemolytic anemia, pemphigus and pemphigoid, antiphospholipid syndrome, cold agglutinin disease, severe thrombocytopenia, macular degeneration, uveitis, ANCA-associated vasculitis, atherosclerosis, mood disorders, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, sepsis, acute respiratory distress syndrome (ARDS), cerebral malaria, psoriatic arthritis, dermatomyositis, osteoarthritis, dementia, glaucoma, diabetic angiopathy, myocardial infarction, stroke, after bypass surgery, multiple trauma, nerve trauma, antiphospholipid syndrome, preeclampsia, or hemodialysis. In certain embodiments, treatment comprises reducing inflammation or reducing or inhibiting an inflammatory response as a result of the disease or disorder.

[0114] AAV gene therapy has already been demonstrated to be very promising in the revolution of disease management, but there are several important barriers. The immune response to AAV administration results in 1) potential serious adverse events and 2) the production of AAV neutralizing antibodies, preventing re-administration of AAV therapy. The immune response to AAV and related pathologies have not yet been fully elucidated, but the innate immune system, particularly complement activation, has emerged as an important facilitator. Therefore, therapeutically modulating complement during AAV administration may address both of these issues, improving the safety of AAV therapy and significantly expanding its potential by enabling multiple administrations. Regarding safety, several adverse events have been clinically observed in AAV therapy, some of which have led to clinical holds by the FDA. Adverse events include atypical hemolytic uremic syndrome (aHUS) and other thrombotic microangiopathies (TMA), which can result in fatal outcomes. Complement activation has emerged as a major facilitator of these harmful immune responses.

[0115] In certain embodiments, provided herein is a method of attenuating an immune response, such as an innate immune response, to AAV gene therapy, the method comprising administering to a subject in need thereof an effective amount of a chemically modified heparin described herein or a composition comprising the same. In certain embodiments, the method reduces neutralizing antibody production and / or complement-mediated adverse reactions to AAV gene therapy.

[0116] In certain embodiments, provided herein is a chemically modified heparin for attenuating an immune response, such as an innate immune response, to AAV gene therapy. In certain embodiments, the chemically modified heparin reduces the production of neutralizing antibodies (NAbs) and / or complement-mediated adverse reactions to AAV gene therapy.

[0117] In certain embodiments, provided herein is the use of the chemically modified heparin disclosed herein in AAV gene therapy to attenuate the innate immune response, thereby 1) preventing or treating adverse events such as aHUS and TMA, 2) reducing the production of neutralizing antibodies, and enabling readministration of AAV.

[0118] In certain embodiments, provided is a method of treating a hemolytic disorder, or one or more adverse effects of gene therapy, described herein, the method comprising administering to a subject in need thereof an effective amount of a chemically modified heparin disclosed herein, or a heparin having reduced anticoagulant activity compared to unfractionated porcine heparin, such as, but not limited to, glycol-split heparin (e.g., certoparin, tafoxiparin, nemiparin, etc.), 2-O,3-O desulfated heparin (also referred to as ODSH or DSTAT), or N-acetylated glycol-split heparin (e.g., roneparstat). In certain embodiments, the hemolytic disorder is cancer, blood cancer, melanoma, leukemia, multiple myeloma, beta thalassemia, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), a neurological disorder, amyotrophic lateral sclerosis (ALS), sickle cell disease (including, but not limited to, vaso-occlusive crises), an immune response in gene therapy by adeno-associated virus (AAV), acute respiratory distress syndrome (ARDS), a cardiovascular disorder, an ophthalmic disorder or condition, a kidney disorder, thrombotic microangiopathy (TMA), hereditary angioedema, thrombotic thrombocytopenic purpura (TTP), Shiga toxin-positive HUS, post-infectious HUS, thrombotic microangiopathy, membranoproliferative glomerulonephritis (MPGN), primary MPGN, C3 glomerulopathy (C3G), transplant rejection, delayed kidney transplant rejection, antibody-mediated kidney transplant rejection, kidney transplant reperfusion injury, kidney transplant in CAPS patients, neuromyelitis optica, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, lupus nephritis, IgA nephropathy, rheumatoid arthritis, Crohn's disease, ulcerative colitis, hemolytic anemia, autoimmune hemolytic anemia, pemphigus and pemphigoid, antiphospholipid syndrome, cold agglutinin disease, severe thrombocytopenia, macular degeneration, uveitis, ANCA-associated vasculitis, atherosclerosis, mood disorder, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, sepsis, cerebral malaria, psoriatic arthritis, dermatomyositis, osteoarthritis, dementia, glaucoma, diabetic angiopathy, myocardial infarction, stroke, post-bypass surgery, multiple trauma, nerve trauma, antiphospholipid syndrome, preeclampsia, or hemodialysis.In certain embodiments, the hemolytic disease is selected from hemolytic uremic syndrome (HUS), beta thalassemia, atypical HUS (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), sickle cell disease, thrombotic microangiopathy, hemolytic anemia, autoimmune hemolytic anemia, and other conditions that cause hemolysis (such as hemodialysis).

[0119] Administration and Dosage The compounds and compositions disclosed herein can be used in the manufacture of pharmaceuticals and for treating humans and other animals by administration according to conventional procedures, such as an active ingredient in a pharmaceutical composition.

[0120] The compounds of the present disclosure can be administered for treatment by any suitable route, specifically oral or parenteral (including subcutaneous, intramuscular, intravenous, intravitreal, intrathecal, and intradermal). It will also be understood that the preferred route of administration will vary depending on the condition and age of the subject, as well as the disease being treated.

[0121] In one embodiment, chemically modified heparin is administered in the composition. The present disclosure provides a composition comprising chemically modified heparin and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers known to those skilled in the art can be used, including water or physiological saline. As is known in the art, the components and their relative amounts are determined by the intended use and delivery method. The diluent or carrier used in the composition can be selected so as not to reduce the desired effect of the chemically modified heparin. Examples of suitable compositions include aqueous solutions, such as isotonic saline, solutions in 5% glucose. Other well-known pharmaceutically acceptable liquid carriers, such as alcohols, glycols, esters, and amides, can be used. In certain embodiments, the composition further comprises one or more excipients, such as, but not limited to, ion strength modifiers, osmotic pressure modifiers, solubility enhancers, sugars such as mannitol or sorbitol, pH buffers, surfactants, stabilizing polymers, preservatives, and / or co-solvents.

