Heparanase inhibitors and methods of use

Abstract

Repeating structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] n ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n ) and at least one sulfur moiety is attached to at least one hydroxyl of the repeating structure, a polymer is disclosed. Methods for producing and using these polymers are also disclosed.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 63 / 355,144, filed on June 24, 2022. The entire content of the patent application referenced above is hereby expressly incorporated by reference into this specification.

Background Art

[0002] Glycosaminoglycans (GAGs) are essential components of the extracellular matrix (ECM) and are linear, negatively charged heteropolysaccharides that contribute to their biological and biomechanical properties (1). One of the most studied classes of GAGs is heparin / heparan sulfate (HS), which has a backbone containing disaccharide repeats composed of (i) hexosamine residues, glucosamine (GlcN) with either an N - acetyl (Ac) or N - sulfo group, and (ii) uronic acid residues of either glucuronic acid (GlcUA) or iduronic acid (2). The specific biological roles of GAGs, such as the regulation of cell - cell interactions, enzyme activities, and cell proliferation during various processes, are related to their backbone structure, post - polymerization modifications (e.g., site - specific sulfation, epimerization), chain size, and their cellular localization (3). Several methods are available and have been used in the past to synthesize various natural and artificial GAG structures with biological activity (4 - 6, 15).

[0003] HS is degraded by heparanase, an endo - β - glucuronidase. The overexpression of heparanase significantly correlates with cancer metastasis. Thus, heparanase has become a therapeutic target in the treatment of metastatic cancer. Heparosan (HEP; [→4]) - α - D - GlcUA - (1→4) - β - D - GlcNAc(1→] n ) is the non - sulfated biosynthetic precursor to HS and heparin in animals, as well as the capsular polysaccharide of certain pathogenic microorganisms (Figure 1). New drugs with improved efficacy and / or selectivity against heparanase are desired.

[0004] Heparin and some of its derivatives act as heparanase inhibitors. Thus, the first approach in the art was to use modified polymers similar to the HS substrate of heparanase. The drug heparin (a highly sulfated version of HS) is a potent heparanase inhibitor, but due to its strong anticoagulant activity, it must be "inactivated" by chemical treatment (i.e., periodate, desulfurization) when used in the treatment of cancer to avoid hemorrhagic side effects. Several heparin-like derivatives, including muparfostat (a mixture of disaccharides to hexasaccharides that are sulfated), PG545 (a highly sulfated hexasaccharide with a lipophilic group), lonapalstat (fully N-acetylated glycol-split heparin; see, for example, U.S. Patent No. 7,781,416), and necoparanib (glycol-split low molecular weight heparin), are being tested in clinical trials for the treatment of cancer. However, these heparin-like derivatives are complex heterogeneous mixtures derived from natural sources (i.e., 1 - 3 sulfates / repeating unit, variable acetylation and epimerization, etc.), and thus, QC methods for characterization are complex and costly to monitor for batch-to-batch or seasonal variations. Additionally, materials derived from animals (e.g., porcine intestinal mucosa) have security issues in the supply chain. For example, in 2008, a large amount of heparin was intentionally contaminated, resulting in deaths and fatalities.

[0005] Furthermore, both PG545 and necoparanib also retain significant anticoagulant activity, and lonapalstat requires high dosing levels due to its short half-life. All of that is a liability that can limit their clinical use. PG545 (currently one of the most potent of the described inhibitors) lacks significant anticoagulant activity and cytotoxicity but has other undesirable effects (some inhibitory, some stimulatory), including cell-type-dependent immunocyte effects.

Prior Art Documents

Patent Documents

[0006] [Patent Document 1] U.S. Patent No. 7,781,416 [Non-Patent Document]

[0007] [Non-Patent Document 1] Kowitsch, A., Zhou, G. & Groth, T., Medical application of glycosaminoglycans: a review. J. Tissue. Eng. Regen. Med. 12, e23 - e41 (2018) [Non-Patent Document 2] Lindahl, U., Couchman, J., Kimata, K. & Esko, J.D. Proteoglycans and Sulfated Glycosaminoglycans in Essentials of Glycobiology, 3rd edition, A. Varki, Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2017) Chapter 17 [Non-Patent Document 3] Esko, J.D., Prestegard, J.H. & Linhardt, R.J. Proteins That Bind Sulfated Glycosaminoglycans Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015 - 2017. in Essentials of Glycobiology, 3rd edition, A. Varki, Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2017) Chapter 38 [Non-Patent Document 4] DeAngelis, P. L., Liu, J. & Linhardt, R. J. Chemoenzymatic synthesis of glycosaminoglycans: Re-creating, re-modeling and re-designing nature’s longest or most complex carbohydrate chains, Glycobiology 23, 764-777 (2013)

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

[0008] Most current heparin mimetics are not specific for heparanase and can interact with various heparin-binding proteins, with the drawback of resulting in unknown outcomes and off-target effects. Furthermore, 3 out of 4 mimetics in clinical trials had heterogeneous structures, further increasing the uncertainty as an executable drug for human use. A number of heparanase-inhibiting small molecules have been reported, but none have entered clinical trials.

[0009] Therefore, there is a need in the art for a new and improved composition made using a defined and safe precursor having heparanase inhibitory activity while avoiding the side effects of current heparin-like derivatives.

Brief Description of the Drawings

[0010]

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[0011] A novel class of sulfated or sulfonated testosteronan (also referred to herein as s-testosteronan or sTestan) is described herein. Testosteronan (also referred to herein as Testan and first disclosed in WO 2012 / 16353) has a repeating structure [-4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] n ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n) is a polysaccharide. Sulfated or sulfonated sTestan of the present disclosure is resistant to digestion by heparanase, an enzyme important in human health and disease. Further, sTestan acts as a competitive inhibitor of heparanase and can thus be useful for the treatment of cancer or other diseases involving heparanase, such as (but not limited to) diabetes, diabetes complications (e.g., cardiomyopathy), atherosclerosis, thrombosis, viral infections (e.g., herpes simplex), etc. Further, sTestan can be used as a selective modulator of glycoprotein interactions due to its unique structural differences from polymers naturally present in the body.

[0012] Before more particularly describing various embodiments of the compositions and methods of the present disclosure as illustrative explanations, examples, and results, it should be understood that the embodiments of the present disclosure are not limited in their application to the details of the methods and compositions described in the following description. Embodiments of the compositions and methods of the present disclosure can be implemented or executed in various ways not explicitly described herein. Accordingly, the language used herein is intended to be given the broadest possible scope and meaning, and the embodiments are intended to be illustrative and not exhaustive. Also, it should be understood that the expressions and terms used herein are for purposes of explanation and should not be regarded as limiting unless otherwise specified. Further, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the present disclosure. However, it will be apparent to those skilled in the art that embodiments of the present disclosure can be practiced without these specific details. In other instances, features well known to those skilled in the art are not described in detail to avoid unnecessary complication of the description. Although the compositions and methods of the present disclosure have been described with respect to specific embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods, and the steps or order of steps of the methods described herein, without departing from the concepts, spirit, and scope of the invention as described herein. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit and scope of the concepts of the invention disclosed herein.

[0013] All patents, published patent applications, and non-patent publications referred to or mentioned in any part of this specification, including but not limited to U.S. Patent No. 9,695,427 and U.S. Patent No. 10,273,517, indicate the level of skill in the art to which the present disclosure pertains, and each such patent or publication is hereby expressly incorporated by reference in its entirety to the same extent as if each individual patent or publication were specifically and individually incorporated herein.

[0014] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include singulars.

[0015] When used in accordance with the methods and compositions of the present disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated.

[0016] The use of the words "a" or "an" when used in combination with the term "comprising" in the claims and / or the specification may mean "one", but this also is consistent with the meaning of "one or more", "at least one", and "one or more than one". The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. However, the present disclosure supports definitions that refer to alternatives only and "and / or". The use of the term "at least one" is understood to include any quantity greater than one, including one and any integers including 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or more. The term "at least one" can be extended up to 100 or 1000 or more, depending on the term with which it is associated. Further, amounts of 100 / 1000 should not be considered limiting as higher limits may also yield satisfactory results. Further, the use of the term "at least one of X, Y, and Z" is understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0017] As used in this specification and the claims, the word "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (any form of "having", such as "have" and "has"), "including" (any form of "including", such as "includes" and "include") or "containing" (any form of "containing", such as "contains" and "contain") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0018] As used herein, the term "or combinations thereof" refers to all replacements and combinations of the listed items preceding that term. For example, "A, B, C, or combinations thereof" includes at least one of A, B, C, AB, AC, BC, or ABC, and where order is important in a particular situation, is also intended to include BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more of the items or terms such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. are explicitly included. Those skilled in the art will understand that typically there is no limit to the number of items or terms in any combination, unless it is apparent from the context.

