Chemically modified heparin

Abstract

Provided is a bovine heparin chemically modified with l-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide, having an anti-factor IIA activity of from about 20 IU / mg to about 30 IU / mg, where the modified bovine heparin exhibits an anti-factor IIa activity of between 20% and 50% of that of a non-chemically modified bovine heparin, and a method for preparing the bovine heparin chemically modified with l-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide, the bovine heparin chemically modified with l-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide. And pharmaceutical compositions comprising the chemically modified bovine enteric heparin, compositions, and methods of making and using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 349,395, filed on June 6, 2022, which is hereby incorporated by reference in its entirety. Background Art

[0003] Sickle cell disease (SCD) is a devastating disease that affects more than 100,000 people in the United States and more than 6 million people worldwide. It is associated with incapacitating pain and chronic progressive ischemic damage to nearly every organ in the body, plummeting life expectancy by more than 20 years. SCD is characterized by vaso-occlusive crises (VOCs). VOCs are acute events of extreme pain and serve as a precursor to serious complications such as acute chest syndrome (ACS), a type of acute lung injury and a leading cause of death in SCD patients. The resulting impact on patients is profound and affects all aspects of life. Although several preventive drugs such as anti-P-selectin antibodies (crizanlizumab), hydroxyurea or L-glutamine have shown promise in at least partially reducing VOCs, no disease-specific acute VOC therapies have been approved, representing a major treatment gap. Summary of the invention

[0004] In one embodiment, the present disclosure provides a chemically modified bovine intestinal heparin, wherein at least a portion of the free carboxylic acid moieties on the non-chemically modified bovine intestinal heparin have been converted to N-acyl urea amides, such that the chemically modified bovine intestinal heparin exhibits between 20% and 50% of the anti-factor IIa activity of the non-chemically modified bovine intestinal heparin; and wherein the complement inhibition activity of the chemically modified bovine intestinal heparin is substantially the same as the complement inhibition activity of the non-chemically modified bovine intestinal heparin, and a pharmaceutical composition comprising the chemically modified bovine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity is not less than 25% of the potency of the non-chemically modified bovine intestinal heparin. The chemically modified bovine intestinal heparin disclosed herein and the composition comprising the same have reduced anticoagulant activity and are optimized for selectin and complement inhibition, thereby allowing effective therapeutic effects when administered to subjects in need thereof, and the risk of adverse bleeding is limited.

[0005] In certain embodiments, a method for 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 as disclosed herein. In certain embodiments, the subject is in an early stage of a vaso-occlusive crisis, such as a prodromal stage. In certain embodiments, the subject is in an established vaso-occlusive crisis (VOC).

[0006] In certain embodiments, provided is a method for preventing or reversing cell adhesion in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition as disclosed herein.

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

[0008] In certain embodiments, a method for 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 as disclosed herein. In certain embodiments, the solid tumor expresses sLex or sLea (Sialyl Lewis x or Sialyl Lewis a ). In certain embodiments, the solid tumor is a gastrointestinal tumor, a breast tumor, a prostate tumor, an ovarian tumor, a colorectal tumor, a liver tumor, a lung tumor, a cervical tumor, a head tumor, a neck tumor, an esophageal tumor, a brain tumor, or a pancreatic tumor.

[0009] Also provided is a method for treating a disease or condition mediated at least in part by inhibiting cell binding to P-selectin and / or inhibiting complement activation pathways in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chemically modified bovine enteroheparin as described herein or a composition comprising the same, wherein the disease or condition is, but is not limited to, cancer, blood cancer, melanoma, leukemia, multiple myeloma, chemotherapy-induced peripheral neuropathy (CIPN), beta thalassemia, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), a nervous system disease, amyotrophic lateral sclerosis (ALS), sickle cell disease (including but not limited to vaso-occlusive crisis), immune response in gene therapy using adeno-associated virus (AAV), acute respiratory distress syndrome (ARDS), cardiovascular disease, ophthalmic disease or condition, renal disease, thrombotic microangiopathy (TMA), hereditary angioedema, thrombotic thrombocytopenic purpura (TTP), Shiga Toxin-positive HUS, postinfectious HUS, thrombotic microangiopathy, membranoproliferative glomerulonephritis (MPGN), primary MPGN, C3 glomerulopathy (C3G), transplant rejection, delayed renal transplant rejection, antibody-mediated renal transplant rejection, renal transplant reperfusion injury, 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 In some embodiments, the treatment includes reducing inflammation or reducing or inhibiting the inflammatory response caused by the disease or disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown are the effects of test compounds on neutrophil binding to immobilized P-selectin in an in vitro model.

[0011] Figure 2 Complement-mediated hemolysis data for unmodified porcine heparin, Compound A, and Compound C are shown.

[0012] Figure 3 It is shown that all groups gained weight throughout the 70-day study period.

[0013] Figure 4 It was shown that tumor growth was inhibited by combined treatment with a sensitizing dose of gemcitabine and the test compound.

[0014] Figure 5 Ex vivo imaging of metastasis at day 70 is shown. DETAILED DESCRIPTION

[0015] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight (including ranges) are approximate and vary in increments of 0.1% or 10% (+) or (-). It should be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about". It should also be understood that, although not always explicitly stated, the reagents described herein are exemplary only and that equivalents thereof are known in the art.

[0016] "Non-chemically modified" bovine intestinal heparin refers to natural bovine intestinal heparin that has not been modified by chemical methods. Exemplary heparins that have been modified by chemical methods include, but are not limited to, LMWH derived from natural heparin, heparin sulfate, heparin from biotechnology sources, synthetic heparin, or other heparin analogs. Exemplary chemical modifications include, but are not limited to, one or more of the following: 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-, O-desulfation followed by N-resulfation), sulfation, etc.

[0017] "Chemically modified" bovine enteroheparin refers to non-chemically modified bovine enteroheparin or native bovine enteroheparin that has been modified to include a covalent bond to 1-(3-dimethylaminopropyl)-3-ethylurea.

[0018] 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 heparin). The reaction of EDAC with a carboxyl group is usually carried out by adding a free carboxylate to one of the double bonds of the diimide system to obtain an O-acylurea product. In the presence of a nucleophile, an acyl-nucleophile product and a urea of ​​a carbodiimide are formed. In the absence of a nucleophile, the O-acylurea is rearranged to a more stable N-acylurea isomer by intramolecular acyl transfer, as shown below:

[0019] The wavy lines indicate covalent bonds to the heparin backbone.

[0020] In many cases, the chemically modified bovine enteroheparin compounds of the present disclosure are capable of forming acid and / or base salts by virtue of the presence of sulfoxide and / or carboxyl groups or groups similar thereto. In certain embodiments, salts, compositions, dosage forms or other materials useful for preparing pharmaceutical compositions suitable for veterinary or human pharmaceutical use are provided.

[0021] A "pharmaceutical composition" is intended to include an active agent in combination with an inert or active carrier, such that the composition is suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0022] A "subject" of diagnosis or treatment is an animal, such as a mammal, including a human.

[0023] "Effective amount" refers to the amount of an agent sufficient to induce a desired biological and / or therapeutic result. The result may be alleviation of signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

[0024] As used herein, the terms "treating", "treatment" and the like 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 therapeutic in terms of partially or completely curing a disorder and / or adverse effects attributable to the disorder.

[0025] "Administration" can be performed in one dose continuously or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the pharmaceutical composition used for treatment, the purpose of the treatment, the target cell being treated, and the subject being treated. Single or multiple administrations can be performed with a dosage level and pattern selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art.

[0026] Chemically modified bovine enteroheparin

[0027] Chemically modified bovine intestinal heparin and compositions comprising the same are provided herein. Heparin is a naturally occurring glycosaminoglycan. Glycosaminoglycan (GAG) or mucopolysaccharide is a long linear polysaccharide consisting of repeating disaccharide units. Except for keratin, the repeating unit is composed of amino sugars and uronic acid or galactose. The molecular weight range of natural heparin is 3 to 30kDa. Various molecular weight heparins can be used herein, such as a single disaccharide unit from about 650kDa to 700Da to a polysaccharide of about 50kDa. In certain embodiments, heparin is about 10kDa to about 20kDa. In certain embodiments, heparin is about 15kDa to about 20kDa. In certain embodiments, heparin is at most about 15kDa, or about 16kDa, or about 17kDa, or about 18kDa, or about 19kDa, or about 20kDa.

[0028] 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-pyranoglucopyranosyl; GlcNS represents 2-deoxy-2-sulfamido-α-D-pyranoglucopyranosyl; and GlcNS(6S) represents 2-deoxy-2-sulfamido-α-D-pyranoglucopyranosyl-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).

[0029] Heparin compounds and compositions with reduced anticoagulant activity can allow higher doses of heparin to be administered to subjects in which anticoagulant activity is contraindicated (e.g., subjects taking aspirin, ibuprofen or other anti-inflammatory drugs (e.g., NSAIDs) or drugs containing these ingredients). The anticoagulant activity of heparin can also be measured according to its activity of inhibiting factor Xa (fXa) or factor IIa (thrombin). Examples can be found, for example, in Stuart, M, Johnson, L, Hanigan, S, Pipe, SW, Li, SH. 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 examples disclosed herein. Bovine enteroheparin as used herein is derived from bovine intestine.

[0030] In certain embodiments, a chemically modified bovine enteroheparin is provided, wherein at least a portion of the free carboxylic acid moieties on the non-chemically modified bovine enteroheparin have been converted to N-acyl urea amides, such that the chemically modified bovine enteroheparin exhibits between 20% and 50%, or about 20% to about 40%, or about 25% to 40%, or about 25% to 35%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% of the anti-Factor IIa activity of the non-chemically modified bovine enteroheparin; and wherein the complement inhibition activity of the chemically modified bovine enteroheparin is substantially the same as the complement inhibition activity of the non-chemically modified bovine enteroheparin.