[0122] Suitable ion strength modifiers and / or osmotic pressure modifiers include, for example, glycerin, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

[0123] In certain embodiments, the composition is an aqueous solution. Aqueous solutions are suitable for use in formulating compositions based on ease of formulation and the ability to readily administer such compositions by injection into a solution. In certain embodiments, the composition is a suspension, a viscous or semi-viscous gel, or other types of solid or semi-solid compositions. In certain embodiments, the composition is a solution that is applied directly to or contacts the inner wall of a vein or artery. In some embodiments, the composition comprises a polymeric matrix. In other embodiments, the composition is absorbent. In certain embodiments, the composition comprises a pH buffer. In some embodiments, the composition comprises a lubricity enhancer.

[0124] In certain embodiments, it may be necessary to enhance the solubility of chemically modified heparin. In such cases, solubility can be enhanced by using appropriate formulation techniques such as incorporation of mannitol, ethanol, glycerin, polyethylene glycol, propylene glycol, poloxamer, and other solubility enhancing compositions known in the art.

[0125] In certain embodiments, the composition comprises a lubricity enhancer. As used herein, the lubricity enhancer refers to one or more pharma- ceutically acceptable polymeric materials that can modify the viscosity of the pharma-ceutically acceptable carrier. Suitable polymeric materials include ionic and non-ionic water-soluble polymers; hyaluronic acid and its salts, chondroitin sulfate and its salts, dextran, gelatin, chitosan, gellan, bioconjugates or polysaccharides, or any combination thereof; cellulosic polymers and cellulosic polymer derivatives, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose; collagen and modified collagen; galactomannans such as guar gum, locust bean gum, and tara gum, as well as the aforementioned natural gums and malic acids. Examples of suitable polysaccharides include, but are not limited to, polysaccharides derived from similar natural or synthetic gums containing glycerol and / or galactose moieties as the primary structural components (e.g., hydroxypropyl guar); gums such as tragacanth gum and xanthan gum; gellan gum; alginates and sodium alginate; chitosan; vinyl polymers; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers, and polyvinyl alcohol; carboxyvinyl polymers or crosslinked acrylic acid polymers such as the "carbomer" family of polymers, e.g., carboxypolyalkylenes commercially available under the Carbopol™ trademark; and various other viscous or viscoelastic materials.

[0126] Examples of pH buffers suitable for use in the compositions of the present invention include, for example, acetate, borate, carbonate, citrate, and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethylbarbituric acid, and proteins, as well as various biological buffers such as TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, cacodylate, or MES. In certain embodiments, a suitable buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate, or boric acid) is added to the composition to prevent pH drift under storage conditions. In some embodiments, the buffer is a phosphate buffered saline (PBS) solution (i.e., containing sodium phosphate, sodium chloride, and in some formulations potassium chloride and potassium phosphate). Specific concentrations vary depending on the agent being used. In certain embodiments, a pH buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate or boric acid) is added to maintain the pH within the range of about pH 4 to about pH 8, or about pH 5 to about pH 8, or about pH 6 to about pH 8, or about pH 7 to about pH 8. In some embodiments, the buffer is selected to maintain the pH within the range of about pH 4 to about pH 8. In some embodiments, the pH is about pH 5 to about pH 8. In some embodiments, the buffer is a saline buffer. In certain embodiments, the pH is about pH 4 to about pH 8, or about pH 3 to about pH 8, or about pH 4 to about pH 7.

[0127] Chemically modified heparin or a composition containing the same may be sterilized to remove unwanted contaminants including, but not limited to, endotoxins and infectious agents. Sterilization techniques that do not adversely affect the structure and biotropic properties of the chemically modified heparin can be used. In certain embodiments, the chemically modified heparin can be disinfected and / or sterilized using conventional sterilization techniques including, but not limited to, treatment with propylene oxide or ethylene oxide, sterile filtration, gas plasma sterilization, gamma irradiation, electron beam, and / or peracids such as peracetic acid. In one embodiment, the chemically modified heparin can undergo one or more sterilization processes. Alternatively, the chemically modified heparin can be wrapped in any type of container including plastic wrap or foil wrap and further sterilized.

[0128] In some embodiments, the chemically modified heparin or a composition containing the same needs to be administered separately or sequentially to facilitate delivery. In certain embodiments, the chemically modified heparin or a composition containing the same can be administered at different dosing frequencies or intervals. Further, as will be apparent to those skilled in the art, the chemically modified heparin or a composition containing the same can be administered using the same or different routes of administration.

[0129] In one embodiment, the treatment method can further include the administration of an effective amount of another agent. In some embodiments, the other agent is an anti-spike protein antibody or fragment. In some embodiments, the second agent is co-administered simultaneously or sequentially with the antibody or fragment thereof.

[0130] In some embodiments, the second agent is effective to reduce or inhibit a cytokine release storm. In some embodiments, the second agent is a corticosteroid. Non-limiting examples include methylprednisolone (especially in patients with rheumatic diseases) and dexamethasone (especially in patients with FHLH).

[0131] In some embodiments, the second agent is a cell depletion therapy. Non-limiting examples include cyclophosphamide (especially in JIA and MAS patients), etoposide (especially in FHLH patients), rituximab (especially in Epstein-Barr virus (EBV)-associated HLH), antithymocyte globulin (especially in patients at the bone marrow transplantation stage of FHLH therapy), and alemtuzumab (especially in FHLH or SLE-associated MAS patients).

[0132] In some embodiments, the second agent is a T cell modulator. Non-limiting examples include calcineurin, which inhibits the production of IL-2 (e.g., cyclosporine), and abatacept, which inhibits CD28 signaling in T cells. In some embodiments, the second agent is an anti-GM-CSF inhibitor or antibody.

[0133] In some embodiments, the second agent is a cytokine inhibitor, and such inhibitors target INFγ, IL-1β, IL-18, IL-33, IL-6, and / or TNF.