[0019] Throughout this application, the terms “about” and “approximately” are used to indicate that a value includes the inherent variability of error for the quantity, the method used to administer the composition, or the variation that exists between an object or subject. As used herein, the modifier “about” or “approximately” not only includes the exact value, amount, degree, orientation, or other modified characteristic or value, but also, for example, includes some minor variations due to measurement error, observer error, wear and tear, and combinations thereof. When the terms “about” or “approximately” are used herein to refer to a measurable value such as an amount, percentage, duration, etc., it means including a variation of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, such variations being appropriate to carry out the disclosed method and understood by a person skilled in the art. As used herein, the term “substantially” means that the event or situation described subsequently occurs completely, or that the event or situation described subsequently occurs to a significant extent or degree. For example, the term “substantially” means that the event or situation described subsequently occurs at least 90%, or at least 95%, or at least 98% of that time.

[0020] As used herein, any reference to “one embodiment” or “an embodiment” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment and may be included in other embodiments. The phrase “in one embodiment” appearing in various places in this specification does not necessarily refer to the same embodiment, nor is it necessarily limited to a single or particular embodiment. Further, any reference to one or more embodiments or examples should be construed as not limiting the claims.

[0021] As used herein, all numerical values or ranges include the values of the numbers and integers between such numbers within such ranges, and the numbers between the integers within such ranges, unless the context clearly indicates otherwise. Thus, by way of example, reference to a numerical range such as 1 to 10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Thus, reference to a range of 1 to 50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and the range is not limited to only integers. Reference to a series of ranges includes ranges that combine the boundary values of different ranges within the series of ranges. Thus, by way of example, to illustrate reference to a range of 1 to 1,000, the range of 1 to 1,000 includes, for example, 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500 to 750, 750 to 1,000, and includes ranges of 1 to 20, 10 to 50, 50 to 100, 100 to 500, and 500 to 1,000. Thus, a range of 100 units to 2,000 units includes, for example, without limitation, 100 units to 1,000 units, 100 units to 500 units, 200 units to 1,000 units, 300 units to 1,500 units, 400 units to 2,000 units, 500 units to 2,000 units, 500 units to 1,000 units, 250 units to 1,750 units, 250 units to 1,200 units, 750 units to 2,000 units, 150 units to 1,500 units, 100 units to 1,250 units, and 800 units to 1,200 units, and refers to all values or ranges of values of the units, as well as the numbers between the values of the units and the integers within the range, and includes. Thus, any two values within the range of about 100 units to about 2,000 units can be used to set the lower and upper limits of the range according to embodiments of the present disclosure.

[0022] As used herein, the term "GlcNAc" refers to N-acetylglucosamine. The terms "GlcA" and "GlcUA" as used herein are interchangeable and refer to glucuronic acid. The terms "UDP-GlcNAc" and "UDP-GlcUA" refer to the uridine diphosphate sugar precursors of GlcNAc and GlcUA, respectively. These compounds are used by glycosyltransferases to transfer GlcNAc / GlcUA residues to substrates. "[-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n " In the formula, n can be in the range of, for example, 1 to 100, or 2 to 100, or 1 to 200, or 2 to 200, or 1 to 500, 2 to 500, or 1 to 1000, or any range including them.

[0023] The term "pharmaceutically acceptable" refers to compounds and compositions suitable for administration to humans and / or animals without undue adverse side effects such as toxicity, irritation, and / or allergic reactions commensurate with a reasonable benefit / risk ratio.

[0024] As used herein, the term "active agent" is intended to refer to a substance having biological activity related to the present disclosure, and particularly refers to substances for treatment and diagnosis that can be used in the methods described in the present disclosure. "Biological activity" means the ability of an active agent to act on or modify an organic or inorganic molecule, or the molecular, biochemical, or physiological system of a cell, tissue, or organism, regardless of how the active agent has its effect. "Biological activity" refers to any biological property of an active agent.

[0025] As used herein, "pure", "substantially pure" or "isolated" means that the species of interest is the predominant species present (i.e., is more abundant on a molar basis than any other species of interest in the composition), and in particular, a substantially purified fraction is a composition in which the species of interest constitutes at least about 50% (on a molar basis) of all the polymeric species present. Generally, a substantially pure composition constitutes more than about 80%, more specifically more than about 85%, more than about 90%, more than about 95%, or more than about 99% of all the polymeric species present in the composition. The terms "pure" or "substantially pure" also refer to preparations in which the species of interest (e.g., a peptide compound) is at least 60% (w / w) pure, or at least 70% (w / w) pure, or at least 75% (w / w) pure, or at least 80% (w / w) pure, or at least 85% (w / w) pure, or at least 90% (w / w) pure, or at least 92% (w / w) pure, or at least 95% (w / w) pure, or at least 96% (w / w) pure, or at least 97% (w / w) pure, or at least 98% (w / w) pure, or at least 99% (w / w) pure, or 100% (w / w) pure. As used herein, the term "high specificity" refers to a specificity of at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%. As used herein, the term "high sensitivity" refers to a sensitivity of at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%.

[0026] The terms "subject" and "patient" are used interchangeably herein and are understood to refer to an organism to which the compositions of the present disclosure are applied and used, such as a vertebrate, or more specifically a warm-blooded animal, such as a mammal. Non-limiting examples of animals within the scope and meaning of this term include dogs, cats, rats, mice, guinea pigs, chinchillas, horses, goats, cows, sheep, llamas, zoo animals, Old World monkeys and New World monkeys, non-human primates, and humans.

[0027] "Treatment" refers to a therapeutic treatment, such as for the healing or recovery of damaged tissue. The term "treating" refers to administering a composition to a patient for such therapeutic purposes, which may result in an improvement in a condition or disease.

[0028] The terms "therapeutic composition" and "pharmaceutical composition" refer to an active pharmaceutical composition that can be administered to a subject by any method known in the art or otherwise contemplated herein, such as the hydrogel compositions described herein, and administration of the composition results in a therapeutic effect described elsewhere herein. Further, certain compositions of the present disclosure can be designed to provide targeting, delayed, controlled, extended, and / or sustained release using formulation techniques well known in the art.

[0029] The term "effective amount" refers to an amount of an active agent sufficient to exhibit a detectable biochemical and / or therapeutic effect, for example, when used in the manner of the present disclosure, without undue adverse side effects (such as toxicity, irritation, allergic reactions, etc.) commensurate with a reasonable benefit / risk ratio. The effective amount for a patient depends on the type of patient, the size and health of the patient, the nature and severity of the condition being treated, the method of administration, the duration of treatment, (if any) the nature of combination therapies, the particular formulation used, etc. For this reason, the exact effective amount cannot be specified in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0030] The term "ameliorate" means a detectable or measurable improvement in the state of the subject or its symptoms. Detectable or measurable improvements include subjective or objective decreases, reductions, inhibitions, suppressions, limitations, or controls in the occurrence, frequency, severity, progression, or duration of a state, or improvements in the symptoms, or the underlying cause or consequence of a state, or reversal of symptoms. The outcome of a successful treatment can result in a "therapeutic effect" or "benefit" that improves, decreases, reduces, inhibits, suppresses, limits, controls, or prevents the occurrence, frequency, severity, progression, or duration of a state in the subject, or the consequences of the state.

[0031] A decrease or reduction in worsening, such as stabilization of a state, is also an outcome of a successful treatment. Thus, the benefit of treatment need not be complete disappearance or recovery of a state, or any one, most, or all of the adverse symptoms, complications, consequences, or underlying causes associated with the state. Thus, a satisfactory endpoint can be achieved when there is a gradual improvement, such as a partial decrease, reduction, inhibition, suppression, limitation, control, or prevention in occurrence, frequency, severity, progression, or duration, or suppression or reversal (e.g., stabilization) of a state over a short or long period (e.g., seconds, minutes, hours).