[0031] In certain embodiments, a pharmaceutical composition is provided, comprising a chemically modified bovine enteroheparin and a pharmaceutically acceptable excipient, wherein at least a portion of the free carboxylic acid moieties on the non-chemically modified bovine enteroheparin have been converted to N-acyl urea amides such that the chemically modified bovine enteroheparin exhibits between 20% and 50%, or about 20% to about 40%, or about 25% to 40%, or about 25% to 35%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% of the anti-Factor IIa activity of the non-chemically modified bovine enteroheparin; and wherein the complement inhibition activity of the chemically modified bovine enteroheparin is substantially the same as the complement inhibition activity of the non-chemically modified bovine enteroheparin.

[0032] In certain embodiments, "chemically modified" heparin and "non-chemically modified" heparin are derived from the same source when compared herein. In certain embodiments, "chemically modified" heparin and "non-chemically modified" heparin are not derived from the same source when compared herein.

[0033] In certain embodiments, the non-chemically modified bovine enteroheparin has an anti-factor IIa activity of greater than 80 U / mg. In certain embodiments, the non-chemically modified bovine enteroheparin has an anti-factor IIa activity of greater than 90 U / mg. In certain embodiments, the non-chemically modified bovine enteroheparin has an anti-factor IIa activity of about 100 U / mg, or about 90 U / mg to about 135 U / mg.

[0034] In certain embodiments, the chemically modified bovine enteroheparin has an anti-Factor IIa activity of less than 135 U / mg.

[0035] In certain embodiments, the chemically modified bovine enteroheparin has an anti-factor IIa activity between 80 U / mg and 135 U / mg. In certain embodiments, the chemically modified bovine enteroheparin has an anti-factor IIa activity between 90 U / mg and 135 U / mg.

[0036] In certain embodiments, the chemically modified bovine enteroheparin exhibits between 20% and 50%, or about 20% to about 40%, or about 25% to 40%, or about 25% to 35%, or about 25% to 30%, or about 30% to 35%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% of the anti-Factor IIa activity of non-chemically modified bovine enteroheparin.

[0037] In certain embodiments, the chemically modified bovine enteroheparin comprises one or more chemically modified sugar units of Formula I:

[0038]

[0039] in:

[0040] Each R 1 Independently and

[0041] R 2 Hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; wherein M is a cation.

[0042] In certain embodiments, R 2 Hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2ONa.

[0043] In certain embodiments, the chemically modified bovine enteroheparin comprises one or more chemically modified sugar units of Formula IIA:

[0044]

[0045] Each R 1 Independently

[0046] In certain embodiments, the chemically modified bovine enteroheparin comprises one or more chemically modified sugar units of Formula IIB:

[0047]

[0048] Each R 1 Independently

[0049] In certain embodiments, R 1 for

[0050] It is contemplated that the chemically modified bovine enteroheparin disclosed herein comprises a mixture of the above-mentioned EDU-amine isomers.

[0051] In some embodiments, the anti-factor IIa activity of non-chemically modified bovine enteroheparin is 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 or 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 between about 80 U / mg and 135 U / mg, or between about 90 U / mg and 135 U / mg, or about 90 U / mg. to 120 U / mg, or about 90 U / mg to 110 U / mg, or about 80 U / mg to 120 U / mg, or about 80 U / mg to 110 U / mg, or about 100 U / mg to 135 U / mg, or about 100 U / mg to 130 U / mg, or about 100 U / mg to 125 U / mg, or about 100 U / mg to 120 U / mg, or about 100 U / mg to 130 U / mg mg, or about 100 U / mg to 120 U / mg, or about 100 U / mg to 110 U / mg, or about 110 U / mg to 140 U / mg, or about 110 U / mg to 130 U / mg, or about 110 U / mg to 120 U / mg, or about 120 U / mg to 140 U / mg, or about 120 U / mg to 130 U / mg, or about 130 U / mg to 140 U / mg.

[0052] In certain embodiments, the chemically modified heparin exhibits an anti-Factor IIa activity of about 40 U / mg, or about 35 U / mg, or about 30 U / mg, or about 29 U / mg, or about 28 U / mg, or about 27 U / mg, or about 26 U / mg, or about 25 U / mg, or about 20 U / mg.

[0053] In certain embodiments, the chemically modified heparin exhibits an anti-Factor IIa activity of about 25 U / mg to 35 U / mg. In certain embodiments, the chemically modified heparin exhibits an anti-Factor IIa activity of about 25 U / mg to 30 U / mg.

[0054] In certain embodiments, provided herein is a chemically modified bovine enteroheparin, wherein at least a portion of the free carboxylic acid moieties on a non-chemically modified heparin having an anti-Factor IIa activity greater than 90 U / mg has been converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide, such that the chemically modified bovine enteroheparin exhibits about 20% to 30% or about 25% to 30% of the anti-Factor IIa activity of the non-chemically modified bovine enteroheparin.

[0055] In certain embodiments, provided herein is a pharmaceutical composition comprising a chemically modified bovine enteroheparin and a pharmaceutically acceptable excipient, wherein at least a portion of the free carboxylic acid moieties on the non-chemically modified heparin having an anti-Factor IIa activity greater than 90 U / mg has been converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide, such that the pharmaceutical composition exhibits about 20% to 30% or about 25% to 30% of the anti-Factor IIa activity of the non-chemically modified bovine enteroheparin.

[0056] Also provided herein is a pharmaceutical composition comprising a chemically modified heparin and a pharmaceutically acceptable excipient, wherein at least a portion of the free carboxylic acid moieties on the non-chemically modified heparin having an anti-Factor IIa activity greater than 90 U / mg has been converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide, such that the pharmaceutical composition exhibits about 20%, or about 25%, or about 27%, or about 28%, or about 29%, or about 30% of the anti-Factor IIa activity of the non-chemically modified heparin.

[0057] In certain embodiments, the pharmaceutical composition increases P-selectin inhibitory activity compared to non-chemically modified bovine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity of the chemically modified bovine intestinal heparin is about 10% higher than the P-selectin inhibitory activity of the non-chemically modified bovine intestinal heparin. In certain embodiments, the P-selectin inhibitory activity of the chemically modified bovine intestinal heparin is about 15%, or about 20%, or about 30%, or about 40%, or about 50%, or about 70%, or greater than about 100%, or greater than about 150%, or greater than about 200%, or greater than about 250%, or greater than about 300%, or greater than about 400% of the P-selectin inhibitory activity of the non-chemically modified bovine intestinal heparin.

[0058] In certain embodiments, P-selectin inhibitory activity (IC 50 ) is between less than 150% and greater than 150% of the P-selectin inhibitory activity of non-chemically modified bovine enteroheparin. In certain embodiments, the P-selectin inhibitory activity (IC 50 ) is between less than 50% and greater than 50% of the P-selectin inhibitory activity of non-chemically modified bovine enteroheparin.

[0059] In certain embodiments, the P-selectin inhibitory activity is substantially the same as the P-selectin inhibitory activity of non-chemically modified bovine enteroheparin (±50%, or ±40%, or ±30%, or ±20%, or ±10%).

[0060] In certain embodiments, the pharmaceutical composition does not reduce complement inhibitory activity compared to non-chemically modified bovine enteroheparin when measured by a CH50 assay as described herein. In certain embodiments, the complement inhibitory activity of the chemically modified heparin is about 100% of the complement inhibitory activity of non-chemically modified bovine enteroheparin. In certain embodiments, the complement inhibitory activity of the chemically modified bovine enteroheparin is about 95%, or about 90%, or about 85%, or about 80% of the complement inhibitory activity of non-chemically modified bovine enteroheparin.

[0061] In certain embodiments, complement inhibition activity (IC 50 ) has substantially the same complement inhibition activity as that of non-chemically modified bovine enteroheparin. In certain embodiments, the complement inhibition activity (IC 50 ) is up to 150% of the complement inhibitory activity of non-chemically modified bovine enteroheparin.

[0062] In certain embodiments, the complement inhibitory activity (IC 50 ) has a potency that is less than 1.5 times, at most 1.5 times, or less than 2 times, or about 2 times that of chemically modified heparin.

[0063] In certain embodiments, the pharmaceutical composition reduces anticoagulant activity by up to 80% and does not increase or decrease P-selectin inhibitory activity by more than 150% compared to non-chemically modified bovine enteroheparin.

[0064] In certain embodiments, the pharmaceutical composition reduces anticoagulant activity by up to 80% and increases P-selectin inhibitory activity by up to 150% compared to non-chemically modified bovine enteroheparin.

[0065] In certain embodiments, the pharmaceutical composition reduces anticoagulant activity by up to 80% and P-selectin inhibitory activity by up to 150% compared to non-chemically modified bovine enteroheparin.

[0066] In certain embodiments, the pharmaceutical composition reduces anticoagulant activity by up to 80% and reduces complement inhibitory activity by up to 50% compared to non-chemically modified bovine enteroheparin.

[0067] In certain embodiments, the pharmaceutical composition does not increase or decrease P-selectin inhibitory activity by more than 150% and does not decrease complement inhibitory activity by more than 50% compared to non-chemically modified bovine enteroheparin.

[0068] In certain embodiments, the pharmaceutical composition increases P-selectin inhibitory activity by up to 150% and does not decrease complement inhibitory activity by more than 50% compared to non-chemically modified bovine enteroheparin.