[0134] In some embodiments, the second agent is a chemotherapeutic agent. Exemplary chemotherapeutic agents include cisplatin, etoposide, irinotecan, camptothecin, topotecan, paclitaxel, docetaxel, epothilone, taxotere, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH66336, R115777, L778,123, BMS214662, IRESSA® (gefitinib), TARCEVAR® (erlotinib hydrochloride), an antibody against EGFR, GLEEVEC® (imatinib), Intron, ara-C, adriamycin, cytoxan, gemcitabine, uracil mustard, chloromethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, vinblastine, vincristine, vindesine, bleomycin, doxorubicin, dactinomycin, daunorubicin, epirubicin, idarubicin, mitomycin, deoxycoformycin, mitomycin C, L-asparaginase, teniposide, 17α-ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, nabumetone, anastrozole, letrozole, capecitabine, raloxifene, droloxifene, hexamethylmelamine, avastin, herceptin, vecsal, berkeley, zevalin, trisenox, xeloda, vinorelbine, porfimer, Erbitux® (cetuximab), liposome, thiotepa, altretamine, melphalan,Trastuzumab, letrozole, fulvestrant, exemestane, fulvestrant, ifosfamide, rituximab, C225, campus, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agent, paclitaxel, gemcitabine, navelbine, farnesyl protein transferase inhibitor, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate are included, but not limited thereto.,

[0135] In certain embodiments, the second agent is a CDK (cyclin-dependent kinase) inhibitor such as ribociclib, palbociclib, abemaciclib, P1446A-05, trilaciclib, flavopiridol, olomoucine, roscovitine, dinaciclib, PD-0332991, SNS-032, LY-2835219, R547, LEE011, AT7519, AZD5438, or AG-024322.

[0136] In certain embodiments, the second agent is a checkpoint inhibitor. Exemplary checkpoint inhibitors include, but are not limited to, ipilimumab (Yervoy®), nivolumab (Opdivo®), pembrolizumab (Keytruda®), atezolizumab (Tecentriq®), avelumab (Bavencio®), durvalumab (Imfinzi®), and cemiplimab (Libtayo®).

[0137] In certain embodiments, the second agent is a chimeric antigen receptor T cell (CAR-T cell). Exemplary CAR-T cells include, but are not limited to, tisagenlecleucel (Kymriah®), axicabtagene ciloleucel (Yescarta®), brexucabtagene autoleucel (Tecartus®), lisocabtagene maraleucel (Breyanzi®), and idecabtagene vicleucel (Abecma®).

[0138] In some embodiments, the second agent is a viral vector such as those used in gene therapy. Exemplary viral vectors include, but are not limited to, those related to retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses, or hybrids thereof. In some embodiments, the second agent is a bacteriophage (e.g., Qβ, AP205).

[0139] Accordingly, in certain embodiments, provided herein is a method for treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the chemically modified heparin described herein or a composition comprising the same in combination with one or more chemotherapeutic agents.

[0140] Administration of the chemically modified heparin or a composition comprising the same described herein can be performed before or after a second agent or treatment at intervals ranging from minutes to weeks. For example, in certain embodiments, one or more agents can be administered within about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 28 hours, about 31 hours, about 35 hours, about 38 hours, about 42 hours, about 45 hours, or about 48 hours, or more, before and / or after administration of the chemically modified heparin or a composition comprising the same. In certain embodiments, the agent can be administered within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 8 days, about 9 days, about 12 days, about 15 days, about 16 days, about 18 days, about 20 days, or about 21 days before and / or after administration of the chemically modified heparin or a composition comprising the same. However, depending on the situation, if several weeks (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, or about 8 weeks, or more) elapse between each administration, it may be desirable to significantly extend the treatment period.

[0141] In some embodiments, the second agent targets a potential disease or condition such as SARS-CoV-2 infection. Non-limiting examples include lopinavir, ritonavir, oseltamivir (Tamiflu), favipiravir, fingolimod, methylprednisolone, bevacizumab, chloroquine phosphate, chloroquine, hydroxychloroquine sulfate, and remdesivir.

[0142] In another aspect, the present disclosure provides a pharmaceutical composition comprising the heparin of the present disclosure formulated with a pharmaceutically acceptable carrier. Optionally, it may contain one or more additional pharmaceutically active ingredients such as heparin or a drug. The pharmaceutical composition of the present disclosure can also be administered, for example, in combination therapy with an antiviral agent or a vaccine.

[0143] The pharmaceutical composition can contain any number of excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of appropriate excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference. In certain embodiments, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, intravitreal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound can be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the phrase "parenteral administration" means a route of administration other than enteral and topical administration, usually by injection, which includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intravitreal, and intrathoracic injections and infusions. Alternatively, the heparin of the present disclosure can be administered via routes other than parenteral, such as topical, epidermal, or mucosal administration routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0144] For administration by inhalation or the intranasal route, the chemically modified heparin can usually be delivered in the form of a solution, suspension, emulsion, or semi-solid aerosol from a pressurized pack or nebulizer using a propellant (e.g., halogenated carbons derived from methane and ethane, carbon dioxide, or any other suitable gas). For topical aerosols, hydrocarbons such as butane, isobutene, and pentane are useful. For pressurized aerosols, appropriate dosage units can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, can be formulated for use in inhalers and injectors. These usually contain a powder mixture of the compound with a suitable powder base such as lactose or starch.

[0145] The pharmaceutical composition can be in the form of a sterile aqueous solution or dispersion. It can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0146] The amount of the active ingredient that can be combined with the carrier substances to produce a single dosage form will vary depending on the subject to be treated and the particular method of administration and will generally be the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.01% to about 99% of the active ingredient, or from about 0.1% to about 70% of the active ingredient, or from about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0147] The dosing regimen is adjusted so as to obtain the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally decreased or increased depending on the urgency of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the parenteral composition in unit dosage form. A unit dosage form for use with chemically modified heparin refers to physically discrete units suitable as a unit dose for the subject to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, chemically modified heparin can be administered as a sustained release formulation, in which case the required dosing frequency is reduced.