[0032] As used herein, the term "combination therapy" is used interchangeably with the terms "combined therapy" and "adjunctive therapy" and is understood to mean that a patient in need of treatment is treated in combination with a novel active agent of the present disclosure (e.g., sTestan) or is given another drug for the disease. This combination therapy can be a sequential therapy where the patient is first treated with one drug and then the other, or the two drugs are administered simultaneously. In certain embodiments, the subject can be administered additional therapeutic and / or diagnostic agents in combination with sTestan. The additional agents can be administered simultaneously within the same or different compositions, or sequentially. For example, sTestan can be administered first and the additional agent second. Or, sTestan can be administered after the additional agent has been administered.

[0033] As used herein, the term "chemotherapeutic agent" (or "chemotherapeutic") can refer to a therapeutic agent utilized for preventing or treating cancer.

[0034] As used herein, the term "molecular weight" can generally refer to the mass or average mass of a material. In the case of a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weights of polymers and oligomers can be estimated or characterized by various methods, including gel permeation chromatography (GPC) using light scattering detection, or electrophoresis of an appropriate standard. The GPC molecular weight is reported as the number average molecular weight (M n ), as opposed to the weight average molecular weight (M w ).

[0035] As used herein, the term "polysaccharide" is understood to refer to a large carbohydrate molecule containing from about 20 to thousands of sugar units (i.e., monosaccharide residues). The term "oligosaccharide" as used herein is understood to refer to a smaller carbohydrate molecule containing less than about 20 sugar units. The term "polymer" as used herein is understood to refer to a naturally occurring or synthetic compound composed of repeating units. The term "polymer" encompasses both oligosaccharide structures and polysaccharide structures.

[0036] As used herein, the term "polydisperse" refers to a polymer having chain lengths that vary over a wide range of molecular weights such that there is a heterogeneity in molecular weight. The terms "monodisperse", "substantially monodisperse" and "quasi-monodisperse" as used herein are understood to refer to defined glycosaminoglycan polymers having a narrow size distribution. Further, the polydispersity value or index of heterogeneity is a measure of the molecular weight distribution of a given polymer sample. The calculated polydispersity value is the weight average molecular weight divided by the number average molecular weight. This indicates the distribution of the individual molecular weights in a batch of polymer. The polydispersity value has a value of 1 or greater, but approaches 1 as the polymer chains approach a uniform chain length.

[0037] Active agents disclosed herein (e.g., sulfated or sulfonated testosteronan) can be formulated into compositions for delivery to a subject. The compositions can be administered alone and / or in admixture with a pharmaceutically acceptable medium or excipient. Suitable media are, for example (but not limited to), water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Further, the medium can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, biocompatible solvents, pH buffering agents, or adjuvants (not limited to these). The compositions of the present disclosure can also contain auxiliary substances such as, for example (but not limited to), pharmacological agents, cytokines, or other biological response modifiers.

[0038] The active agent, alone or as a pharmaceutical composition, can be delivered by any means known in the art, such as (but not limited to) systemically, regionally, or locally, subcutaneously, intratracheally (e.g., by aerosol), or transmucosally (e.g., buccal mucosa, bladder mucosa, vaginal mucosa, uterine mucosa, rectal mucosa, nasal mucosa), intraarterially, intrathecally (IT), intravenously (IV), parenterally, intrapleurally, locally, orally, transdermally, or topically, or directly localized within a tumor. Administration by intravenous or subcutaneous administration to the systemic circulation is typical. Intravenous administration can be, for example (but not limited to), by infusion over a period such as 30 - 90 minutes, or by single bolus injection.

[0039] Furthermore, the composition can be formulated into a composition in either neutral or salt form. Pharmaceutically acceptable salts include (but are not limited to) acid addition salts (formed with the free amino groups of the active polypeptide), formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2 - ethylaminoethanol, histidine, and procaine.

[0040] The composition for treatment can be administered in a single-dose treatment or a multi-dose treatment according to a schedule and duration suitable for the age, weight, and condition of the subject, the specific composition used, and the route of administration. In one non-limiting embodiment, a single dose of the composition according to the present disclosure is administered. In other non-limiting embodiments, multiple doses are administered. The frequency of administration can vary depending on any of a variety of factors, such as the severity of the symptoms, or whether the composition is used for prophylactic or curative purposes. For example, in certain non-limiting embodiments, the composition is administered once a month, twice a month, three times a month, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, every other day, daily, twice a day, or three times a day. The treatment period (i.e., the period during which the composition is administered) can vary depending on any of a variety of factors, such as the response of the subject. For example, the composition can be administered over a period ranging from about 1 day to about 1 week, about 2 weeks to about 4 weeks, about 1 month to about 2 months, about 2 months to about 4 months, about 4 months to about 6 months, about 6 months to about 8 months, about 8 months to about 1 year, about 1 year to about 2 years, or about 2 years to about 4 years, or longer.

[0041] The composition can form a pharmacological composition in combination with a pharmaceutically acceptable carrier (excipient). The pharmaceutically acceptable carrier can contain, for example, but not limited to, a physiologically acceptable compound that acts to stabilize, increase, or decrease the absorption rate or clearance rate of the pharmaceutical composition. Examples of physiologically acceptable compounds include, but are not limited to, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposome carriers; excipients; or other stabilizers and / or buffers. Other physiologically acceptable compounds include, but are not limited to, wetting agents, emulsifying agents, dispersing agents, or preservatives.

[0042] When administered orally, the composition can be protected from digestion. This can be achieved by combining the active agent with the composition to render it resistant to acidic and enzymatic hydrolysis, or by packaging the active agent in a suitably resistant carrier such as, for example (but not limited to), liposomes as shown in U.S. Patent No. 5,391,377.

[0043] For transmucosal or transdermal administration, suitable penetration enhancers can be used in the formulation for the barrier to be penetrated. Such penetration enhancers are generally known in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. Further, detergents can be used to enhance permeation. Transmucosal administration can be by nasal spray or using suppositories. For topical transdermal administration, the drug is formulated into ointments, creams, plasters, powders, and gels. Transdermal delivery systems can also include (but are not limited to, for example) patches. The composition can also be administered by sustained delivery or sustained release. For example, biodegradable microspheres or capsules capable of sustained delivery of peptides or the composition of other biodegradable polymers can be included herein.

[0044] For inhalation, the composition can be delivered using any system known in the art including (but not limited to) dry powder aerosols, liquid delivery systems, air jet nebulizers, propellant systems, etc. For example (but not limited to), pharmaceutical formulations can be administered in the form of aerosols or mists. In the case of aerosol administration, the formulation can be supplied in a finely divided form together with a surfactant and a propellant. In another aspect, the device for delivering the formulation to the respiratory tissue is an inhaler in which the formulation is vaporized. Other liquid delivery systems include (without limitation, for example) air jet nebulizers.

[0045] In one aspect, the composition is formulated as a sustained release formulation that includes a carrier that protects the active agent from rapid elimination from the body, such as (but not limited to) implants and microencapsulation delivery systems. Biodegradable biocompatible polymers such as (but not limited to) ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0046] The active agent can generally be formulated to obtain a composition that includes one or more pharmaceutically suitable excipients, surfactants, polyols, buffers, salts, amino acids, or additional components, or some combination thereof. This can be achieved by known methods for preparing pharmaceutically useful dosages, whereby the active compound is mixed in a mixture with one or more pharmaceutically suitable excipients. Sterile phosphate buffered saline is one non-limiting example of a pharmaceutically appropriate excipient.

[0047] For parenteral administration, the composition is formulated in injectable form in unit dosages such as (but not limited to) solutions, suspensions, or emulsions in combination with pharmaceutically acceptable excipients. Such excipients are essentially non-toxic and non-therapeutic. Non-limiting examples of such excipients include saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous excipients such as (but not limited to) fixed oils and ethyl oleate can also be used. An alternative non-limiting excipient is 5% dextrose in saline. The excipients can contain small amounts of additives such as (but not limited to) substances that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0048] The formulated composition containing the active agent can be used for subcutaneous, intramuscular, or transdermal administration (for example, but not limited to). The composition can be provided in unit dosage forms with preservatives, such as ampoules or multi-dose containers. The composition can also take forms such as suspensions, solutions, or emulsions in an oily or aqueous medium, and can contain formulating agents (but not limited to these) such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the composition can be in powder form for constitution with a suitable medium, such as sterile pyrogen-free water, before use.