[0069] In certain embodiments, the pharmaceutical composition reduces P-selectin inhibitory activity by up to 150% and reduces complement inhibitory activity by no more than 50% compared to non-chemically modified bovine enteroheparin.

[0070] In certain embodiments, the pharmaceutical composition reduces anticoagulant activity by up to 80%, does not increase or decrease P-selectin inhibitory activity by more than 150%, and does not decrease complement inhibitory activity by more than 50%, compared to non-chemically modified bovine enteroheparin.

[0071] In certain embodiments, the pharmaceutical composition reduces anticoagulant activity by up to 80%, increases P-selectin inhibitory activity by up to 150%, and does not reduce complement inhibitory activity by more than 50%, compared to non-chemically modified bovine enteroheparin, when measured by the CH50 assay as described herein.

[0072] In certain embodiments, the pharmaceutical composition reduces anticoagulant activity by up to 80%, reduces P-selectin inhibitory activity by up to 150%, and does not reduce complement inhibitory activity by more than 50%, compared to non-chemically modified bovine enteroheparin.

[0073] In certain embodiments, the chemically modified bovine enteroheparin is unfractionated bovine enteroheparin.

[0074] In certain embodiments, the chemically modified bovine enteroheparin disclosed herein is composed of heparin that has been reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, EDAC or EDCI) in the absence of a nucleophile, and thus EDAC is not used to conjugate the heparin to another compound or material. In addition, the degree or level of sulfation on the bovine heparin is not reduced or altered by the chemical modification (i.e., EDU modification). In addition, the molecular weight of the bovine heparin is not reduced or altered by the chemical modification (i.e., EDU modification).

[0075] In certain embodiments, a chemically modified bovine enteroheparin is provided, comprising about 15 to about 90 disaccharide units, wherein at most about 15%, or about 2.5% to 15%, or about 2.5% to 14% of the disaccharide units comprise 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide.

[0076] In certain embodiments, a chemically modified bovine enteroheparin is provided, comprising about 15 to about 90 disaccharide units, wherein up to about 15%, or about 2.5% to 15%, of the disaccharide units comprise 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide; and the anti-factor IIA activity is about 20 IU / mg to about 30 IU / mg.

[0077] In certain embodiments, the anti-Factor IIA activity is greater than 20 IU / mg and less than the anti-Factor IIA activity of non-chemically modified bovine enteroheparin.

[0078] In certain embodiments, the anti-Factor IIA activity is between 17 IU / mg and 50 IU / mg, or between 20 IU / mg and 50 IU / mg, or between 20 IU / mg and 45 IU / mg, or between 20 IU / mg and 40 IU / mg, or between 20 IU / mg and 35 IU / mg, or between 20 IU / mg and 30 IU / mg, or about 25 IU / mg, 26 IU / mg, 27 IU / mg, or 28 IU / mg.

[0079] In certain embodiments, the P-selectin activity of the chemically modified bovine enteropeptidase is not significantly different from the P-selectin inhibitory activity of the parent non-chemically modified bovine enteropeptidase.

[0080] In certain embodiments, the chemically modified bovine enteroheparin P-selectin IC 50 Less than about 3 μg / mL.

[0081] In certain embodiments, a chemically modified bovine enteroheparin of Formula IA is provided:

[0082]

[0083] or a salt thereof, wherein:

[0084] n is 26 to 30;

[0085] Each R 1 are independently -OH,

[0086] Each R 2 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM;

[0087] Each R 3 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM;

[0088] Each R 4independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; and

[0089] Each M is independently a cation;

[0090] Where R 1 Less than 15% of the portion is

[0091] In certain embodiments, a composition comprising the chemically modified bovine enteroheparin described herein is provided.

[0092] In certain embodiments, a chemically modified bovine enteroheparin consisting of bovine enteroheparin is provided, wherein less than 15%, or 1% to 15%, or 2.5% to 15%, or 2.5% to 14% of the carboxylic acid functional groups on the bovine enteroheparin have 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide moieties covalently bonded thereto. In certain embodiments, about 2.5% to 15%, or 5% to 15%, or 5% to 14%, or 8% to 14%, or 9% to 14%, or 10% to 15%, or about 13% of the carboxylic acid functional groups on the bovine enteroheparin have 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide moieties covalently bonded thereto.

[0093] In any embodiments described herein, the number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups per heparin is an average, wherein certain chemically modified bovine enteroheparin compounds in the composition may have more 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups per heparin and other compounds have fewer 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups per heparin. Thus, in certain embodiments, the number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups as described herein is an average in a chemically modified bovine enteroheparin composition.

[0094] For example, in certain embodiments, the chemically modified bovine enteroheparin is a composition wherein the average number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide groups / heparin is about 3 to 5, or about 3 to 4, or about 3, or about 3.3, or about 3.6, or about 3.9, or about 4, or about 4.3, or about 4.5, or about 5, or 3.3 to 3.9, wherein the average molecular weight of the heparin is about 16 kDa. 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 3 to 5, or about 3 to 4, or about 3, or about 3.3, or about 3.6, or about 3.9, or about 4, or about 4.3, or about 4.5, or about 5, or 3.3 to 3.9.

[0095] In certain embodiments, the chemically modified bovine enteroheparin can be characterized by the number of carboxylic acid functional groups that have been converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide (i.e., the degree of substitution (DOS) or the percentage of functionalization). The degree of substitution (DOS) or the percentage of functionalization is based on the percentage of disaccharide units in the heparin that are functionalized with 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide on the heparin backbone.

[0096] 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 Da to 600 Da, or about 575 Da) by the molecular weight of the glycan (e.g., about 15 Da to 17 kDa, or about 16 kDa). In certain embodiments, a chemically modified bovine enteroheparin is provided, wherein the degree of substitution (DOS) or percentage of functionalization is less than 15%, or is between about 1% and 15%, or between about 1.5% and 15%, or between about 2% and 15%, or between about 2.5% and 15%, or between about 3% and 14%, or between about 4% and 14%, or between about 5% and 14%, or between about 5% and about 13.5%, or between about 6% and about 13.5%, or between about 7% and about 13.5%, or between about 8% and about 13.5%, or between about 9% and about 13.5%, or between about 10% and about 13.5%, or about 11%, or about 13.5%, or about 13%.

[0097] In certain embodiments, the number of carboxylic acid functional groups that have been converted to 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide can be described as the "functionalization percentage by mass" based on the number of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide units on the heparin backbone. In certain embodiments, a chemically modified bovine enteroheparin is provided, wherein the functionalization percentage by mass is less than 5%, about 0.1% to 4.5%, or about 1% to 4.5%, or about 2% to 4%, or about 3% to 4%, or about 3.5% to 4%, or about 3%, or about 3.5%, or about 3.6%, or about 3.7%, or about 3.8%, or about 4%.

[0098] In certain embodiments, a chemically modified heparin of Formula IA is provided:

[0099]

[0100] or a salt thereof, wherein:

[0101] n is 15 to 90;

[0102] R 1 As defined herein;

[0103] Each R 2 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM;

[0104] Each R 3 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM;

[0105] Each R 4 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; and

[0106] Each M is independently a cation.

[0107] In certain embodiments, a chemically modified heparin is provided, comprising about 15 to about 90 disaccharide units, wherein about 1% to less than 15% of the disaccharide units comprise 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide; and the anti-Factor IIA activity is about 20 IU / mg to about 30 IU / mg.

[0108] In certain embodiments, heparin is 9KDa to 50KDa, or about 9KDa to 35KDa, or about 9KDa, or about 12KDa, or about 16KDa, or about 20KDa, or about 35KDa, or about 50KDa. In certain embodiments, n is about 15 to 87, or about 20 to 65, or about 25 to 35, or about 25 to 30.

[0109] In certain embodiments, a chemically modified bovine enteroheparin of Formula IA is provided:

[0110]

[0111] or a salt thereof, wherein:

[0112] n is 26 to 30;

[0113] R 1 As defined herein;

[0114] R 2 Hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM;

[0115] Each R 3 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM;

[0116] Each R 4 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; and

[0117] Each M is independently a cation.

[0118] In certain embodiments, a chemically modified bovine enteroheparin of Formula IA is provided:

[0119]

[0120] or a salt thereof, wherein:

[0121] n is 26 to 30;

[0122] Each R 1 are independently -OH,

[0123] R 2 Hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM;

[0124] Each R 3 independently hydrogen, -S(O)2O -, -S(O)2OH or -S(O)2OM;

[0125] Each R 4 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; and

[0126] Each M is independently a cation; wherein:

[0127] i)R 1 Less than 15%, or about 1% to 15%, or about 1.5% to 15%, or about 2% to 15%, or about 2.5% to 15%, or about 3% to 14%, or about 4% to 14%, or about 5% to 14%, or about 5% to about 13.5%, or about 6% to about 13.5%, or about 7% to about 13.5%, or about 8% to about 13.5%, or about 9% to about 13.5%, or about 10% to about 13.5%, or about 11%, or about 13.5%, or about 13% selected from

[0128] ii) R 1 Less than 5, or about 1 to 5, or about 2 to 5, or about 2.5 to 4.5, or about 2.5, or about 3, or about 3.5, or about 4, or about 4.5 of the parts are selected together.

[0129] iii) R 1 About 7 / 8 of the xn in the moiety are -OH.