[0148] In the case of administering a chemically modified heparin, the dosage ranges from about 0.0001 to 100 mg / kg of the host body weight, or about 1 to 12 mg / kg, or about 1 to 6 mg / kg, or 0.01 to 5 mg / kg. For example, the dosage can be within the range of 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or 1 to 10 mg / kg. Exemplary treatment schedules include administration once a day, twice a day, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Exemplary dosing regimens of the chemically modified heparin of the present disclosure include 1 mg / kg body weight, 3 mg / kg body weight, or up to 6 mg / kg body weight by intravenous or subcutaneous administration. Exemplary dosing regimens of the chemically modified heparin of the present disclosure include 1 mg / kg body weight, 3 mg / kg body weight, or up to 6 mg / kg body weight by intravenous administration, and the chemically modified heparin is administered using one of the following dosing schedules: (i) Administer 6 times every 4 weeks and then administer every 3 months. (ii) Administer every 3 weeks. (iii) Administer 3 mg / kg body weight once and then administer 1 mg / kg body weight every 3 weeks. In some methods, the dosage is adjusted to achieve a plasma heparin concentration of about 1 to 1000 μg / mL, and in some methods, about 25 to 300 μg / mL.

[0149] Exemplary dosing regimens of the chemically modified heparin of the present invention for a subject with vascular occlusive crisis include administering 3 mg / kg (e.g., 1 to 6 mg / kg) twice a day via the subcutaneous route for 3 to 10 days during the vascular occlusive crisis.

[0150] In some embodiments, the appropriate dosage of the chemically modified heparin of the present disclosure for a human patient is 5 mg to 1200 mg, 10 mg to 1000 mg, 20 mg to 500 mg, 20 mg to 300 mg, 20 mg to 200 mg, 50 mg to 150 mg, 70 mg to 120 mg per day. In some embodiments, the appropriate dosage of the heparin of the present disclosure for a human patient is about 5 mg, 10 mg, 20 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg per day. In some embodiments, the appropriate dosage of the chemically modified heparin of the present disclosure for a subject is about 100 mg per day.

[0151] In some embodiments, the chemically modified heparin is administered to a subject by continuous infusion at 0.2 mg / Kg / hour to 50 mg / Kg / hour, 0.4 mg / Kg / hour to 40 mg / Kg / hour, 0.8 mg / Kg / hour to 30 mg / Kg / hour, 2 mg / Kg / hour to 30 mg / Kg / hour, 4 mg / Kg / hour to 30 mg / Kg / hour, 6 mg / Kg / hour to 30 mg / Kg / hour, 8 mg to 25 mg / Kg / hour, 12 mg / Kg / hour to 20 mg / Kg / hour, 0.2 mg to 25 mg / Kg / hour, 0.2 mg / Kg / hour to 20 mg / Kg / hour, 0.2 mg / Kg / hour to 15 mg / Kg / hour, 0.2 mg / Kg / hour to 12 mg / Kg / hour, 0.2 mg / Kg / hour to 8 mg / Kg / hour, 0.2 mg / Kg / hour to 5 mg / Kg / hour, or 0.2 mg / Kg / hour to 2 U / Kg / hour. In some embodiments, the appropriate dosage of the heparin of the present disclosure for a human patient is about 0.1 mg / Kg / hour, 0.2 mg / Kg / hour, 0.3 mg / Kg / hour, 0.4 mg / Kg / hour, 0.5 mg / Kg / hour, 0.6 mg / Kg / hour, 0.7 mg / Kg / hour, 0.8 mg / Kg / hour, 0.9 mg / Kg / hour, 1 mg / Kg / hour, 2 mg / Kg / hour, 3 mg / Kg / hour, 4 mg / Kg / hour, 5 mg / Kg / hour, 6 mg / Kg / hour, 7 mg / Kg / hour, 8 mg / Kg / hour, 9 mg / Kg / hour, 10 mg / Kg / hour, 15 mg / Kg / hour, or 20 mg / Kg / hour by continuous infusion. In some embodiments, the chemically modified heparin is administered to a subject by continuous infusion at 0.2 U / Kg / hour to 2 U / Kg / hour.

[0152] In some embodiments, the dosing regimen includes a loading dose and a subsequent maintenance dose (mg / kg / hour) to achieve an appropriate dosage range (e.g., 1 - 6 mg / kg / day). In some embodiments, the loading dose is administered intravenously as a bolus. In some embodiments, the loading dose is administered intravenously as a drip.

[0153] In some embodiments, the dosing regimen includes an intravenous loading dose of about 0.1 to 100 mg / kg, followed by a continuous dosing rate of about 0.01 to 10 mg / kg / h.

[0154] In some embodiments, the administration is once, twice, or three times a day. In some embodiments, the administration is once a week or once a month.

Example

[0155] Example 1. Chemically Modified Heparin Using carbodiimide chemistry, the carboxyl groups on the iduronic acid / glucuronic acid sugars of heparin were chemically modified as described below. The reaction of carbodiimide with a carboxyl group generally proceeds by adding the free carboxylate to one of the double bonds of the diimide to form an O-acylurea product. In the absence of a nucleophile, the O-acylurea rearranges to a more stable N-acylurea by intramolecular acyl transfer.

[0156] Bovine intestinal heparin was reacted with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) at various molar ratios of EDAC to the carboxylate groups (-COOH) on heparin. Information regarding heparin, EDAC, and the reaction buffer is shown in Table 1.

Table 1

[0157] Heparin was dissolved in MES buffer at 20 mg / mL. The EDAC dry powder was added directly to the heparin solution and dissolved by vortexing at the molar ratios listed in Table 2. The reaction was then carried out at room temperature (74°F) for 1 hour. Next, the modified heparin was purified by tangential flow filtration using a 10 kDa MWCO mPES filter (Repligen). In the purification process, at least 3 volumes of 300 mM NaCl were exchanged, followed by at least 10 volumes of water, to obtain a chemically modified heparin compound purified in water.

[0158] The concentration of the test compound was measured by SEC-HPLC under the operating conditions described in Heparin USP. The anticoagulant activity of the compound was measured by anti-Factor IIa activity according to the USP method. Briefly, the anti-Factor IIa activity was measured as follows. Each sample / standard was run in duplicate. 50 μL of standard / sample / blank (reaction buffer, 0.05 M Tris) was pipetted into each well. 100 μL of standard solution reagent R1 (antithrombin) was added to each well and incubated at 37 °C and 900 rpm for 2 minutes. 25 μL of standard solution reagent R2 (thrombin) was added to each well and incubated at 37 °C and 900 rpm for 2 minutes. 50 μL of standard solution reagent R3 - (chromogenic substrate) was added to each well and incubated at 37 °C and 900 rpm for 1 minute. 50 μL of stop solution was added to each well and incubated at 37 °C and 900 rpm for 1 minute. The absorbance of the sample / standard was measured against the blank using a plate reader (405 nm).