[0049] The composition can be administered in solution state. The formulation can be a solution having a suitable pharmaceutically acceptable buffer (but not limited to) such as phosphate, tris(hydroxymethyl)aminomethane-HCl, or citrate. The concentration of the buffer should be in the range of 1 to 100 mM. The formulated solution can also contain salts, such as (but not limited to) sodium chloride or potassium chloride, at a concentration of 50 to 150 mM. An effective amount of a stabilizer such as mannitol, trehalose, sorbitol, glycerol, albumin, globulin, detergent, gelatin, protamine, or a salt of protamine (but not limited to these) can also be included.

[0050] Exemplary non-limiting ranges of therapeutically or prophylactically effective amounts of the active agent include, for example, without limitation, ranges from about 0.001 mg / kg to about 500 mg / kg per kg of the subject's body weight, such as, for example, ranges from about 0.01 mg / kg to about 250 mg / kg, ranges from about 0.1 mg / kg to about 100 mg / kg, ranges from about 0.1 mg / kg to about 50 mg / kg, ranges from about 1 mg / kg to about 30 mg / kg, ranges from about 1 mg / kg to about 25 mg / kg, ranges from about 2 mg / kg to about 30 mg / kg, ranges from about 2 mg / kg to about 20 mg / kg, ranges from about 2 mg / kg to about 15 mg / kg, ranges from about 2 mg / kg to about 12 mg / kg, ranges from about 2 mg / kg to about 10 mg / kg, ranges from about 3 mg / kg to about 30 mg / kg, ranges from about 3 mg / kg to about 20 mg / kg, ranges from about 3 mg / kg to about 15 mg / kg, ranges from about 3 mg / kg to about 12 mg / kg, or ranges from about 3 mg / kg to about 10 mg / kg, or ranges from about 10 mg to about 1500 mg, as fixed dosages.

[0051] The composition is formulated to contain an effective amount of the active agent, the amount of which depends on the subject being treated and the severity of the subject's condition. In certain non-limiting embodiments, the active agent can be administered in dosages in the range of 0.001 mg to about 10 g, about 0.01 mg to about 10 g, about 0.1 mg to about 10 g, about 1 mg to about 10 g, about 1 mg to about 9 g, about 1 mg to about 8 g, about 1 mg to about 7 g, about 1 mg to about 6 g, about 1 mg to about 5 g, about 10 mg to about 10 g, about 50 mg to about 5 g, about 50 mg to about 5 g, about 50 mg to about 2 g, about 0.05 μg to about 1.5 mg, about 10 μg to about 1 mg of protein, about 30 μg to about 500 μg, about 40 μg to about 300 μg, about 0.1 μg to about 200 mg, about 0.1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 25 μg, about 25 μg to about 50 μg, about 50 μg to about 100 μg, about 100 μg to about 500 μg, about 500 μg to about 1 mg, or about 1 mg to about 2 mg. The specific dosage level for any particular subject depends on a variety of factors including, but not limited to, the activity of the specific active agent, age, body weight, general health, gender, diet, time of administration, route of administration, and rate of excretion, drug combination, and the severity of the disease of the subject being treated.

[0052] The dosage of the active agent administered to humans varies according to factors such as the patient's age, weight, height, gender, general medical condition, and medical history (but not limited to these). In certain non-limiting embodiments, the recipient is supplied with a dosage of the active agent in the range of about 1 mg to about 1000 mg as a single infusion or as a single or multiple injections, although lower or higher dosages may also be administered. In certain non-limiting embodiments, the dosage may be in the range of about 25 mg to about 100 mg per square meter of the typical adult body surface area (m 2 ) but lower or higher dosages may also be administered. Non-limiting examples of dosages that may be administered to a human subject further include 1 - 500 mg, 1 - 70 mg, or 1 - 20 mg, although higher or lower dosages may be used. The dosage can be repeated, if necessary, for example (but not limited to), once a week over 4 - 10 weeks, once a week over 8 weeks, or once a week over 4 weeks. It can also be given less frequently, for example (but not limited to), bi-weekly over several months, or more frequently, for example, twice a week, or by continuous infusion.

[0053] In some non-limiting embodiments, the amount of the active agent is about 1 nM, about 5 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 500 nM, about 550 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 60 μM, about 70 μM, about 75 μM, about 80 μM, about 90 μM, about 100 μM, about 125 μM, about 150 μM, about 175 μM, about 200 μM, about 250 μM, about 300 μM, about 350 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 750 μM, about 800 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, about 10 M, about 15 M, about 20 M, about 25 M, about 30 M, about 35 M, about 40 M, about 45 M, about 50 M, about 75 M, about 100 M concentration, or any range between any two of the aforementioned concentrations, such as the two concentrations as the endpoints of the range, or any number between any two of the aforementioned concentrations.

[0054] The number of administrations to be given depends on the severity of the condition and the response to treatment (e.g., whether presenting acute or chronic symptoms), and the treatment can be repeated for recurrence or acute exacerbation of an acute disorder. In the case of a chronic disorder, the active agent can be administered at regular intervals such as weekly, every two weeks, monthly, every three months, every six months, etc. (but not limited to these) for at least one year, five years, or ten years, or for the lifetime of the patient if the condition is chronic.

[0055] In certain non-limiting embodiments, the pharmaceutical composition for parenteral administration is sterile, substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., the dosage for a single administration). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. In the case of injection, the active agent can be formulated in an aqueous solution, for example (but not limited to) a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline, or an acetate buffer (to reduce discomfort at the injection site). The solution can contain formulating agents such as (but not limited to) suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active agent can be in a lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0056] Some non-limiting embodiments provided herein include kits. In some non-limiting embodiments, the kit can include an active agent in an amount described herein or otherwise contemplated. In some non-limiting embodiments, the active agent is lyophilized. In some non-limiting embodiments, the active agent is present in an aqueous solution or other carrier described herein. In some non-limiting embodiments, the kit includes a pharmaceutical carrier for administering the active agent. Certain non-limiting embodiments of the present disclosure include kits containing components suitable for treatment or diagnosis. Exemplary kits can contain at least one active agent. In some non-limiting embodiments, the kit may include a device for delivering the components of the kit by injection, such as a syringe for subcutaneous injection. When transdermal administration is used, in some non-limiting embodiments, a delivery device such as a hollow microneedle delivery device can be included in the kit. Exemplary transdermal delivery devices such as, but not limited to, hollow microstructure transdermal systems (e.g., 3M) are known in the art and any such known device can be used. The components of the kit may be packaged together or separated into two or more containers. In some non-limiting embodiments, the container can be a vial containing a sterile lyophilized formulation of a composition suitable for reconstitution. The kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Alternatively, the active agent can be delivered and stored as a liquid formulation. Other containers that can be used include, but are not limited to, pouches, trays, boxes, tubes, etc. The components of the kit can be packaged inside the container and maintained sterile. Another component that can be included is instructions for use of the kit for treatment.

[0057] The active agents of the present disclosure can be combined in synergistic formulations or treatments. As used herein, the terms "synergy," "synergistic," or "synergistic effect" refer to a therapeutic effect or result that is greater than the additive effect of each active agent used individually. The presence or absence of a synergistic effect for a particular combination of treatment substances can be quantified by using the combination index (CI) (e.g., Chou, Pharmacol Rev, 2006.58(3):621-81), wherein a CI value less than 1 indicates a synergistic effect and a value greater than 1 means an antagonistic effect. Combinations of inhibitors and antagonists of the present disclosure can be tested in vitro for synergistic cell growth inhibition using standard cell lines for a particular cancer, or in vivo using standard animal cancer models. The synergistic effect of the combinations described herein can, in some embodiments, enable the use of lower doses of one or more of the components of the combination. The synergistic effect can also, in some embodiments, enable less frequent dosing of at least one of the active agents administered. Such lower doses and reduced dosing frequencies can reduce the toxicity associated with at least one administration of the treatment to the subject without reducing the effectiveness of the treatment.

[0058] The term "co-administration" refers to the administration of two or more active agents, such as a heparanase inhibitor and an anti-cancer agent. The timing of co-administration is dependent on the combination and composition being administered and can include administration simultaneous with, immediately before, or immediately after the performance of one or more additional therapies. Co-administration is intended to include co-administering the compounds and / or compositions individually or in combination either simultaneously or sequentially. Thus, the formulations can also be combined with other active substances, if desired (e.g., to reduce metabolic degradation). For example, the compositions described herein can be used in combination with each other or with other active agents known to be useful in the treatment of cancer.