[0130] In certain embodiments of Formula IA, each R 1 are independently -OH, The condition is R 1 Less than 15%, or about 1% to 15%, or about 1.5% to 15%, or about 2% to 15%, or about 2.5% to 15%, or about 3% to 14%, or about 4% to 14%, or about 5% to 14%, or about 5% to about 13.5%, or about 6% to about 13.5%, or about 7% to about 13.5%, or about 8% to about 13.5%, or about 9% to about 13.5%, or about 10% to about 13.5%, or about 11%, or about 13.5%, or about 13% selected from

[0131] In certain embodiments of Formula IA, each R 1 are independently -OH, The condition is R 1Less than 5, or about 1 to 5, or about 2 to 5, or about 2.5 to 4.5, or about 2.5, or about 3, or about 3.5, or about 4, or about 4.5 of the parts are selected from

[0132] In certain embodiments of Formula IA, R 1 About 7 / 8 of the xn in the moiety are -OH.

[0133] In some embodiments, n is 26. In some embodiments, n is 27. In some embodiments, n is 28. In some embodiments, n is 29. In some embodiments, n is 30.

[0134] In certain embodiments, each R 1 are independently -OH or

[0135] method

[0136] Heparin and various other chemically or biochemically modified heparins have been shown to be useful for many diseases and disorders. Chemically modified heparins as described herein and compositions comprising chemically modified heparins as described herein (whose antithrombin activity is reduced to none) can be used for many diseases and disorders for which heparin is administered, as well as diseases and disorders for which heparin compositions having some or significant anti-factor IIa activity are contraindicated.

[0137] In certain embodiments, a method for 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.

[0138] TNF-α is a hallmark cytokine of inflammation and an important therapeutic target (see, e.g., Esposito et al. Current medicinal chemistry 16.24 (2009): 3152-3167). It is expected that the chemically modified bovine enteroheparin described herein will be able to reduce the production of TNF-α. Therefore, in certain embodiments, a method for 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.

[0139] In certain embodiments, a method for reducing anti-inflammatory properties in a subject is provided, such as reducing recruitment of leukocytes (such as neutrophils) to the endothelium by P- and L-selectin blockade, attenuating cytokine production by NF-kB, inhibiting complement activation, or modulating neutrophil extracellular traps (NETs) in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chemically modified heparin as described herein.

[0140] In certain embodiments, a method for treating or alleviating 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 a symptom of one or more of sepsis, SARS-CoV-2 infection, aspiration of vomitus, drowning events, severe pneumonia, physical injury to the lungs, physical injury to the part of the brain that controls breathing, pancreatitis (inflammation of the pancreas), massive blood transfusion, burns, or inhalation of smoke or chemical fumes.

[0141] In certain embodiments, provided is a method for treating sepsis in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chemically modified heparin as described herein.

[0142] In one embodiment, the subject is hospitalized. In one embodiment, the subject is treated with mechanical ventilation. In one embodiment, the subject suffers from cytokine release syndrome.

[0143] P-selectin is an important therapeutic target for a variety of 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 (such as to LPS) (see, e.g., Mayadas et al. Cell 74.3 (1993): 541-554).

[0144] In certain embodiments, a method for 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 a chemically modified heparin as 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.

[0145] In certain embodiments, a method for 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 a chemically modified heparin as 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.

[0146] In certain embodiments, the cancer is a cancer with high expression of the selectin ligand Sialyl Lewis, Sialyl Lewis X, or Sialyl Lewis A (also known as CA19-9). In certain embodiments, a method for treating cancer in a subject in need thereof is provided, wherein the subject exhibits expression or overexpression of Sialyl Lewis X or Sialyl Lewis A. Overexpression of Sialyl Lewis X or Sialyl Lewis A can be detected by measuring various biomarkers such as antibodies.

[0147] In certain embodiments, the solid tumor is a CNS cancer, such as but not limited to glioblastoma.

[0148] In certain embodiments, the tumor is a colorectal tumor, a liver tumor, a pancreatic tumor, a lung tumor, a breast tumor, a prostate tumor, an ovarian tumor, a head and neck tumor, an esophageal tumor, or other tumor type.

[0149] In certain embodiments, a method for inhibiting 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 a chemically modified heparin as described herein. In certain embodiments, the cancer cells are melanoma cells. Melanoma is the most aggressive form of skin cancer and is generally incurable once the cancer has metastasized. It has been shown that melanoma can metastasize via the blood or lymphatic system. In certain embodiments, a method for inhibiting metastasis of cancer cells to the mesentery is provided. In certain embodiments, a method for inhibiting metastasis of cancer cells to the lung is provided.

[0150] In certain embodiments, a method for 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 a chemically modified heparin as described herein. Exemplary cancers include, but are not limited to, blood cancers, such as leukemia, multiple myeloma, and the like.

[0151] In certain embodiments, a method for treating a disease or condition mediated at least in part by the binding of inhibitory cells to P-selectin 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, treatment comprises reducing inflammation or reducing or inhibiting an inflammatory response caused by the disease or condition. In certain embodiments, treatment comprises reducing P-selectin mediated sickle cell vaso-occlusive crisis.

[0152] In certain embodiments, the disease or disorder is cancer (e.g., a blood cancer such as leukemia, multiple myeloma, etc., or a metastatic cancer such as melanoma, etc.), chemotherapy-induced peripheral neuropathy (CIPN), beta thalassemia, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), a neurological disease such as amyotrophic lateral sclerosis (ALS), sickle cell disease (including but not limited to vaso-occlusive crisis), immune response in gene therapy using adeno-associated virus (AAV), acute respiratory distress syndrome (ARDS), cardiovascular disease (e.g., after myocardial infarction or interventional treatment), ophthalmic disease or disorder, renal disease, or thrombotic microangiopathy (TMA).

[0153] In certain embodiments, a method for treating a 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 chemically modified heparin as described herein. In certain embodiments, the subject is in the early stages of VOC, such as the prodromal stage. In certain embodiments, during the early stages of VOC, administration is performed via subcutaneous administration (such as at home or in a pharmacy). The early stages of VOC are generally one or more days earlier than the determined VOC, such as 1 day, 2 days, 3 days, 4 days, 5 days, 1 to 5 days, 1 to 4 days, 2 to 5 days, 2 to 4 days, 1 to 3 days, or 2 to 3 days. In certain embodiments, administration is during VOC, or in the early symptoms of VOC, or in the determined stage of VOC. Administration can alleviate or prevent one or more symptoms of VOC, such as, but not limited to, pain crisis, tissue damage, or hospitalization.

[0154] In certain embodiments, a method for 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 a chemically modified heparin as described herein. In certain embodiments, the subject is in a vaso-occlusive crisis. In certain embodiments, the subject in need thereof is in the prodromal or early stages of a vaso-occlusive crisis (VOC).

[0155] In certain embodiments, the term administration includes self-administration, including self-administration outside of a healthcare setting, such as in a home setting.

[0156] In certain embodiments, a method for treating a disease or disorder mediated at least in part by inhibition of a complement activation pathway 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. 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.

[0157] Exemplary diseases or conditions 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 transplant in CAPS patients, neuromyelitis optica, multiple sclerosis, Guillain-Barré syndrome, severe Myasthenia, lupus nephritis, IgA nephropathy, rheumatoid arthritis, Crohn's disease, ulcerative colitis, hemolytic anemia, autoimmune hemolytic anemia, pemphigus and pemphigoid, anti-phospholipid syndrome, cold agglutinin disease, severe thrombocytopenia, macular degeneration, uveitis, ANCA-associated vasculitis, atherosclerosis, mood disorders, asthma, chronic obstructive pulmonary disease (COPD), allergies, sepsis, acute respiratory distress syndrome (ARDS), cerebral malaria, psoriatic arthritis, dermatomyositis, osteoarthritis, dementia, glaucoma, diabetic vasculopathy, myocardial infarction, stroke, post-bypass surgery, multiple trauma, neurotrauma, antiphospholipid syndrome, preeclampsia or hemodialysis. In certain embodiments, treatment includes reducing inflammation or reducing or inhibiting the inflammatory response caused by the disease or disorder.

[0158] AAV gene therapy has shown great promise in transforming disease management, but several key barriers remain. Immune responses to AAV administration can result in 1) potentially significant adverse events, and 2) the production of AAV neutralizing antibodies, which prevent re-administration of AAV therapy. While the immune response to AAV and the associated pathologies have not been fully elucidated, the innate immune system, particularly complement activation, has emerged as a key driver. Therefore, therapeutic modulation of complement during AAV administration has the potential to address both of these issues, thereby improving the safety of AAV therapy while also greatly expanding its potential by allowing multiple dosing. In terms of safety, several adverse events have been observed with AAV therapy in the clinic, some of which have resulted in FDA clinical holds. Adverse events include atypical hemolytic uremic syndrome (aHUS) or other thrombotic microangiopathy (TMA), which can have fatal consequences. Complement activation is emerging as a key driver of these adverse immune responses.

[0159] In certain embodiments, the present invention provides a method for reducing an immune response such as an innate immune response to AAV gene therapy, the method comprising administering an effective amount of a chemically modified heparin as described herein or a composition comprising the same to a subject in need thereof. In certain embodiments, the method reduces the production of neutralizing antibodies and / or complement-mediated adverse responses to AAV gene therapy.

[0160] In certain embodiments, a chemically modified heparin is provided herein for attenuating an immune response, such as an innate immune response, to AAV gene therapy. In certain embodiments, the chemically modified heparin reduces neutralizing antibody (NAb) production and / or complement-mediated adverse responses to AAV gene therapy.

[0161] In certain embodiments, provided herein is the use of a chemically modified heparin as disclosed herein for attenuating the innate immune response in AAV gene therapy, thereby 1) preventing or treating adverse events such as aHUS and TMA, and 2) reducing neutralizing antibody production and allowing AAV re-administration.