[0159] Since the potency of the test compound sample was low, the dilution of the sample was adjusted. The heparin concentration was pre-measured using HPLC-RI.

[0160] The samples were analyzed using a microtiter plate method with endpoint measurement based on USP<208>.

[0161] As shown in Table 2, the anticoagulant activity of the chemically modified heparin decreased by 0% - 7.5% compared to the unmodified parent bovine intestinal heparin.

Table 2

[0162] The chemically modified heparin unexpectedly showed a greater decrease in anticoagulant activity, and complete removal of the anticoagulant effect was achieved at a 2:1 molar ratio (EDAC:hep-COOH).

[0163] Since no unbound EDAC or unbound EDAC by-products were observed, the functionalization was confirmed by HPLC-RI. HPLC conditions: HPLC: Agilent 1100 HPLC with refractive index detector. Column: TSKgel G3000SW XL, 7.8×300 mm, 5 μm (Tosoh Bioscience, 08541) + TSKgel G4000SW XL, 7.8×300 mm, 8 μm (Tosoh Bioscience, 08542). Guard column: TSKgel G2000SWxl-G4000SWxl, and QC-PAK GFC guard column for 7.8 mm ID column, 7 μm (Tosoh Bioscience, 08543). Mobile phase: 0.1 M ammonium acetate in 0.02% azide. Detection: refractive index. Column temperature: 30 °C. Flow rate: 0.6 mL / min. Injection volume: 20 μL.

Table A

[0164] The calculation of the degree of functionalization (or degree of substitution) of the modified N-acylurea-modified heparin, Compound A and Compound B was completed using NMR by comparing the anomeric proton region with the distinct signals of the modified side chains on the polymer backbone.

[0165] Nuclear magnetic resonance spectroscopy (NMR) was completed at 500 MHz (30 °C and 70 °C) using D2O as the solvent. The spectra were referenced to the HDO peak (proton 4.70 ppm (30 °C)). For the 2D spectra described here (without carbon reference signals in the carbon domain), the methyl acetate resonance was found to resonate at 13 C δ 22.0 ppm.

[0166] For use in the comparison, the entire anomeric region was integrated. The anomeric region was integrated using the region of approximately δ 4.94 - 6.0 ppm, and the value of 2 protons, i.e., one disaccharide unit, was assigned.

[0167] The integration of the resonance at δ 2.7 - 3.0 ppm is assigned as two methyl resonances on the ammonium species (terminating the side chain), and thus these six protons appear as a singlet. The integration in the anomeric region is normalized to two protons representing the two protons on the anomeric center of each disaccharide, and thus the degree of substitution (DOS), i.e., the number of dimethyl-containing side chains per disaccharide, can be calculated by dividing the methyl resonance by six.

[0168] The degrees of substitution of Compound A and Compound B are shown in Table 3.

Table 3

[0169] The data indicate that for Compound B, approximately one-fourth of the carboxyls reacted, and for Compound A, slightly more than half of the carboxyls were substituted. The assignment of the important resonances of the acylurea side chain was confirmed by 2D NMR spectroscopy. The HSQC experimental data confirmed that the anomeric region does not seem to be perturbed as the degree of substitution changes.

[0170] Example 2. Mouse Model with LPS-Activated Inflammation The following is an in vivo mouse model for evaluating test compounds for TNF-α and C5a inhibition.

[0171] Male Swiss Webster mice, 7 - 8 weeks old, are acclimated for at least 7 days prior to the start of the study. On Test Day - 1, the animals are weighed and randomized by weight. On the night of Test Day - 1, the animals are administered a vehicle or a test substance as shown in Table 3. On Test Day 0, 2 minutes after intravenous administration of the vehicle / test substance, the animals are administered saline (Group 1) or LPS (1 mg / kg) intraperitoneally. At 2 hours after LPS administration (at corresponding 1-minute intervals), the animals are sacrificed by inhalation of isoflurane anesthesia, exsanguination, followed by cervical dislocation to confirm euthanasia. Serum is collected in 3 x 60 μL aliquots for each animal and stored at -80 °C until further testing. The TNF-α and C5a concentrations from the serum samples are measured by ELISA.

[0172] Example 3. P - selectin - mediated cell binding The following example shows that the compounds disclosed herein inhibit P - selectin, as demonstrated by inhibition of neutrophil - like cell binding.

[0173] In vitro test design A 96 - well plate was coated overnight with 10 μg / mL of protein A and then blocked with 2% FBS for 1 hour. 2 μg / mL of P - selectin / Fc chimera was bound to protein A at 4°C for 3 hours. Next, HL - 60 cells (2e5 cells / well, CMFDA - labeled) were layered on P - selectin and allowed to bind at room temperature for 1 hour. The wells were treated with test samples simultaneously to measure inhibitory binding activity. After 1 hour, unbound cells were washed, and bound cells were lysed with 1% Triton® - X solution and read at 480 / 520 nm on a fluorometer.

[0174] As shown in Figure 1, the compounds provided herein inhibit P - selectin - mediated neutrophil (HL - 60) cell binding. The P - selectin IC 50 values, anti - factor IIA values, and complement values of unfractionated unmodified porcine heparin, unmodified bovine intestinal heparin, enoxaparin, compound A, compound B, and compound D are shown in Table 4 below. Figure 8 shows the P - selectin IC 50 and anti - factor IIA for unmodified bovine intestinal heparin and various chemically modified bovine intestinal heparins. As shown in Figure 8, the claimed chemically modified bovine intestinal heparin exhibits unique P - selectin IC 50 and anti - factor IIA activities compared to unmodified bovine intestinal heparin and highly chemically modified bovine intestinal heparin. [Table 4]

[0175] Example 4. In vitro complement inhibition test Complement, an important effector mechanism of the immune system, is an enzymatic cascade of approximately 30 serum proteins that leads to the amplification of specific humoral responses. It can be activated through the classical pathway or the alternative pathway, or the mannose-binding lectin pathway. Deficiencies or exacerbations of complement activation cause diseases of varying severity, and pharmacological inhibition of the complement system is considered a therapeutic strategy to improve the inflammatory effects caused by exacerbation of complement activation.