[0059] In particular, by way of non-limiting example, the active agents of the present disclosure can be combined with another cargo molecule, such as a liposome equipped with an anti-cancer agent. Liposomes can contain amphiphilic agents such as lipids that aggregate in aqueous solution in the form of micelles, insoluble monolayers, liquid crystals, or lamellar layers, in addition to other pharmaceutically acceptable carriers. Lipids suitable for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysophosphatidylcholine, phospholipids, saponins, bile acids, combinations thereof, and the like. The preparation of such liposome formulations is well within the ordinary skill level in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, the entire contents of each of which are incorporated herein by reference. As used herein, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in one or more spherical bilayers. Liposomes are single- or multi-layer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion can contain the active agent to be delivered. Liposomes can be made from phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC) or other similar lipids. Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as (but not limited to) soy PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0060] Returning to the consideration of various specific embodiments of the present disclosure, as previously described, sTestan is the polysaccharide Testan[-4-D-GlcUA-α1,4-D-GlcNAc-α1-] nIt is a heparosan (HEP) analog synthesized by microbial GAG synthase that generates it and the enzyme CtTS derived from the opportunistic pathogen Comamonas testosteroni. Testan contains the same sugars as biomedically relevant GAG HEP (or N-acetyl-heparosan), but there are differences in the bonds (glycosidic bonds) between GlcUA-GlcNAc in the copolymer chain. In Testan, GlcUA-GlcNAc is in an α-linkage, while in HEP it is in a β-linkage (Figure 1). The other bonds between the GlcNAc-GlcUA sites of the copolymer remain the same, i.e., they are in an α-linkage. The nature of the glycosidic bonds of carbohydrates is widely known to have important implications for their biological activities. For example, at the molecular level, both cellulose (wood and paper) and starch (e.g., bread) are 1→4-glucose polymers having either β-linkages or α-linkages, respectively. Thus, Testan and its derivatives have unique properties useful for medical applications. The structure and synthesis method of Testan are shown in U.S. Patent Nos. 9,695,427 and 10,273,517, each of which is hereby expressly incorporated by reference in its entirety.

[0061] Certain non-limiting embodiments of the present disclosure have a repeating structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] n ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n) include polymers in which at least one sulfur moiety is linked to the repeating structure, where n ranges from about 2 to about 500. For example, without limitation, n can be about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500. Additionally, the scope of the present disclosure explicitly includes n values in ranges formed from any two of the above values (e.g., ranges from about 2 to about 500, from about 2 to about 200, from about 10 to about 100, from about 10 to about 300, etc.).

[0062] In one non-limiting embodiment of the present disclosure, sTestan is described above as an enzyme that metabolizes heparan sulfate (HS) in animals and forms the basis for a new class of competitive inhibitors of heparanase, which is an important therapeutic target in the treatment of cancer (18 - 19). As an example of the chemical sulfation of sTestan, sulfur trioxide-trimethylamine complex in basic aqueous solution produced O-sulfated Testan (Figure 2). In another example, sulfur trioxide-trimethylamine complex is used in anhydrous (dry) aprotic solvents (e.g., dimethylformamide, formamide) (Figure 4). O-sulfated heparosan-based polymers have modifications recognized by human heparanase (13).

[0063] As used herein with respect to sTestan, the term "sulfate" refers to a moiety in which the oxygen-containing portion of GlcNAc or GlcUA is bonded to an SO3 group, i.e., R-O-SO3. R = monosaccharide. In other words, the hydroxyl of the monosaccharide is sulfonated such that a sulfate group is introduced. Thus, with respect to sTestan having O-linked sulfur, as used herein, the terms "sulfated" and "sulfonated" are interchangeable.

[0064] In certain embodiments, the active agent of the present disclosure is sTestan linked to a heparosan chain to form an sTestan-heparosan chimera. For example (but not limited to), the sTestan moiety is the actual inhibitory moiety, while the heparosan moiety is used to assist or direct binding to the heparanase target or to alter pharmacokinetic behavior (e.g., extend half-life, control access to tissues, etc.).

[0065] Recombinant human heparanase cleaves O-sulfoheparosan and native HS species, but not sTestan (Figure 3). Furthermore, co-incubation of sTestan with an O-sulfoheparosan substrate inhibited digestion of the substrate (Figures 6 and 7). Similarly, sTestan acts as an inhibitor, protecting native HS from human heparanase digestion (Figure 8), thereby demonstrating that this novel polymer acts as a competitive inhibitor against heparanase and thus has oncological applications. sTestan was at least about 100-fold more active on a molar basis than the heparosan substrate based on IC 50 This result indicates that heparanase binding to sTestan, which has α-α linkages, is stronger than to polysaccharides having only the natural alternating β-α linkages of the HS family. Furthermore, in Figure 7, sTestan was shown to be a potent competitive inhibitor of human heparanase, similar to the known heparanase inhibitor, generic roneparstat (U.S. Patent No. 7,781,416).

[0066] Regarding a higher level of human heparanase inhibition than predicted by the sTestan polymer, although not wishing to be bound by theory, it was predicted that the α-linked bond mimics some aspects of the transition state of the heparan sulfate chain undergoing cleavage at the heparanase active site (13). The glycosidic bond emerging from the GlcUA residue of sTestan occupies an axial position rather than the typical equatorial position found in native HS chains. Thus, these polymers at the heparanase-targeted, readily cleavable bond could be aligned with the catalytic nucleophile of the enzyme active site, as seen in the standard glycosidase mechanism that they retain (12). However, the enzyme cannot cleave the strongly bound chain, thereby preventing other HS chains from approaching the active site and acting as a competitive enzyme inhibitor.

[0067] It is well known that transition state analogs are better inhibitors than strict substrate structure analogs of unperturbed bonds (12). Thus, compared to other types of heparanase inhibitors (10, 14) previously used clinically, the sTestan polymers disclosed herein represent a new class of therapeutic agents.

[0068] Furthermore, the sTestan polysaccharide structure can enhance or decrease interactions with various GAG-binding proteins depending on the proteins and polysaccharides being studied. Thus, sTestan has improved selectivity compared to natural HS-based sugars that bind to numerous proteins (e.g., heparin binds to over 200 different proteins in human plasma), thereby resulting in numerous useful biological activities. For example, selectivity is possible when only a subset of the proteins of "heparisomes" interact with the sTestan family of polymers, potentially enabling a reduction in side effects for patients. In one example, sTestan has been shown not to be an anticoagulant. Since sTestan is at least about 20,000-fold less effective than heparin (Figure 9), the thrombin / antithrombin III interaction is not affected by sTestan. Furthermore, sTestan should not have the side effect of excessive bleeding when used as a drug in the human body and thus is a safer therapeutic agent.

[0069] (i) The size of the polymer chain and (ii) the parameters of the level and pattern of sulfation can be varied to suit the desired biological activity of the sTestan class of molecules. The utility of sTestan promises to expand the possibilities of selective and improved therapies.

Examples

[0070] Examples are presented below. However, it should be understood that the present disclosure is not limited in its application to the specific experiments, results, and experimental procedures disclosed below in this specification. Rather, the examples are presented merely as one of various embodiments, are illustrative, and are not intended to be exhaustive.

[0071] All reagents were purchased from Sigma-Aldrich or other commercial vendors and used without further purification, unless otherwise specified. The source of the Testan backbone polymer, or the method of manufacture, may be, for example, (i) using a natural or recombinant microorganism having the CtTs gene or an active variant or derivative in vivo, or (ii) using a recombinant microorganism having the CtTs enzyme or an active variant or derivative in vitro, and using an extract with the appropriate precursor under appropriate conditions (e.g., UDP-sugar, reaction buffer containing divalent cations, etc.). See, for example, Otto, op. cit., as well as U.S. Patent Nos. 9,695,427 and 10,273,517.

[0072] For example, in one non-limiting embodiment, the Testan backbone polymer is produced by fermenting the native bacterium C. testosteroni KF-1 in a chemically defined medium as described in Otto, N.J., Solakyildirim, K., Linhardt, R.J., DeAngelis, P.L. Comamonas testosteronan synthase, a bifunctional glycosyltransferase that produces a unique heparosan polysaccharide analog. Glycobiology. 21(10):1331-1340 (2011). However, the source of the starting backbone is not limited to use in producing the compounds of the present disclosure.