[0162] In certain embodiments, provided herein is a method for treating a hemolytic disease as described herein or one or more adverse effects from gene therapy, the method comprising administering to a patient in need thereof an effective amount of a chemically modified heparin as disclosed herein, or a heparin having reduced anticoagulant activity relative to porcine unfractionated heparin, such as but not limited to glycol-cleaved heparin (e.g., sevuparin, tafoxiparin, necuparanib, etc.), 2-O,3-O desulfurized heparin (also known as ODSH or DSTAT), or N-acetylated glycol-cleaved heparin (e.g., roneparstat). In certain embodiments, the hemolytic disease is cancer, a blood cancer, melanoma, leukemia, multiple myeloma, beta thalassemia, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), a neurological disease, amyotrophic lateral sclerosis (ALS), sickle cell disease (including but not limited to vaso-occlusive crisis), immune response in gene therapy using adeno-associated virus (AAV), acute respiratory distress syndrome (ARDS), cardiovascular disease, ophthalmic disease or disorder, renal 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 renal allograft rejection, delayed renal allograft rejection, antibody-mediated renal allograft rejection, renal allograft reperfusion injury, renal transplant 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 pemphigoid, anti-phospholipid syndrome, cold agglutinin disease, severe thrombocytopenia, macular degeneration, uveitis, ANCA-associated vasculitis, atherosclerosis, mood disorders, asthma, chronic obstructive pulmonary disease (COPD), allergies, sepsis, cerebral malaria, psoriatic arthritis, dermatomyositis, osteoarthritis, dementia, glaucoma, diabetic vasculopathy, myocardial infarction, stroke, post-bypass surgery, polytrauma, neurotrauma, antiphospholipid syndrome, preeclampsia, or hemodialysis. In certain embodiments, the hemolytic disease is selected from hemolytic uremic syndrome (HUS), 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).

[0163] Administration and Dosage

[0164] The compounds and compositions disclosed herein can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration according to conventional procedures, such as active ingredients in pharmaceutical compositions.

[0165] The compounds of the present disclosure can be administered by any suitable route for treatment, particularly by oral or parenteral (including subcutaneous, intramuscular, intravenous, intravitreal, intrathecal and intradermal) administration. It should also be understood that the preferred route will vary with the condition and age of the subject and the disease being treated.

[0166] In one embodiment, the chemically modified heparin is administered in the form of a composition. The present disclosure provides a composition comprising a chemically modified heparin and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers known to those of ordinary skill in the art can be used, including water or saline. As known in the art, 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 that they do not reduce the desired effect of the chemically modified heparin. Examples of suitable compositions include aqueous solutions, such as 5% glucose isotonic saline solutions. Other known pharmaceutically acceptable liquid carriers can be used, such as alcohols, glycols, esters and amides. In certain embodiments, the composition further comprises one or more excipients, such as but not limited to ionic strength regulators, osmotic pressure regulators, solubility enhancers, sugars such as mannitol or sorbitol, pH buffers, surfactants, stabilizing polymers, preservatives and / or cosolvents.

[0167] Suitable ionic strength adjusters and / or osmotic pressure adjusters include, for example, glycerol, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

[0168] In certain embodiments, the composition is an aqueous solution. Based on the ease of preparation and the ability to easily apply such compositions by instillation in the solution, the aqueous solution is suitable for use in the composition formulation. In certain embodiments, the composition is a suspension, a viscous or semi-viscous gel or other types of solid or semisolid compositions. In certain embodiments, the composition is a solution directly applied to or contacting the inner wall of a vein or artery. In certain embodiments, the composition comprises a polymer matrix. In other embodiments, the composition is absorbable. In certain embodiments, the composition comprises a pH buffer. In certain embodiments, the composition contains a lubricity enhancer.

[0169] In certain embodiments, it may be desirable to enhance the solubility of the chemically modified heparin. In such cases, the solubility may be increased by using appropriate formulation techniques, such as incorporation of solubility enhancing compositions, such as mannitol, ethanol, glycerol, polyethylene glycol, propylene glycol, poloxamer, and other compositions known in the art.

[0170] In certain embodiments, the composition contains a lubricity enhancer. As used herein, a lubricity enhancer refers to one or more pharmaceutically acceptable polymeric materials that are capable of changing the viscosity of a pharmaceutically acceptable carrier. Suitable polymeric materials include, but are not limited to: ionic and non-ionic water-soluble polymers; hyaluronic acid and its salts, chondroitin sulfate and its salts, dextran, gelatin, chitosan, gellan gum, bioconjugates or polysaccharides, or any combination thereof; cellulose polymers and cellulose polymer derivatives, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose; collagen and modified collagen; galactomannan, such as guar gum. , locust bean gum and tara gum, and polysaccharides derived from the above natural gums and similar natural or synthetic gums containing mannose and / or galactose moieties as major structural components (e.g., hydroxypropyl guar gum); gums such as tragacanth 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 cross-linked acrylic acid polymers, such as the "carbomer" family of polymers, for example, which can be found in the market as Carbopol TM Carboxypolyalkylenes available commercially under the trademark HYDROXYL®; and various other viscous or viscoelastic materials.

[0171] Suitable pH buffers for compositions herein include, for example, acetate, borate, carbonate, citrate and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, potassium dihydrogen phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethyl barbituric acid and protein, and various biological buffers, such as TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, cacodylate or MES. In certain embodiments, an appropriate 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 certain embodiments, the buffer is a phosphate buffered saline (PBS) solution (i.e., containing sodium phosphate, sodium chloride and, in some preparations, potassium chloride and potassium phosphate). The specific concentration will vary depending on the reagent 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 in 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 in 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.

[0172] Chemically modified heparin or a composition comprising it can be sterilized to remove unwanted contaminants, including but not limited to endotoxins and pathogenic factors. Sterilization techniques that will not adversely affect the structure and biogenic properties of chemically modified heparin can be used. In certain embodiments, chemically modified heparin can be disinfected and / or sterilized using conventional sterilization techniques, which include propylene oxide or ethylene oxide treatment, sterile filtration, gas plasma sterilization, gamma radiation, electron beams, and / or sterilization with peracids such as peracetic acid. In one embodiment, chemically modified heparin can undergo one or more sterilization processes. Alternatively, chemically modified heparin can be wrapped in any type of container, including plastic film or foil, and can be further sterilized.

[0173] In some embodiments, it is necessary to administer chemically modified heparin or a composition comprising it separately or sequentially to facilitate delivery. In certain embodiments, chemically modified heparin or a composition comprising it can be administered with different dosing frequencies or intervals. In addition, as will be apparent to those skilled in the art, chemically modified heparin or a composition comprising it can be administered using the same route of administration or different routes of administration.

[0174] In one embodiment, the method of treatment may further include administering 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 with the antibody or fragment thereof simultaneously or sequentially.

[0175] In some embodiments, the second agent is effective to reduce or inhibit the cytokine release storm. In some embodiments, the second agent is a corticosteroid. Non-limiting examples include methylprednisolone (particularly in patients with rheumatic diseases) and dexamethasone (particularly in patients with FHLH).

[0176] In some embodiments, the second agent is a cell ablative therapy. Non-limiting examples include cyclophosphamide (particularly in patients with JIA and MAS), etoposide (particularly in patients with FHLH), rituximab (particularly in Epstein-Barr virus (EBV)-associated HLH), anti-thymocyte globulin (particularly for patients in the bone marrow transplantation phase of FHLH therapy), and alemtuzumab (particularly in patients with FHLH or SLE-associated MAS).

[0177] In some embodiments, the second agent is a T cell modulator. Non-limiting examples include calcineurin (e.g., cyclosporin) to prevent IL-2 production and abatacept to inhibit CD28 signaling of T cells. In some embodiments, the second agent is an anti-GM-CSF inhibitor or antibody.

[0178] In some embodiments, the second agent is a cytokine inhibitor that targets INFγ, IL-1β, IL-18, IL-33, IL-6, and / or TNF.

[0179] In some embodiments, the second agent is a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, cisplatin, etoposide, irinotecan, camptostar, topotecan, paclitaxel, docetaxel, epothilone, taxotere, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH 66336, R115777, L778,123, BMS 214662, (gefitinib), (erlotinib hydrochloride), anti-EGFR antibodies, (imatinib), intron, ara-C, doxorubicin, cytoxan, gemcitabine, uracil mustard, nitrogen mustard, ifosfamide, melphalan, chlorambucil, pipobroman, triethylene melamine, triethylene thiophosphamide, busulfan, carmustine, lomustine, streptozotocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thio Guanine, fludarabine phosphate, pentostatin, vinblastine, vincristine, vindesine, bleomycin, doxorubicin, dactinomycin, daunomycin, epirubicin, idarubicin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide, 17α-ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drostanolone propionate, testolactone, methyl acetate medroxyprogesterone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorpheniramine, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, navelbine, anastrozole, letrozole, capecitabine, raloxafene (reloxafine), duloxetine (droloxafine), hexamethylmelamine, Avastin (Avastin), herceptin (herceptin), Bexxar (Bexxar), Velcade (Velcade), Zevalin (Zevalin), Trisenox, Xeloda (Xeloda), Vinorelbine (Vinorelbine), Porfimer sodium (Porfimer), (cetuximab), liposomes, thiotepa, altretamine, melphalan, trastuzumab, letrozole, fulvestrant, exemestane, ifosfomide, rituximab, C225, Campath, carboplatin, procarbazine, dichloromethyl diethylamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunomycin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate.