[0176] The compounds disclosed herein were tested for complement inhibition using the total complement assay, CH50, as described in Oberkersch et al. Thrombosis research 125.5 (2010): e240-e245. Briefly, normal serum pooled from healthy subjects was incubated with the test compound while increasing the concentration of the test compound in the presence of sensitized sheep red blood cells (EA). The total complement activity in the sample was measured spectrophotometrically by measuring the degree of hemolysis. The test compound was evaluated in triplicate at five different concentrations to determine the degree of hemolysis. The experiment was performed in three independent runs to evaluate the effect of the test compound. The IC 50 values for the test substances for inhibition of the classical complement pathway were calculated from the dose-response curves plotted using test substance concentration versus % complement-induced hemolysis. The IC 50 of complement inhibition of unmodified porcine heparin, non-chemically modified bovine intestinal heparin, Compound A, and Compound B are shown in Table 5.

Table 5

[0177] Figure 2 shows the hemolysis data of unmodified porcine heparin, Compound A, and Compound C. As shown in this example, the complement inhibition measured by the above CH50 assay for non-chemically modified bovine intestinal heparin decreased.

[0178] Example 5: In Vivo Inhibition of Melanoma Metastasis The in vivo inhibition of melanoma metastasis was evaluated as follows. Six-week-old female C57 / BL6 mice (Charles River) were intravenously injected via the tail vein with 3x10 5 cells in 100 μL of luciferase-expressing mouse melanoma cells (B16 / F10-luc). Thirty minutes prior to the administration of the melanoma cells, the mice were pretreated with either saline (control) or 100 μL of compound B by intraperitoneal injection. 10 mg / kg of D-luciferin was administered and in vivo imaging was used with the bioluminescence of luciferase reporter cells to measure metastasis and tumor formation in the lungs of the mice at various time points. Fluorescence was quantified by measuring the region of interest in the lungs. Data are shown from 7 days after the administration of the melanoma cells, and significant inhibition of fluorescence, and thus inhibition of lung metastasis, was observed with compound B treatment (Figure 3).

[0179] Example 6: HL-60 cell assay HUVECs were cultured on custom flow channels coated with fibronectin. Following the formation of a confluent layer (24 - 48 hours), the HUVECs were treated with TNF-α (10 ng / mL) for 4 hours. Next, HL-60 cells were injected and perfused for 10 - 15 minutes to allow adhesion to activated endothelium. Compound B was added to the HUVECs at 100 μg / mL during the incubation period with TNF-α, and cells with only medium were used as a negative control. Next, the attached HL-60 cells were quantified by microscopic imaging and compared to the control channel. The experiment was performed in independent triplicates. As shown in Figure 4, compound B inhibited cell binding to inflamed endothelium by approximately 70%, which supports the possibility that it inhibits selectin-mediated cell binding to selectin-expressing cells such as endothelium or inflamed endothelium. This mechanism supports the possibility that compound B prevents the adhesion of sickle red blood cells, and thus supports the use of the compounds disclosed herein for the treatment of sickle cell disease and as an emergency medicine for patients with sickle cell disease in the prodromal stage of vaso-occlusive crisis.

[0180] Example 7: Rat PD test Studies were conducted to evaluate the pharmacodynamics of a test substance (Compound B) administered subcutaneously (SC) or intravenously (IV) to male and female Sprague Dawley rats. On study day -1, animals were randomized to treatment groups by body weight. On study day 0, animals were administered a single (1x) intravenous or subcutaneous dose of 30 mg / kg (15 mg / mL) of Compound B. Blood samples were collected from the animals via the tail vein at eight time points (from pre-dose to 8 hours post-dose) to obtain serum, and then finally by descending aorta blood collection, and the animals were euthanized at 8 hours post-dose (Group 1, intravenous administration) or 24 hours post-dose (Group 2, subcutaneous administration). Serum samples were analyzed for HL-60 binding to P-selectin using a custom HL-60 / P-selectin binding assay (described herein), and the readout was the fluorescence intensity from the labeled cells.

[0181] All animals survived until the end of the study. Compound B inhibited HL-60 binding to P-selectin with both intravenous and subcutaneous administrations. Administration via the subcutaneous route showed higher activity, with a maximum inhibition level of approximately 45%, reaching a peak at the 30-minute time point. This inhibitory activity decreased to approximately 30% at 8 hours and then returned to near baseline at 24 hours. Inhibition with the intravenous administration route was observed within 1 minute, reached a plateau over 2 hours, and then a gradual increase in inhibition was observed, returning to baseline at 8 hours.

[0182] Example 8: Pancreatic cancer BxPC3 model Studies were conducted to evaluate the anti-cancer activity of Compound A and Compound B in a mouse model of pancreatic cancer. Human pancreatic cancer cells BxPC3 were subcutaneously implanted into 6-week-old female nude mice (5 per group) (1x10 in 100 μL of Matrigel). 6cells). On the 7th day after transplantation, treatment was initiated with either gemcitabine (100 mg / kg, intraperitoneally, every 3 days) or compound A (5 mg / kg, subcutaneously, twice a day) or compound B (5 mg / kg, subcutaneously, twice a day). Tumor volume was measured weekly with calipers. On the 56th day, the dosages of both compound A and compound B were increased to 10 mg / kg, subcutaneously twice a day. On the 70th day, the animals were sacrificed, the tumors were excised and fixed, and stored for further tests.

[0183] On the 70th day, gemcitabine, compound A, and compound B decreased the tumor volume by approximately 58%, 41%, and 52%, respectively.

[0184] Example 9: Acute lung injury model The inventors investigated the activity of compound D in reducing harmful neutrophil infiltration using an established rat model of acute lung injury. [Fernandez - Bustamante, 2015] Acute lung injury was induced in Sprague - Dawley rats (3 - 4 months old) randomized into a control group or a group pretreated with compound D. The rats were anesthetized and a small ventral neck incision was made to expose the trachea. Lung injury was induced by intratracheal injection of interleukin - 1 (IL - 1) and lipopolysaccharide (LPS). A single dose of 50 ng of recombinant IL - 1 diluted in 0.5 mL of saline was injected intratracheally using a 24 - gauge IV catheter and the catheter was immediately removed. One hour later, another bolus of 5 mg / kg of Escherichia coli LPS 0111:B4 in 0.5 mL of saline was injected intratracheally. Then the neck incision was sutured closed, and the rats were allowed to breathe spontaneously and recover from anesthesia. The treated rats received 30 mg / kg of compound D via the subcutaneous route 30 minutes before IL - 1 injection. Control rats were treated as above but not with compound D.