[0073] Quantification of polysaccharides.

[0074] The amount of polymer was measured by the carbazole assay using a GlcUA standard (13). Every other sugar residue of the sTestan-based strand is GlcUA.

[0075] Sulfation of polysaccharides

[0076] For sulfation and sulfonation of Testan, various methods can be used that employ either chemical reagents (e.g., sulfur trioxide complexes, chlorosulfonic acid) or recombinant GAG sulfotransferases, either in vivo or in vitro. Non-limiting examples of such methods are shown below.

[0077] Synthesis of sTestan using chemical routes:

[0078] A variety of sulfation methods can be used (16, 17). In one non-limiting example, to create an sTestan prototype, a solution of Testan polymer dissolved in water was adjusted with concentrated NaOH to a final concentration of 0.05 - 4 M NaOH and 1 - 15 mg / ml carbohydrate. Then, a sulfating reagent (e.g., solid sulfur trioxide:trimethylamine complex; Aldrich) was added to the reactants while mixing at a desired ratio (e.g., reagent / polymer ratio 0.1:1 - 100:1 w / w). The suspension was rotated or inverted at a temperature in the range of 2°C to 40°C for a period in the range of 10 minutes to 2 days. The remaining solid sulfating reagent was then removed by centrifugation (e.g., 1,000 - 20,000×g, 1 - 10 minutes), and the solution was neutralized with HCl while mixing. The material was then subjected to ultrafiltration against water 6 - 7 times using a spin unit (e.g., 3 kDa MWCO), and the resulting concentrate containing sTestan was recovered. Further purification by strong anion exchange chromatography using NaCl elution (e.g., Sepharose Q) and / or reverse phase extraction (e.g., C18 resin, solvents, etc.) was performed to increase the purity level and remove unwanted contaminants.

[0079] As an alternative solvent system using the same sulfur trioxide:trimethylamine complex sulfating reagent or other sulfating reagents (e.g., chlorosulfonic acid), dried formamide (using a 3 Å molecular sieve) was used to dissolve lyophilized (frozen and dried) Testan. This solution was reacted with the sulfating reagent in various ratios and then incubated at 0 - 40 °C for 0.1 - 48 hours. For the anhydrous sulfation reaction, purification was achieved by the same process as for the reaction containing water. In some cases, alcohol precipitation (e.g., 2.5 - 4 volumes of ethanol or isopropanol, 20 - 120 minutes, -20 - 22 °C, followed by recovery by centrifugation) was performed prior to ultrafiltration or chromatography for removing the reaction solvent.

[0080] Synthesis of sTestan using the enzymatic sulfation pathway:

[0081] Testan can also be converted to sTestan using an enzyme that can convert heparosan to heparan sulfate (Li, G., Masuko, S., Green, D. E., Xu, Y., Li, L., Zhang, F., Xue, C., Liu, J., DeAngelis, P. L., Linhardt, R. J. N-sulfotestosteronan, a novel substrate for heparan sulfate 6-O-sulfotransferases and its analysis by oxidative degradation. Biopolymers. 99(10):675 - 685(2013)). However, since there are many sulfotransferases, different catalysts can be used as long as the sulfation pattern and level result in sTestan having the desired biological activity or inhibitor activity.

[0082] Overall, by combining in vivo controlled fermentation of microorganisms (natural or recombinant) or chemoenzymatic synthesis in vitro with a chemical or enzymatic sulfation step, a more defined and reproducible product was obtained than current heparin-derived products. Furthermore, the envisioned sTestan supply chain is safer and less prone to adulteration than sourcing from animals. Compared to current low molecular weight compounds made by organic synthesis, the various routes for synthesizing sTestan involve less toxic reagents and fewer dangerous by-products and are more "green".

[0083] Analysis of sulfated polysaccharides

[0084] The conversion from Testan to sTestan was analyzed using agarose gel (1 - 1.5%, 1×TAE) or polyacrylamide gel electrophoresis (PAGE; 6 - 20% gel, 1×TBE) analysis. To visualize the bands, the polymer was stained with Stains-All dye. Testan was stained as a slow-moving blue band, while sTestan was stained as a fast-moving purple band (Figure 2). The latter polymer has a higher charge density due to sulfate addition, and thus the molecule moves faster and the color output is shifted.

[0085] Figure 4 includes another gel analysis of Testan (T) and various sulfated Testans (sT) generated using controlled sulfation levels (using reagent stoichiometry) and a positional control (solvent system). The observed color change from blue through purple to yellow / orange indicates an increase in sulfation level. The exact position of sulfation can shift the hydrodynamic radius of Testan depending on the position of the sugar ring being modified, even if the overall density of sulfate is the same. Thus, the hydrated and anhydrous sTestan polymers have different migration rates even at similar sulfation levels.

[0086] The sulfated Testan aqueous solution was also examined by NMR (Figure 5) and LC-MS. 1D and 2D NMR and LC-MS indicated that the 2-OH of the GlcA sugar was highly modified under these conditions to form the "sTestan aqueous solution". This finding was unexpected because when the same sulfur trioxide complex was used in an organic solvent, the 6-OH of GlcNAc was the most typically sulfated position in heparosan and HS derivatives as the major hydroxyl functional group.

[0087] Heparanase challenge

[0088] Recombinant human heparanase (73 ng; R&D Systems; Minneapolis, MN) was incubated with sTestan polysaccharide (180 ng) at 30 °C for 24 h in 50 mM sodium acetate, pH 5, 1 mg / ml acetylated bovine serum albumin (Promega; Madison, WI). The reaction was analyzed by polyacrylamide gel electrophoresis (20% gel, 1×TBE) and Stains-All detection. As seen in Figure 3, recombinant human heparanase cleaves O-sulfoheparosan and native HS species, but not sTestan.

[0089] Heparanase enzyme inhibition assay

[0090] Gel-based assays were used to monitor the catalysis of recombinant human heparanase (R&D Systems; Minneapolis, MN) on polysaccharide substrates. In these assays, cleavage of the fluorescent chain was monitored, and the disappearance of the parent substrate band was readily recognizable. Since each chain contains multiple overlapping heparanase cleavage sites, the molecular weight gradually decreases over time. This simple and defined assay directly monitors the digestion of polysaccharides that approximate naturally occurring substrates (i.e., glycans > about 20 monosaccharide units).

[0091] To track heparanase activity, two types of substrates were used: (i) fluorescein end-labeled synthetic O-sulfated O-linked heparosan, or (ii) polyacrylamide gel electrophoresis-purified rhodamine-labeled HS. Heparanase (final 1.3 ng / μl) was incubated with the substrates (synthetic, 57 ng / μl; HS, 3.3 ng / μl) in 50 mM sodium acetate, pH 5, 1 mg / ml acetylated bovine serum albumin (Promega; Madison, Wisconsin) at 30 °C for various times (typically about 0.2 - 5 hours). Aliquots of the reaction were analyzed by polyacrylamide gel electrophoresis (2 μl of reaction / lane; 20% gel, 1×TBE) and fluorescence imaging (exposure for 5 - 120 seconds; ChemiDoc MP). Then, Stains-All detection was used to track the overall size range of the polysaccharide fragments. For inhibition studies, the sTestan preparation was titrated against the fluorescent substrate at various molar ratios. Parallel control digestion reactions with 25% (1 / 4) or 50% (1 / 2) of the enzyme were used to help calibrate the progress or inhibition level of the reaction and as a comparison for evaluating the IC 50 in the experimental samples.

[0092] Co-incubation of sTestan with the synthetic O-sulfoheparosan substrate inhibited substrate digestion (Figure 6). Similarly, sTestan acted as an inhibitor, protecting native HS from human heparanase digestion (Figure 8), thereby demonstrating that this novel polymer acts as a competitive inhibitor against heparanase and thus has oncological applications. sTestan was at least about 100-fold more active on a molar basis than the heparosan substrate based on the IC 50 This finding indicates that heparanase binds more strongly to sTestan, which has α-α linkages, than to polysaccharides having only the natural alternating β-α linkages of the HS family.

[0093] Furthermore, sTestan was shown to be as potent as a known heparanase inhibitor (generic roneparstat) as a competitive inhibitor of human heparanase (Figure 7).