[0180] In certain embodiments, the second agent is a CDK (cyclin-dependent kinase) inhibitor, such as ribociclib, palbociclib, abemaciclib, P1446A-05, tremaciclib, favopiridol, olomucine, roscovitine, dinixib, PD-0332991, SNS-032, LY-2835219, R547, LEE011, AT7519, AZD5438, or AG-024322.

[0181] In certain embodiments, the second agent is a checkpoint inhibitor. Exemplary checkpoint inhibitors include, but are not limited to, ipilimumab Nivolumab Pembrolizumab Atezolizumab Avelumab Durvalumab and cimiprilimab

[0182] 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, tesalencyclase Akilum brexucabtageneautoleucel Leakey Myron and idecabtagene vicleucel

[0183] In some embodiments, the second agent is a viral vector, such as those used for gene therapy. Exemplary viral vectors include, but are not limited to, those associated with 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).

[0184] Thus, in certain embodiments, provided herein is a method for treating cancer, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a chemically modified heparin or a composition comprising a chemically modified heparin as described herein in combination with one or more chemotherapeutic agents.

[0185] As described herein, chemically modified heparin or the application of the composition comprising it can be before or after the second medicament or treatment, and the interval range is a few minutes to a few weeks. For example, in some aspects, chemically modified heparin or the application of the composition comprising it can be before and / or after 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, to about 48 hours or longer time, one or more medicaments are applied. In certain embodiments, the agent may 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, to about 21 days before and / or after administration of a chemically modified heparin or a composition comprising the same. However, in some cases, it may be desirable to significantly extend the treatment period with a few weeks (e.g., about 1, about 2, about 3, about 4, about 6, or about 8 weeks or longer) between respective administrations.

[0186] In some embodiments, the second agent targets an underlying 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.

[0187] On the other hand, the present disclosure provides a pharmaceutical composition comprising the heparin of the present disclosure formulated with a pharmaceutically acceptable carrier. It may optionally contain one or more additional pharmaceutically active ingredients, such as heparin or a drug. The pharmaceutical composition of the present disclosure may also be administered in a combination therapy with, for example, an antiviral agent or a vaccine.

[0188] The pharmaceutical composition can include any number of excipients. Available excipients include carriers, surfactants, thickeners or emulsifiers, solid adhesives, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents and combinations thereof. The selection and use of suitable excipients are edited in Gennaro, Remington: The Science and Practice of Pharmacy, the 20th edition (Lippincott Williams & Wilkins 2003) has instruction, and its disclosure is incorporated herein by reference. In certain embodiments, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, intravitreal or epidermal administration (for example, by injection or infusion). According to the route of administration, the active compound can be coated in the material to protect it from the effect of acid and other natural conditions that may inactivate it. As used herein, the phrase "parenteral administration" means other modes of administration than enteral and topical administration (usually administered by injection), and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intravitreal, and intrasternal injection and infusion. Alternatively, the heparin of the present disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal administration route, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

[0189] For administration by inhalation or intranasal route, chemically modified heparin can be delivered from a pressurized bag or sprayer in the form of a solution, suspension, emulsion or semisolid aerosol, usually using a propellant, such as a halocarbon derived from methane and ethane, carbon dioxide or any other suitable gas. For topical aerosols, hydrocarbons such as butane, isobutylene and pentane are useful. In the case of pressurized aerosols, a suitable dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges such as gelatin can be formulated for use in an inhaler or insufflator. These usually contain a powder mixture of the compound and a suitable powder base (such as lactose or starch).

[0190] The pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated as microemulsions, liposomes, or other ordered structures suitable for high drug concentration.

[0191] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration, and is generally that amount of the composition that produces a therapeutic effect. Generally speaking, in one hundred percent, in combination with a pharmaceutically acceptable carrier, the amount ranges from about 0.01% to about 99% of the active ingredient, or about 0.1% to about 70% of the active ingredient, or about 1% to about 30% of the active ingredient.

[0192] The dosage regimen is adjusted to provide the best desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. The dosage unit form used for chemically modified heparin refers to physically discrete units suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. Alternatively, chemically modified heparin may be administered as a sustained release formulation, in which case a lower frequency of administration is required.

[0193] For the administration of chemically modified heparin, the dosage range is about 0.0001mg / kg host weight to 100mg / kg host weight or about 1mg / kg host weight to 12mg / kg host weight or about 1mg / kg host weight to 6mg / kg host weight or 0.01mg / kg host weight to 5mg / kg host weight. For example, the dosage can be 0.3mg / kg body weight, 1mg / kg body weight, 3mg / kg body weight, 5mg / kg body weight or 10mg / kg body weight or within the range of 1mg / kg to 10mg / kg. Exemplary treatment regimens require 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 3 months or once every 3 to 6 months. The exemplary dosage regimen of chemically modified heparin disclosed herein includes 1mg / kg body weight, 3mg / kg body weight or up to 6mg / kg body weight via intravenous or subcutaneous administration. An exemplary dosage regimen of the chemically modified heparin disclosed herein includes 1 mg / kg body weight, 3 mg / kg body weight, or up to 6 mg / kg body weight administered intravenously, wherein the chemically modified heparin is administered using one of the following dosing schedules: (i) once every four weeks for a total of six doses, then once every three months; (ii) once every three weeks; (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight once every three weeks. In some methods, the dosage is adjusted to achieve a plasma heparin concentration of about 1 μg / mL to 1000 μg / mL, and in some methods about 25 μg / mL to 300 μg / mL.

[0194] An exemplary dosage regimen of the chemically modified heparin of the present disclosure for a subject in vaso-occlusive crisis includes 3 mg / kg (eg, 1 mg / kg to 6 mg / kg) twice daily via the sc route and continued for 3 to 10 days during the vaso-occlusive crisis.

[0195] In some methods, the dosage is contemplated to be administered twice daily via subcutaneous delivery. In some methods, the dosage is contemplated to be administered twice daily via subcutaneous delivery at 10 mg / kg for about 3 to 5 days, or up to 10 days (e.g., for sickle cell disease), or long-term for up to several months (e.g., for cancer).

[0196] In some embodiments, the suitable dosage of chemically modified heparin disclosed herein for human patients is 5mg to 1200mg, 10mg to 1000mg, 20mg to 500mg, 20mg to 300mg, 20mg to 200mg, 50mg to 150mg, 70mg to 120mg per day. In some embodiments, the suitable dosage of heparin disclosed herein for human patients is about 5mg, 10mg, 20mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, or 200mg per day. In some embodiments, the suitable dosage of chemically modified heparin disclosed herein for subjects is about 100mg per day.

[0197] In some embodiments, the chemically modified heparin administered to the subject is 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 via continuous infusion. Hour, 12mg / Kg / hour to 20mg / Kg / hour, 0.2.mg to 25mg / Kg / hour, 0.2mg / Kg / hour to 20mg / Kg / hour, 0.2mg / Kg / hour to 15mg / Kg / hour, 0.2mg / Kg / hour to 12mg / Kg / hour, 0.2mg / Kg / hour to 8mg / Kg / hour, 0.2mg / Kg / hour to 5mg / Kg / hour, or 0.2mg / Kg / hour to 2U / Kg / hour. In some embodiments, the suitable dosage of heparin of the present disclosure for human patients 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 via continuous infusion. In some embodiments, chemically modified heparin is administered to a subject by continuous infusion of 0.2 U / Kg / hour to 2 U / Kg / hour.

[0198] In some embodiments, the dosage regimen includes a loading dose followed by a maintenance dose (mg / kg / h) to achieve an appropriate dosage range (e.g., 1 mg / kg / day to 12 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 an infusion.

[0199] In some embodiments, the dosage regimen comprises an intravenous loading dose of about 0.1 mg / kg to 100 mg / kg, followed by continuous doses of about 0.01 mg / kg / h to 10 mg / kg / h.

[0200] In some embodiments, administration is once, twice, or three times a day. In some embodiments, administration is once a week or once a month.

[0201] Examples

[0202] Example 1. Chemically modified heparin

[0203] The carboxyl groups on the iduronic acid / glucuronic acid sugars of heparin are chemically modified as described below using carbodiimide chemistry. The reaction of carbodiimide with carboxyl groups is usually carried out by adding a free carboxylate to one of the double bonds of the imide to give an O-acylurea product. In the absence of a nucleophile, the O-acylurea rearranges to a more stable N-acylurea by intramolecular acyl transfer.

[0204] Bovine enteroheparin was reacted with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) at different molar ratios of EDAC to carboxylic acid groups (-COOH) on heparin. Information on heparin, EDAC and reaction buffer is shown in Table 1.

[0205] Table 1. EDAC and heparin reactive materials

[0206]

[0207] Unfractionated, non-chemically modified bovine enteroheparin

[0208] Heparin was dissolved in MES buffer at 20 mg / mL. EDAC dry powder was added directly to the heparin solution and dissolved by vortexing at the molar ratio listed in Table 2. The reaction was then allowed to proceed for 1 hour at room temperature (74 ° F). The modified heparin was then purified by tangential flow filtration using a 10 kDa MWCO mPES filter (Repligen). At least 3 volumes of 300 mM NaCl were exchanged during the purification process, followed by at least 10 volumes of water to produce purified chemically modified heparin compounds in water.