[0185] After 24 hours, all rats were anesthetized again, and the neck incision was reopened to perform tracheotomy, laparotomy, and thoracotomy. The lungs were lavaged with a total of 17 mL of saline through tracheotomy. Subsequently, using this bronchoalveolar lavage fluid (BAL), the total cell count and white blood cell count were measured with a hemocytometer. The neutrophil fraction was obtained using a Wright-stained cytospin specimen of the BAL sample.

[0186] Compound D reduced neutrophil infiltration into the lungs by 60% (n = 6 rats / group) (Figure 9).

[0187] The present disclosure is not limited in scope by the specific embodiments described as single examples of individual aspects of the present disclosure, and any composition or method that is functionally equivalent is within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and changes can be made to the methods and compositions of the present disclosure without departing from the spirit or scope thereof. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they come within the scope of the appended claims and their equivalents. All publications and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A chemically modified bovine intestinal heparin containing approximately 15 to 90 disaccharide units, wherein approximately 15% to 50% of the disaccharide units contain 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide. Chemically modified bovine intestinal heparin with anti-factor IIA activity of less than approximately 15 IU / mg.

2. The chemically modified bovine intestinal heparin according to claim 1, wherein the anti-factor IIA activity is less than about 12 IU / mg, less than 10 IU / mg, or 1 to 15 IU / mg, or 1 to 12 IU / mg, or 1 to 10 IU / mg.

3. The chemically modified bovine intestinal heparin according to claim 1, wherein the P-selectin activity is substantially the same as that of unchemically unmodified bovine intestinal heparin.

4. P-selectin IC ５０ The chemically modified bovine intestinal heparin according to claim 1, wherein the concentration is less than approximately 5 μg / mL.

5. A chemically modified bovine intestinal heparin of formula IA or a salt thereof, 【Transformation 36】 IA During the ceremony, n is between 26 and 30. Each R １ is independently -OH, 【Chemistry 37】 、 or 【Transformation 38】 And, Each R ２ These are hydrogen and -S(O) independently. ２ O － , -S(O) ２ OH, or -S(O) ２ OM, Each R ３ is independently hydrogen, -S(O) ２ O － , -S(O) ２ OH, or -S(O) ２ OM, and Each R ４ These are hydrogen and -S(O) independently. ２ O － , -S(O) ２ OH, or -S(O) ２ OM, Each M is independently a cation, and the R １ Approximately 15% to 50% of the portion 【Chemistry 39】 or 【Chemistry 40】 Chemically modified bovine intestinal heparin or a salt thereof, which is one of the following.

6. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5.

7. A pharmaceutical composition comprising chemically modified bovine intestinal heparin, wherein at least a portion of the free carboxylic acid moiety on chemically unmodified bovine intestinal heparin having anti-factor IIa activity exceeding 90 U / mg is converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide, and as a result, the pharmaceutical composition exhibits 1% to about 8% of the anti-factor IIa activity of the chemically unmodified bovine intestinal heparin.

8. The chemically modified bovine intestinal heparin comprises one or more chemically modified sugar units of formula I, 【Chemistry 41】 I During the ceremony, Each R １ Independent 【Chemistry 42】 or 【Chemistry 43】 And, R ２ is hydrogen, -S(O) ２ O － , -S(O) ２ OH, or -S(O) ２ The pharmaceutical composition according to claim 7, wherein OM is a cation.

9. R ２ is hydrogen, -S(O) ２ O － , -S(O) ２ OH, or -S(O) ２ The pharmaceutical composition according to claim 7, wherein the active ingredient is ONa.

10. The chemically modified bovine intestinal heparin comprises one or more chemically modified sugar units of formula IIA. 【Chemistry 44】 IIA In the formula, each R １ Independent 【Chemistry 45】 or 【Chemistry 46】 The pharmaceutical composition according to claim 7.

11. The chemically modified bovine intestinal heparin comprises one or more chemically modified sugar units of formula IIB, 【Chemistry 47】 IIB In the formula, each R １ Independent 【Chemistry 48】 or 【Chemistry 49】 The pharmaceutical composition according to claim 7.

12. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the chemically modified bovine intestinal heparin has an anti-factor IIa activity of 90 U / mg to 135 U / mg, or the pharmaceutical composition according to claim 7.

13. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, or the pharmaceutical composition according to claim 7, wherein the composition or pharmaceutical composition exhibits about 3% to 8%, or about 4%, or about 7% of the anti-factor IIa activity of the chemically unmodified bovine intestinal heparin.

14. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the chemically modified bovine intestinal heparin is unfractionated bovine intestinal heparin, or the pharmaceutical composition according to claim 7.

15. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the P-selectin inhibitory activity is reduced compared to the chemically unmodified bovine intestinal heparin, or the pharmaceutical composition according to claim 7.

16. P-selectin inhibitory activity (IC ５０ A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the inhibitory activity of the chemically modified bovine intestinal heparin is less than 400% to more than 400% of that of the chemically unmodified bovine intestinal heparin, or the pharmaceutical composition according to claim 7.

17. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the P-selectin inhibitory activity is greater than or substantially the same as that of unmodified bovine intestinal heparin, or the pharmaceutical composition according to claim 7.

18. P-selectin IC ５０ A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the concentration is less than approximately 5 μg / mL, or the pharmaceutical composition according to claim 7.

19. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the anti-factor IIA activity is less than about 15 IU / mg, or the pharmaceutical composition according to claim 7.

20. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the anti-factor IIA activity is less than about 12 IU / mg, less than 10 IU / mg, or 1 to 15 IU / mg, or 1 to 12 IU / mg, or 1 to 10 IU / mg, or the pharmaceutical composition according to claim 7.

21. Complement inhibitory activity (IC ５０ A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, wherein the complement inhibitory activity of the chemically modified bovine intestinal heparin is more than 200% of that of the chemically unmodified bovine intestinal heparin, or the pharmaceutical composition according to claim 7.

22. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, for reducing inflammation in subjects requiring reduction of inflammation.

23. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, for treating or alleviating one or more symptoms of sickle cell disease in a subject requiring treatment or alleviation of one or more symptoms of sickle cell disease.

24. The composition or pharmaceutical composition according to claim 23, wherein the subject requiring treatment or relief of one or more symptoms of sickle cell disease is in a vascular occlusive crisis or in the prodromal or early stage of a vascular occlusive crisis.

25. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, for preventing or reversing cell adhesion in subjects requiring prevention or reversal of cell adhesion.

26. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, for preventing or reversing complement activation in subjects requiring prevention or reversal of complement activation.

27. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, for treating solid tumors in subjects requiring treatment of solid tumors.

28. The aforementioned solid tumor is sialyl Lewis ｘ or Sial Lewis ａ The composition or pharmaceutical composition according to claim 27, which expresses at least one of (sLex or sLea).

29. The composition or pharmaceutical composition according to claim 28, wherein the solid tumor is a gastrointestinal tumor, breast tumor, prostate tumor, ovarian tumor, colorectal tumor, liver tumor, lung tumor, cervical tumor, head tumor, neck tumor, esophageal tumor, brain tumor, or pancreatic tumor.

30. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, for treating a disease or disorder in a subject requiring treatment that is at least partially mediated by inhibition of cell binding to P-selectin and / or inhibition of the complement activation pathway.

31. The aforementioned diseases or disorders include cancer, hematological malignancies, melanoma, leukemia, multiple myeloma, chemotherapy-induced peripheral neuropathy (CIPN), beta-thalassemia, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), neurological disorders, amyotrophic lateral sclerosis (ALS), sickle cell disease, vaso-occlusive crisis, immune response to adeno-associated virus (AAV) gene therapy, acute respiratory distress syndrome (ARDS), cardiovascular diseases, ophthalmic diseases or disorders, kidney diseases, thrombotic microangiopathy (TMA), hereditary angioedema, thrombotic thrombocytopenic purpura (TTP), Shiga toxin-positive HUS, post-infectious HUS, thrombotic microangiopathy, membranoproliferative glomerulonephritis (MPGN), primary MPGN, C3 glomerulopathy (C3G), transplant rejection, delayed kidney transplant rejection, and antibody-mediated kidney transplantation. The composition or pharmaceutical composition according to claim 30, which is a rejection reaction, reperfusion injury after renal transplantation, renal transplantation in patients with CAPS, neuromyelitis optica, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, lupus nephritis, IgA nephropathy, rheumatoid arthritis, Crohn's disease, ulcerative colitis, hemolytic anemia, autoimmune hemolytic anemia, pemphigus and bullous pemphigoid, antiphospholipid syndrome, cold agglutinin disease, severe thrombocytopenia, macular degeneration, uveitis, ANCA-associated vasculitis, atherosclerosis, mood disorders, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, sepsis, cerebral malaria, psoriatic arthritis, dermatomyositis, osteoarthritis, dementia, glaucoma, diabetic vasculopathy, myocardial infarction, stroke, post-bypass surgery, multiple trauma, neurotrauma, antiphospholipid syndrome, pre-eclampsia, or hemodialysis.

32. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5 for reducing inflammation in a subject requiring reduction of inflammation, for treating or reducing one or more symptoms of sickle cell disease in a subject requiring treatment or reduction of one or more symptoms of sickle cell disease, for preventing or reversing cell adhesion in a subject requiring prevention or reversal of cell adhesion, for preventing or reversing complement activation in a subject requiring prevention or reversal of complement activation, for treating a solid tumor in a subject requiring treatment of a solid tumor, or for treating a disease or disorder at least partially mediated by inhibition of cell binding to P-selectin and / or inhibition of the complement activation pathway in a subject requiring treatment of a disease or disorder at least partially mediated by inhibition of cell binding to P-selectin and / or inhibition of the complement activation pathway, or a pharmaceutical composition according to claim 7, wherein the subject is receiving anticoagulant therapy.

33. A composition comprising chemically modified bovine intestinal heparin according to any one of claims 1 to 5 for reducing inflammation in a subject requiring reduction of inflammation; for treating or reducing one or more symptoms of sickle cell disease in a subject requiring treatment or reduction of one or more symptoms of sickle cell disease; for preventing or reversing cell adhesion in a subject requiring prevention or reversal of cell adhesion; for preventing or reversing complement activation in a subject requiring prevention or reversal of complement activation; for treating solid tumors in a subject requiring treatment of solid tumors; or a pharmaceutical composition according to claim 7, wherein the subject is human.

34. A composition comprising chemically modified bovine enteric heparin according to any one of claims 1 to 5 for reducing inflammation in subjects requiring reduction of inflammation; for treating or reducing one or more symptoms of sickle cell disease in subjects requiring treatment or reduction of one or more symptoms of sickle cell disease; for preventing or reversing cell adhesion in subjects requiring prevention or reversal of cell adhesion; for preventing or reversing complement activation in subjects requiring prevention or reversal of complement activation; for treating solid tumors in subjects requiring treatment of solid tumors; or for treating diseases or disorders at least partially mediated by inhibition of cell binding to P-selectin and / or inhibition of the complement activation pathway in subjects requiring treatment of diseases or disorders at least partially mediated by inhibition of cell binding to P-selectin and / or inhibition of the complement activation pathway, or a pharmaceutical composition according to claim 7, wherein the administration of the composition includes subcutaneous (SC) administration.

35. A composition comprising chemically modified bovine enteric heparin according to any one of claims 1 to 5 for reducing inflammation in subjects requiring reduction of inflammation, for treating or reducing one or more symptoms of sickle cell disease in subjects requiring treatment or reduction of one or more symptoms of sickle cell disease, for preventing or reversing cell adhesion in subjects requiring prevention or reversal of cell adhesion, for preventing or reversing complement activation in subjects requiring prevention or reversal of complement activation, for treating solid tumors in subjects requiring treatment of solid tumors, or for treating diseases or disorders at least partially mediated by inhibition of cell binding to P-selectin and / or inhibition of the complement activation pathway in subjects requiring treatment of diseases or disorders mediated at least partially mediated by inhibition of cell binding to P-selectin and / or inhibition of the complement activation pathway, or a pharmaceutical composition according to claim 7, wherein the administration of the composition includes intravenous (IV) administration.

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