[0094] Anticoagulation assay

[0095] Using the Chromogenix Coatest assay (Diapharma; West Chester, Ohio), the effect of antithrombin III on the inactivation of thrombin (a coagulation factor that converts fibrinogen to fibrin glue) was measured. Typically, heparin (here the international heparin standard) is required to effectively reduce thrombin activity (derived from human plasma) as measured by the absorbance of a chromogenic thrombin substrate (at 415 nm). The sTestan aqueous solution polymer was tested at various concentrations while comparing with the heparin standard curve. As can be seen in Figure 9, sTestan was not an anticoagulant.

[0096] Using a modified diagnostic HIT assay (Zymutest HIA IgG; Aniara Diagnostica, West Chester, Ohio), the side effects of sTestan were evaluated. The titration of sTestan was used to compete with the HIT complex IgG binding sites found in the assay positive control for immobilized heparin. The ELISA method measured the bound antibody as measured by the absorbance (at 415 nm) of the product derived from the enzymatic chromogenic substrate of the secondary antibody probe. Free heparin competes with the plate-bound heparin and reduces the absorbance compared to a control with free sugar polymer. Heparin derivatives treated to reduce anticoagulant activity (non-anticoagulant heparin (NACH) or roneparstat (Rone)) were also tested in the HIT assay. As shown in Figure 10, sTestan (either sulfated in water or anhydrous) appeared to have a lower or similar HIT potential compared to heparin and its derivatives.

[0097] The Transwell migration assay was used to evaluate the effectiveness of sTestan on oncological treatment. Human ovarian cancer cells (OV9; 1×10 in 200 μl of serum-free DMEM 5() was seeded into the upper chamber of a Matrigel-coated Boyden transwell filter together with 20 μM of the sTestan variant or a known inhibitor (generic ronelapstat), or a PBS control. The lower chamber contained 600 μl of DMEM containing 10% FBS. The plates were incubated at 37 °C for 24 hours, then the cells were extracted and assayed for protein. The protein level in the lower compartment was used as a surrogate for the number of invading cells. As shown in Figure 11, sTestan and ronelapstat had similar metastasis-suppressing effects.

[0098] Generation of sTestan-based chimeric polymers

[0099] The sTestan polymers of the present disclosure can be further advantaged by being linked to moieties (e.g., but not limited to, heparosan chains) to assist or induce binding to heparanase targets or to alter pharmacokinetic behavior (e.g., prolong half-life, control tissue access, etc.). Figure 12 shows the production of sTestan-heparosan chimeras using recombinant Pasteurella multocida synthase G (20) to copolymerize monosaccharides from both UDP-sugar donors to the non-reducing end of the sTestan molecule (similar to the process of (15)). As shown in Figure 12, sTestan can be extended with heparosan to create chimeric polysaccharides, as indicated by the production of a larger molecular weight. This new chimeric material has different pharmacokinetics compared to the starting sTestan polymer alone.

[0100] Non-limiting exemplary embodiments Exemplary embodiment 1. Repeating structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] n ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n ) and at least one sulfur moiety is bonded to at least one hydroxyl of the repeating structure, and n ranges from about 2 to about 500.

[0101] Exemplary Embodiment 2. The polymer according to Exemplary Embodiment 1, wherein the sulfur moiety is SO3.

[0102] Exemplary Embodiment 3. The polymer according to Exemplary Embodiment 1 or 2, comprising the structure O-sulfo[-4-GlcUA-α1,4-GlcNAc-α1-] n The polymer according to Exemplary Embodiment 1 or 2, comprising

[0103] Exemplary Embodiment 4. The polymer according to any one of Exemplary Embodiments 1 to 3, wherein the sulfur moiety is O-linked to GlcNAc.

[0104] Exemplary Embodiment 5. The polymer according to any one of Exemplary Embodiments 1 to 4, wherein the sulfur moiety is O-linked to GlcUA.

[0105] Exemplary Embodiment 6. A composition comprising the polymer according to any one of Exemplary Embodiments 1 to 5.

[0106] Exemplary Embodiment 7. The composition according to Exemplary Embodiment 6, which is in an aqueous solution.

[0107] Exemplary Embodiment 7A. The composition according to Exemplary Embodiment 6 or 7, further comprising at least one additional active agent (e.g., an anti-cancer agent or other active agent for co-administration with the polymer).

[0108] Exemplary Embodiment 8. A composition comprising at least one polymer according to any one of Exemplary Embodiments 1 to 5, and a heparosan chain or other polymer linked thereto.

[0109] Exemplary Embodiment 8A. The composition according to any one of Exemplary Embodiments 6 to 8, further comprising at least one additional active agent (e.g., an anti-cancer agent or other active agent for co-administration with the polymer).

[0110] Exemplary Embodiment 9. A method of inhibiting heparanase activity, the method comprising exposing heparanase to at least one polymer according to any one of Exemplary Embodiments 1 to 5.

[0111] Exemplary Embodiment 10. A method of inhibiting heparanase activity, the method comprising exposing heparanase to at least one composition according to any one of Exemplary Embodiments 6 to 8.

[0112] Exemplary Embodiment 11. The method according to Exemplary Embodiment 9 or 10, wherein heparanase activity is inhibited in vitro.

[0113] Exemplary Embodiment 12. The method according to Exemplary Embodiment 9 or 10, wherein heparanase activity is inhibited in vivo.

[0114] Exemplary Embodiment 13. A pharmaceutical composition comprising at least one polymer according to any one of Exemplary Embodiments 1 to 5 and a pharmaceutically acceptable excipient.

[0115] Exemplary Embodiment 13A. The pharmaceutical composition according to claim 13, further defined as a sterile pharmaceutical composition.

[0116] Exemplary Embodiment 13B. The pharmaceutical composition according to Exemplary Embodiment 13 or 13A, further comprising at least one additional active agent (e.g., an anti-cancer agent or other active agent for co-administration with the polymer).

[0117] Exemplary Embodiment 14. A method of treating a subject in need of treatment, the method comprising administering the pharmaceutical composition according to Exemplary Embodiment 13 to the subject in need of treatment.

[0118] Exemplary Embodiment 15. The method according to Exemplary Embodiment 14, wherein the subject has cancer or is susceptible to cancer.

[0119] Exemplary Embodiment 16. The method according to Exemplary Embodiment 14, wherein the subject has at least one disease or condition associated with overexpression, misregulation, or overactivity of heparanase.

[0120] Exemplary Embodiment 17. The method according to Exemplary Embodiment 16, wherein the at least one disease or condition is selected from the group consisting of diabetes, complications of diabetes (e.g., cardiomyopathy), atherosclerosis, thrombosis, viral infections (e.g., herpes simplex), and combinations thereof.

[0121] Exemplary Embodiment 17A. The method according to any one of Exemplary Embodiments 14 to 17, further comprising administering at least one additional active agent (e.g., an anti-cancer agent or another active agent for co-administration with a polymer) simultaneously with, or wholly or partially sequentially with, the pharmaceutical composition.

[0122] Exemplary Embodiment 18. A method for producing a sulfated testan polymer, comprising generating a polymer backbone having a repeating structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] n ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n ) wherein n ranges from about 2 to about 500, and attaching at least one sulfur moiety to at least one hydroxyl of the repeating structure.

[0123] Exemplary Embodiment 19. The method according to Exemplary Embodiment 18, wherein the polymer backbone is produced by natural fermentation or recombinant production using an organism that expresses the testosterone synthase gene.

[0124] Exemplary Embodiment 20. The method according to Exemplary Embodiment 18, wherein the polymer backbone is produced by chemical synthesis or chemoenzymatic synthesis.

[0125] Exemplary Embodiment 21. A kit comprising at least one polymer according to any one of Exemplary Embodiments 1 to 5 and / or at least one composition according to any one of Exemplary Embodiments 6 to 8.

[0126] Exemplary Embodiment 21. The kit according to Exemplary Embodiment 21, further comprising an apparatus for delivering the polymer or composition to a subject.

[0127] Exemplary Embodiment 22. A composition for use in the method according to any one of Exemplary Embodiments 9 to 12 and 14 to 17, comprising a polymer having a repeating structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] n ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n ) and having at least one sulfur moiety bonded to at least one hydroxyl of the repeating structure, wherein n ranges from about 2 to about 500.

[0128] Exemplary Embodiment 23. Use of at least one polymer according to any one of Exemplary Embodiments 1 to 5 and / or at least one composition according to any one of Exemplary Embodiments 6 to 8 in a method of treating a subject in need of treatment.