[0209] The concentration of the test compound is determined by the SEC-HPLC method, and the operating conditions are as described in the USP for heparin. The anticoagulant activity of the compound is measured by anti-factor IIa activity according to the USP method. In short, the anti-factor IIa activity is determined as follows. Each sample / standard is run in duplicate. 50 μL of standard / sample / blank (reaction buffer, 0.05M tris) is moved into each well. 100 μL of working solution reagent R1 (antithrombin) is added to each well and incubated at 37°C for 2 minutes at 900rpm. 25 μL of working solution reagent R2 (thrombin) is added to each well and incubated at 37°C for 2 minutes at 900rpm. 50 μL of working solution reagent R3 (chromogenic substrate) is added to each well and incubated at 37°C for 1 minute at 900rpm. 50 μL of stop solution is added to each well and incubated at 37°C for 1 minute at 900rpm. The absorbance of the sample / standard is measured for the blank using an ELISA reader (405nm).

[0210] Due to the lower potency of the test compound samples, the sample dilution was adjusted. Heparin concentration was previously determined using HPLC-RI.

[0211] Use based on USP <208> The samples were analyzed using the microtiter plate method for endpoint measurement.

[0212] As shown in Table 2, the anticoagulant activity of the chemically modified heparins was reduced compared to the parent non-chemically modified bovine enteroheparin, with complete removal of the anticoagulant effect being achieved at a molar ratio of 2:1 (EDAC:hep-COOH).

[0213] Table 2.

[0214]

[0215] Unfractionated, non-chemically modified bovine enteroheparin

[0216] Functionalization was confirmed by HPLC-RI, and no unconjugated EDAC or unconjugated EDAC byproducts were observed.HPLC conditions: HPLC: Agilent 1100HPLC with a refractive index detector; Column: TSKgel G3000SW XL, 7.8 x 300 mm, 5 microns (Tosoh Bioscience, 08541) + TSKgel G4000SW XL, 7.8 x 300 mm, 8 microns (Tosoh Bioscience, 08542); Guard column: TSKgel G2000SWxl-G4000SWxl, and QC-PAK GFC guard column for 7.8 mm ID column, 7 microns (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.

[0217] sample Retention time (average value, n=2) Compound A 30.08 Compound C 29.85

[0218] Calculation of the degree of functionalization (or substitution) of the modified N-acyl-urea modified heparin, Compound A and Compound C was accomplished using NMR by comparing the anomeric proton region to the clear signals for the modified side chains on the polymer backbone.

[0219] Nuclear magnetic resonance spectroscopy (NMR) was performed at 500 MHz (30°C and 70°C) with D2O as solvent. The spectra were referenced to the HDO peak (protons at 4.70 ppm (30°C)). For the 2-D spectra discussed here (no carbon reference signal for carbon domains), the methyl acetate resonance was found at 13 Cδ resonates at 22.0ppm, no need to adjust the spectral reference.

[0220] The entire anomeric region was integrated for comparison. The anomeric region was integrated using the region between approximately δ 4.94 ppm and 6.0 ppm and assigned a value of 2-protons (ie, one disaccharide unit).

[0221] The resonance integration at δ 2.7 ppm to 3.0 ppm was assigned to the two methyl resonances on the ammonium species (terminating the side chain), and therefore these 6 protons appear as singlets. The integration of the anomeric region has been normalized to 2 protons, representing the two protons on the anomeric center of each disaccharide, so the degree of substitution (DOS) (i.e., the number of side chains containing dimethyl groups per disaccharide) can be calculated by dividing the methyl resonances by 6.

[0222] The substitution degrees of compound A and compound C are shown in Table 3.

[0223] Table 3.

[0224] Compound Degree of substitution (DOS) Compound A 0.57 Compound C 0.13

[0225] The data indicate that for compound C, approximately one eighth of the carboxyl groups have reacted, while for compound A, just over half have been replaced. The assignment of key resonances to the acylurea side chain was confirmed by 2-D NMR spectroscopy. The anomeric region does not appear to be confounded with changes in the degree of substitution, as confirmed by the HSQC experimental data.

[0226] Example 2. Mouse Model of LPS-Activated Inflammation

[0227] The following is an in vivo mouse model used to evaluate the inhibitory effects of test compounds on TNF-α and C5a.

[0228] Male Swiss Webster mice aged 7 to 8 weeks were acclimatized for at least 7 days before the start of the study. On the -1 day of the study, animals were weighed and randomly divided into groups according to body weight. In the evening of the -1 day of the study, the animals were administered with vehicles or test articles as described in Table 3. Then on the 0th day of the study, two minutes after the IV vehicle / test article administration, saline (Group 1) or LPS (1 mg / kg) were administered to the animals IP. 2 hours after the LPS administration (1 minute intervals accordingly), animals were anesthetized by inhalation of isoflurane, bled, and then cervical dislocation was used to kill the animals to confirm euthanasia. The serum of each animal was collected in 3x60 μL aliquots and stored at -80 ° C until further testing. The TNF-α and C5a concentrations in the serum samples were measured by ELISA.

[0229] Example 3. P-selectin-mediated cell binding

[0230] The following examples show that compounds disclosed herein inhibit P-selectin as evidenced by inhibition of neutrophil-like cell binding.

[0231] In vitro study design

[0232] 96-well plates were coated with 10 μg / mL protein A overnight and then blocked with 2% FBS for 1 hour. 2 μg / mL P-selectin / Fc chimera was allowed to bind to protein A for 3 hours at 4°C. HL-60 cells (2e5 cells / well, CMFDA labeled) were then layered onto P-selectin and allowed to bind for 1 hour at room temperature. The wells were simultaneously treated with test samples to determine inhibition of binding activity. After 1 hour, unbound cells were washed and bound cells were lysed in 1% Triton-X solution and read at 480 / 520nm on a fluorometer.

[0233] like Figure 1As shown, the compounds provided herein inhibit P-selectin-mediated neutrophil (HL-60) cell binding. P-selectin IC of unfractionated non-chemically modified porcine heparin, non-chemically modified bovine enteroheparin, enoxaparin, Compound A, and Compound C 50 The values ​​are shown in Table 4 below.

[0234] Table 4.

[0235]

[0236] Unfractionated, non-chemically modified bovine enteroheparin

[0237] Example 4. In vitro complement inhibition studies

[0238] Complement is an important effector mechanism of the immune system and 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 or alternative pathways or through the mannose-binding lectin pathway. Insufficient or exaggerated activation of the complement system can lead to diseases of varying severity, and pharmacological inhibition of the complement system is considered a therapeutic strategy to ameliorate the inflammatory effects of exaggerated complement activation.

[0239] Compounds disclosed herein are tested for complement inhibition using the total complement assay CH50 as described in Oberkersch et al. Thrombosis research 125.5 (2010): e240-e245. Briefly, pooled normal serum from healthy subjects is incubated with increasing concentrations of the test compound in the presence of sensitized sheep erythrocytes (EA). Total complement activity in the samples is determined spectrophotometrically by measuring the extent of hemolysis. Test compounds are evaluated in triplicate at five different concentrations to determine the extent of hemolysis. The experiment is performed in three (3) independent runs to evaluate the effect of the test compounds. The IC corresponding to the test article for inhibition of the classical complement pathway is 50 Values ​​were calculated from dose response curves plotted using test article concentration versus percentage of complement-induced hemolysis. IC of complement inhibition against non-chemically modified porcine heparin, non-chemically modified bovine enteroheparin, Compound A, and Compound C 50 As shown in Table 5.

[0240] Table 5.

[0241]

[0242] Unfractionated, non-chemically modified bovine enteroheparin

[0243] Figure 2Hemolysis data are shown for non-chemically modified porcine heparin, non-chemically modified bovine enteroheparin, Compound A, and Compound C. As shown in this example, complement inhibition measured in the CH50 assay described above was reduced for chemically modified bovine enteroheparin.

[0244] Example 5: In vivo inhibition of melanoma metastasis

[0245] In vivo inhibition of melanoma metastasis was assessed as follows. Six-week-old female C57 / BL6 mice (Charles River) were injected intravenously with 3×10 50 μL of 1% paraformaldehyde in 100 μL of 1% paraformaldehyde. 5 The mouse melanoma cells (B16 / F10-luc) expressing luciferase were injected into individual cells. 30 minutes before melanoma cells were administered, mice were pretreated with 100 μL saline (control) or intraperitoneal injection of test compound. The metastasis and tumor formation of the mouse lungs were measured at different time points by in vivo imaging using the bioluminescence of luciferase reporter cells using 10mg / kg D-luciferin. Fluorescence was quantified by measuring the region of interest of the lungs. The data of 7 days after melanoma cells were administered were presented, wherein the inhibition of fluorescence showed the inhibition of lung metastasis.

[0246] Example 6: HL-60 cell assay

[0247] HUVECs were cultured on custom flow channels coated with fibronectin. After the formation of a confluent layer (24 to 48 hours), HUVECs were treated with TNF-α (10 ng / mL) for 4 hours. HL-60 cells were then injected and perfused for 10 to 15 minutes to allow them to adhere to the activated endothelial cells. During the incubation period with TNF-α, the test compound was added to the HUVEC at 100 μg / mL, while cells containing only medium served as a negative control. The bound HL-60 cells were then quantified by microscopic imaging and compared to the control channel. The experiment was performed independently in triplicate. The inhibition of cell binding to inflamed endothelium supports the potential to inhibit selectin-mediated cell binding to cells expressing selectins, such as endothelium or inflamed endothelium. This mechanism supports the potential of the test compound to prevent sickle cell adhesion and therefore supports the use of the compounds disclosed herein for the treatment of sickle cell disease and as a rescue drug for sickle cell disease patients in the prodromal stage of vaso-occlusive crisis.