[0129] Cited References The following references are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement what is described herein. Also, the following is not intended to be an information disclosure statement. Rather, an information disclosure statement in accordance with the provisions of 37 CFR§1.97 will be separately filed. 1. Kowitsch, A., Zhou, G. & Groth, T., Medical application of glycosaminoglycans: a review. J. Tissue. Eng. Regen. Med. 12, e23 - e41 (2018). 2. Lindahl, U., Couchman, J., Kimata, K. & Esko, J. D. Proteoglycans and Sulfated Glycosaminoglycans in Essentials of Glycobiology, 3rd edition, A. Varki, Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2017) Chapter 17. 3. Esko, J. D., Prestegard, J. H. & Linhardt, R. J. Proteins That Bind Sulfated Glycosaminoglycans Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015 - 2017. in Essentials of Glycobiology, 3rd edition, A. Varki, Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2017) Chapter 38. 4. DeAngelis, P. L., Liu, J. & Linhardt, R. J. Chemoenzymatic synthesis of glycosaminoglycans: Re - creating, re - modeling and re - designing nature’s longest or most complex carbohydrate chains, Glycobiology 23, 764 - 777 (2013). 5. Zhang, X., Lin, L., Huang, H. & Linhardt, R. J. Chemoenzymatic synthesis of glycosaminoglycans, Acc. Chem. Res. 53, 335 - 346 (2020). 6. Liu, J. & Linhardt, R. J. Chemoenzymatic synthesis of heparan sulfate and heparin, Nat. Prod. Rep. 31, 1676 - 1685 (2020). 7. Otto, N. J., Solakyildirim, K., Linhardt, R. J., DeAngelis, P. L. Comamonas testosteronan synthase, a bifunctional glycosyltransferase that produces a unique heparosan polysaccharide analog. Glycobiology. 21(10):1331 - 1340(2011). 8. DeAngelis, P. L., Otto, N. J., Methods of producing testosteronan polymers using testosteronan synthase. United States Patent No. 9,695,427(2017). 9. DeAngelis, P. L., Otto, N. J., Methods of producing testosteronan polymers using testosteronan synthase. United States Patent No. 10,273,517(2019). 10. Jia, L. & Ma, S. Recent advances in the discovery of heparanase inhibitors as anti - cancer agents, Eur. J. Med. Chem. 121, 209 - 220(2016). 11. Heyman, B. & Yang, Y. M. Mechanisms of heparanase inhibitors in cancer therapy, Experimental Hematol. 44, 1002 - 1012(2016). 12.Mayes,H.B.,Broadbelt,L.J.&Beckham,G.T.How sugars pucker:Electronic structure calculations map the kinetic landscape of five biologically paramount monosaccharides and their implications for enzymatic catalysis.J.Am.Chem.Soc.136,1008-1022(2014). 13.Wu,L.Viola,C.M.,Brzozowski,A.M.&Davies,G.J.Structural characterization of human heparanase reveals insights into substrate recognition.Nat.Struct.&Molec.Biol.22(12),1016-1022(2015). 14.Mohan C.D.,Hari,S.,Preetham,H.D.,Rangappa,S.,Barash,U.,Ilan,N.,Nayak,S.C.&Gupta,V.K.,Basappa,Vlodavsky,I.&Rangappa,K.S.Targeting Heparanase in Cancer:Inhibition by Synthetic,Chemically Modified,and Natural Compounds.iScience 15,360-390(2019). 15.Sismey-Ragatz,A.E.,Green,D.E.,Otto,N.J.,Rejzek,M.,Field,R.A.&DeAngelis,P.L.Chemoenzymatic synthesis with distinct Pasteurella heparosan synthases:monodisperse polymers and unnatural structures.J.Biol.Chem.282,28321-28327(2007). 16. Gilbert, E. E. The reactions of sulfur trioxide, and its adducts, with organic compounds. Chem. Rev. 62, 549 - 589 (1962) 17. Minamisawa, T., Asami, N., Fujita, H. & Suzuki, K. Method for sulfating glycosaminoglycans. US Patent 10,259,889 (2016) 18. Li, G., Masuko, S., Green, D. E., Xu, Y., Li, L., Zhang, F., Xue, C., Liu, J., DeAngelis, P. L., Linhardt, R. J. N - sulfotestosteronan, a novel substrate for heparan sulfate 6 - O - sulfotransferases and its analysis by oxidative degradation. Biopolymers. 99(10):675 - 685(2013) 19. Cowman, M. K., Chen, C. C., Pandya, M., Yuan, H., Ramkishun, D., LoBello, J., Bhilocha, S., Russell - Puleri, S., Skendaj, E. & Mijovic J. & Jing, W. Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan. Analytical Biochemistry 417, 50 - 56(2011). 20. Otto NJ, Green DE, Masuko S, Mayer A, Tanner ME, Linhardt RJ, DeAngelis PL. J Biol Chem. 2012 Mar 2;287(10):7203 - 12. doi:10.1074 / jbc.M111.311704. Epub 2012 Jan 10. PMID:22235128. 21. Casu B., Torri G, Naggi AM, Giannini G, Pisano C, Penco S. Derivatives of partially desulphated glycosaminoglycans as heparanase inhibitors, endowed with antiangiogenic activity and devoid of anticoagulating effect. US Patent No. 7,781,416 (2010).

Claims

1. Repeating structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] ｎ ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] ｎ ) and contains at least one sulfur moiety bonded to at least one hydroxyl of the repeating structure, where n ranges from about 2 to about 500, a polymer.

2. wherein the sulfur moiety is SO ３ The polymer according to claim 1, wherein the sulfur moiety is SO

3. Structure O-sulfo[-4-GlcUA-α1,4-GlcNAc-α1-] ｎ The polymer according to claim 1, comprising

4. The polymer according to claim 1, wherein the sulfur moiety is O-linked to the GlcNAc.

5. The polymer according to claim 1, wherein the sulfur moiety is O-linked to the GlcUA.

6. A composition comprising the polymer according to any one of claims 1 to 5.

7. The composition according to claim 6, which is in an aqueous solution.

8. A composition comprising: at least one polymer according to any one of claims 1 to 5, and a heparosan chain or other polymer linked thereto The composition.

9. A method for inhibiting heparanase activity, comprising exposing the heparanase to at least one polymer according to any one of claims 1 to 5.

10. A method for inhibiting heparanase activity, comprising exposing the heparanase to at least one composition according to any one of claims 6 to 8.

11. The method according to claim 9 or 10, wherein the heparanase activity is inhibited in vitro.

12. The method according to claim 9 or 10, wherein the heparanase activity is inhibited in vivo.

13. A pharmaceutical composition comprising: at least one polymer according to any one of claims 1 to 5, and a pharmaceutically acceptable excipient The pharmaceutical composition.

14. A method for treating a subject in need of treatment, comprising: administering the pharmaceutical composition according to claim 13 to the subject in need of treatment The method.

15. The method according to claim 14, wherein the subject has cancer or is predisposed to cancer.

16. The method according to claim 14, wherein the subject has at least one disease or condition associated with overexpression, misregulation, or overactivity of heparanase.

17. The method according to claim 16, wherein the at least one disease or condition is selected from the group consisting of diabetes, diabetic complications, atherosclerosis, thrombosis, viral infections, and combinations thereof.

18. A method for producing a sulfated tester polymer, comprising: A repeating structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] in which n ranges from about 2 to about 500 ｎ ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] ｎ ) to generate a polymer backbone, and binding at least one sulfur moiety to at least one hydroxyl of the repeating structure The method.

19. The method according to claim 18, wherein the polymer backbone is produced by natural fermentation or recombinant production using an organism that expresses the testosteronan synthase gene.

20. The method according to claim 18, wherein the polymer backbone is produced by chemical synthesis or chemoenzymatic synthesis.

21. A kit comprising at least one polymer according to any one of claims 1 to 5 and / or at least one composition according to any one of claims 6 to 8.

22. A composition for use in the method according to any one of claims 9 to 12 and 14 to 17, comprising a polymer containing the repeating structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] ｎ ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] ｎ ) and having at least one sulfur moiety bonded to at least one hydroxyl of said repeating structure, wherein n ranges from about 2 to about 500.

23. Use of at least one polymer according to any one of claims 1 to 5 and / or at least one composition according to any one of claims 6 to 8 in a method of treating a subject in need of treatment.

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