[0248] Example 7: Rat PD study

[0249] The pharmacodynamics of the compounds were evaluated as follows when administered subcutaneously (SC) or intravenously (IV) in 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) IV or SC dose of 30 mg / kg (15 mg / mL) of the test compound. Animals were bled via the tail vein at 8 time points (pre-dose to 8 hours post-dose) to obtain serum, followed by a final bleed via the descending aorta and sacrificed 8 hours post-dose (Group 1, IV dosing) or 24 hours post-dose (Group 2, SC dosing). Serum samples were analyzed for the binding of HL-60 to P-selectin using a custom HL-60 / P-selectin binding assay (as described herein), with the readout being the fluorescence intensity from the labeled cells.

[0250] Example 8: Pancreatic Cancer BxPC3 Model

[0251] The anticancer activity of the compounds in a pancreatic cancer mouse model was evaluated as follows. Human pancreatic cancer cells BxPC3 (1×10 6 6-week-old female nude mice (5 mice per group) were subcutaneously implanted with 100 mg / kg of gemcitabine (e.g., 100 mg / kg ip, once every three days) or test compound (5 mg / kg scbid) starting on day 7 after implantation. Tumor volume was measured weekly with a caliper. On day 56, the dose of test compound was increased to 10 mg / kg scbid. Animals were sacrificed on day 70 and tumors were excised, fixed and stored for further testing.

[0252] Example 9: Gemcitabine-deficient pancreatic cancer model

[0253] Methods: 1F11 cells (1x10 6 ) was injected subcutaneously into the right flank of female NSG mice to generate pancreatic cancer tumors. Tumors were allowed to grow for 7 days before treatment was started. On day 7 after implantation, mice were randomized into treatment groups (n=5 / group) and treatment was started according to the dose route and schedule as described below:

[0254] Group deal with Route of administration 1 Control (PBS) Subcutaneous 2 Compound C Subcutaneous 3 Compound C + gemcitabine subcutaneous + intraperitoneal 4 Gemcitabine Subcutaneous

[0255] A low sensitizing dose of gemcitabine (Gem) was administered by intraperitoneal route, 25 mg / kg QW. Compound C was administered by subcutaneous route, 10 mg / kg BID. Treatments were administered from day 7 to day 70.

[0256] Body weight and tumor volume were measured weekly with calipers. On day 70, imaging was performed by administering 150 mg / kg luciferin and whole body fluorescence was measured by IVIS imaging system. Tumors and organs were then harvested and imaged ex vivo.

[0257] Results: All animals survived the 70-day study period and all groups tolerated the treatment well. All groups gained weight, e.g. Figure 3 shown.

[0258] like Figure 4 As shown, tumor volumes increased in all groups, and in the treated groups, tumor growth was inhibited by about 50% by day 70, with the most significant tumor growth inhibition observed in the combination treatment of gemcitabine + compound C with a low sensitizing dose of gemcitabine. Single agent treatment with compound C also inhibited tumor growth. Gemcitabine alone had no effect on tumor growth.

[0259] Metastasis to other organs was measured by ex vivo fluorescence. Figure 5 As shown, the organs with the highest metastasis were the lung and liver. Metastasis inhibition was observed in all treatment groups, with a trend toward increased metastasis inhibition with Compound C treatment.

[0260] ***

[0261] The present disclosure is not limited to the scope of the specific embodiments described, which are intended to be a single illustration of a separate aspect of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure that fall within the scope of the appended claims and their equivalents.

[0262] All publications and patent applications mentioned in this specification are herein incorporated 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 enteroheparin comprising from about 15 to about 90 disaccharide units, wherein about 2.5% to 14% of the disaccharide units comprise 1-(3-dimethylaminopropyl)-3-ethylurea (EDU)-amide; and The anti-Factor IIA activity is about 20 IU / mg to about 30 IU / mg.

2. The chemically modified bovine enteroheparin according to claim 1, wherein the P-selectin activity is not significantly different from the P-selectin activity of the parent non-chemically modified bovine enteroheparin.

3. The chemically modified bovine enteroheparin according to claim 1, wherein P-selectin IC 50 Less than about 5 μg / mL.

4. A chemically modified bovine enteroheparin of formula IA: or a salt thereof, wherein: n is 26 to 30; Each R 1 are independently -OH, Each R 2 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; Each R 3 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; Each R 4 independently hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; and Each M is independently a cation; wherein the R 1 About 5% to about 13.5% of the portion is 5. A composition comprising the chemically modified bovine enteroheparin according to any preceding claim.

6. A pharmaceutical composition comprising a chemically modified bovine enteroheparin, wherein at least a portion of the free carboxylic acid moieties on a non-chemically modified bovine enteroheparin have been converted to N-acylureaamides, such that the chemically modified bovine enteroheparin exhibits between 20% and 50% of the anti-Factor IIa activity of the non-chemically modified bovine enteroheparin; and The complement inhibition activity of the chemically modified bovine enteroheparin is substantially the same as the complement inhibition activity of the non-chemically modified bovine enteroheparin.

7. The pharmaceutical composition according to claim 6, wherein the chemically modified bovine enteroheparin comprises one or more chemically modified sugar units of formula I: in: Each R 1 Independently and R 2 Hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2OM; wherein M is a cation.

8. The pharmaceutical composition according to claim 7, wherein R 2 Hydrogen, -S(O)2O - , -S(O)2OH or -S(O)2ONa.

9. The pharmaceutical composition according to claim 7, wherein the chemically modified bovine enteroheparin comprises one or more chemically modified sugar units of Formula IIA: Each R 1 Independently 10. The pharmaceutical composition according to claim 7, wherein the chemically modified bovine enteroheparin comprises one or more chemically modified sugar units of formula IIB: Each R 1 Independently 11. The pharmaceutical composition according to any one of claims 6 to 10, wherein the chemically modified bovine enteroheparin has an anti-factor IIa activity between 80 U / mg and 135 U / mg.

12. The pharmaceutical composition according to any one of claims 6 to 11, wherein the pharmaceutical composition exhibits about 25% to about 35% of the anti-Factor IIa activity of the non-chemically modified bovine enteroheparin.

13. The pharmaceutical composition according to any one of claims 6 to 12, wherein the chemically modified bovine enteroheparin is unfractionated bovine enteroheparin.

14. The pharmaceutical composition according to any one of claims 6 to 13, wherein the P-selectin inhibitory activity is not significantly reduced compared to the non-chemically modified bovine enteroheparin.

15. The pharmaceutical composition according to any one of claims 6 to 13, wherein the P-selectin inhibitory activity (IC 50 ) is between less than 150% and greater than 150% of the P-selectin inhibitory activity of the non-chemically modified bovine enteroheparin.

16. The pharmaceutical composition according to any one of claims 6 to 13, wherein the P-selectin inhibitory activity is substantially the same as the P-selectin inhibitory activity of the non-chemically modified bovine enteroheparin.

17. A method for reducing inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 5 or the pharmaceutical composition of any one of claims 6 to 16.

18. A method for treating or alleviating one or more symptoms of sickle cell disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 5 or the pharmaceutical composition of any one of claims 6 to 16.

19. The method of claim 18, wherein the subject in need thereof is in the prodromal or early stages of a vaso-occlusive crisis.

20. A method for preventing or reversing cell adhesion in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 5 or the pharmaceutical composition of any one of claims 6 to 16.

21. A method for preventing or reversing complement activation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 5 or the pharmaceutical composition of any one of claims 6 to 16.

22. A method for treating a solid tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 5 or the pharmaceutical composition of any one of claims 6 to 16.

23. The method of claim 22, wherein the solid tumor expresses SialylLewis x or SialylLewis a At least one of (sLex or sLea).

24. The method of claim 22, wherein the solid tumor is a gastrointestinal tumor, a breast tumor, a prostate tumor, an ovarian tumor, a colorectal tumor, a liver tumor, a lung tumor, a cervical tumor, a head tumor, a neck tumor, an esophageal tumor, a brain tumor, or a pancreatic tumor.

25. A method for treating a disease or condition mediated at least in part by inhibiting cellular binding to P-selectin and / or inhibiting complement activation pathways in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to claim 5 or a pharmaceutical composition according to any one of claims 6 to 16.

26. The method of claim 25, wherein the disease or disorder is cancer, hematological cancer, melanoma, leukemia, multiple myeloma, chemotherapy-induced peripheral neuropathy (CIPN), beta thalassemia, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), a nervous system disease, amyotrophic lateral sclerosis (ALS), sickle cell disease, vaso-occlusive crisis, immune response in gene therapy using adeno-associated virus (AAV), acute respiratory distress syndrome (ARDS), cardiovascular disease, ophthalmic disease or disorder, renal 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 renal glomerulopathy (C3G), transplant rejection, delayed renal transplant rejection, antibody-mediated renal transplant rejection, renal transplant reperfusion injury, renal transplant in CAPS patient, 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, anti-phospholipid syndrome, cold agglutinin disease, severe thrombocytopenia, macular degeneration, uveitis, ANCA-associated vasculitis, atherosclerosis, mood disorders, asthma, chronic obstructive pulmonary disease (COPD), allergies, sepsis, cerebral malaria, psoriatic arthritis, dermatomyositis, osteoarthritis, dementia, glaucoma, diabetic vasculopathy, myocardial infarction, stroke, post-bypass surgery, polytrauma, neurotrauma, antiphospholipid syndrome, preeclampsia, or hemodialysis.

27. The method of any one of claims 17 to 26, wherein the subject is receiving anticoagulant therapy.

28. The method of any one of claims 17 to 27, wherein the subject is a human.

29. The method of any one of claims 17 to 28, wherein the administration comprises subcutaneous (SC) and intravenous (IV) administration.

30. The method of any one of claims 17 to 19, wherein the administering comprises intravenous (IV) administration.

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