Compositions for use in the treatment of disease

Abstract

The present invention relates generally to the field of compositions comprising one or more trehalose conjugates and is directed to methods of use thereof, such as for treating a disease or disorder in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 USC § 119(e) to U.S. Provisional Patent Application No. 63 / 335,978, filed April 28, 2022, entitled "COMPOSITION FOR USE IN THE TREATMENT OF A DISEASE," the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates generally to the field of compositions comprising one or more trehalose conjugates and is directed to methods of use thereof, such as for treating a disease or disorder in a subject. [Background technology]

[0003] Acute pancreatitis (AP) is a common gastrointestinal disease with an increasing incidence worldwide, accounting for approximately 2% of all hospitalized patients. AP is a complex inflammatory syndrome resulting from many etiologies, with gallstones, alcohol, and ERCP being the main causes. Approximately 20% of patients who experience a first AP attack will have a recurrent attack (RAP), and roughly one-third of the latter will progress to end-stage chronic pancreatitis (CP). Despite major advances in medicine, the worldwide mortality rate of AP patients remains high, placing a significant burden on the healthcare system. It is widely accepted that excessive stimulation of the pancreas or direct destructive injury leads to obstruction of zymogen granule outflow, where they are proteolytically activated in acinar cells by lysosomal enzymes, mainly cathepsin B, ultimately causing acute cellular injury. This adverse reaction is further exacerbated by neutrophil enzymes and transcription factors, leading to the production of various pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, and IL-8, along with the conversion of trypsinogen to trypsin, a phenomenon also called autolysis. Furthermore, pro-inflammatory stimuli upregulate cyclooxygenase (COX)-2, a key enzyme that contributes to the production of prostaglandins, leukotrienes, and thromboxanes.

[0004] Given the unclear characterization of the mechanistic pathways involved in AP, therapeutic options targeting specific underlying causes remain elusive, and current treatments rely mainly on analgesics and hydration, with opioids being the most commonly prescribed analgesics for pain relief in AP patients. As AP is secondary to inflammation in the pancreatic parenchyma, nonsteroidal anti-inflammatory drugs (NSAIDs) are also commonly used. Previous studies have revealed a strong involvement of heparanase in the pathogenesis of inflammatory diseases, including AP. Specifically, it has been demonstrated that pancreatic heparanase expression and activity are significantly elevated after caerulein-induced AP. Moreover, pancreatic edema and inflammation as well as the induction of cytokines and signaling molecules after caerulein administration were significantly attenuated by selective heparanase inhibitors PG545 and SST0001, implying that heparanase plays an important role in AP. In particular, all the above characteristics seem to be even more evident in transgenic mice that overexpress heparanase, suggesting that these mice can be used as a highly sensitive model system to elucidate the molecular mechanism by which heparanase functions in AP.Therefore, there is a continuous need to develop new drug therapies for the specific and efficient treatment of AP.

[0005] Inflammatory brain diseases, also called inflammatory diseases of the central nervous system (CNS), are conditions in which the brain and / or spinal cord become inflamed. Brain inflammation can cause irritation and swelling of brain tissue or blood vessels, which can lead to brain damage in the long term.

[0006] Chronic brain inflammation leads to measurable brain shrinkage, especially in areas associated with Alzheimer's disease, the sixth leading cause of death. Chronic brain inflammation leads to mental fatigue, brain fog and memory loss. Chronic brain inflammation is associated with many neurological and psychiatric disorders, including depression, anxiety, substance abuse, schizophrenia, bipolar disorder, Alzheimer's disease and Parkinson's disease.

[0007] Encephalitis is a rare form of acute brain inflammation, usually caused by a viral or bacterial infection. Symptoms include fever, headache, seizures, stiffness in the neck and back, and mental confusion. Encephalitis can cause brain damage and even death.

[0008] Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammation and pain. NSAIDs act by inhibiting cyclooxygenase (COX)-1 and / or COX-2 activity, thereby reducing the intracellular synthesis of prostaglandins associated with inflammation and / or pain. However, most NSAIDs cannot cross the blood-brain barrier (BBB) ​​in sufficient amounts to induce a therapeutic effect. Therefore, there is a continuous need to develop methods to promote the BBB permeability of known NSAIDs, for example for use in the treatment of encephalitis and to reduce COX activity in the brain.

[0009] The foregoing examples of the related art and limitations associated therewith are intended to be illustrative and not exhaustive. Other limitations of the related art will become apparent to those of ordinary skill in the art upon examination of this specification and drawings. Summary of the Invention

[0010] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods that are meant to be representative and illustrative, not limiting in scope.

[0011] In one aspect of the invention there is a composite comprising trehalose covalently bound to a non-steroidal anti-inflammatory drug (NSAID) selected from the group comprising diclofenac, naproxen, diflunisal, salsalate, ibuprofen, indomethacin, mefenamic acid, meclofenamic acid, clonixin, licofelone or combinations thereof.

[0012] In one embodiment, trehalose is covalently attached to a carboxy group of said NSAID.

[0013] In one embodiment, the covalent bond is via a bond selected from an ester, an amide, a thioester, a carbamate, a carbonate, a carbamide, a thiocarbamate, a phosphonate, a phosphodiester, a sulfonate ester, or any combination thereof.

[0014] In one embodiment, trehalose is covalently attached via hydroxy groups at the 2-position, the 6-position, or both.

[0015] In one embodiment, the compound has the formula 1: [ka] Or formula 1a: [ka] Represented by wherein each R independently comprises decarboxylated diclofenac, decarboxylated naproxen, decarboxylated diflunisal, decarboxylated salsalate, decarboxylated paracetamol, decarboxylated ibuprofen, decarboxylated indomethacin, decarboxylated mefenamic acid, decarboxylated meclofenamic acid, decarboxylated clonixin, or decarboxylated licofelone; and each X or X1 independently comprises O, S, or NH.

[0016] In another embodiment, there is a pharmaceutical composition comprising a therapeutically effective amount of a conjugate and a pharma- ceutical acceptable carrier, the pharmaceutical composition comprising trehalose covalently attached to a nonsteroidal anti-inflammatory drug (NSAID).

[0017] In one embodiment, the NSAID is selected from aspirin, diclofenac, naproxen, diflunisal, salsalate, ibuprofen, indomethacin, mefenamic acid, meclofenamic acid, clonixin, licofelone, COX-2 inhibitors, including any pharma- ceutically acceptable salt, pharma-ceutically active derivative, or combination thereof.

[0018] In one embodiment, the composite is a composite of the present invention.

[0019] In one embodiment, the pharmaceutical composition is for use in the prevention or treatment of a disease or disorder in a subject in need thereof.

[0020] In one embodiment, the disease or disorder is or comprises inflammation, pain, or both.

[0021] In one embodiment, the disease or disorder is or comprises pancreatitis.

[0022] In one embodiment, the pharmaceutical composition is for use in inhibiting and / or reducing cyclooxygenase (COX) activity, heparinase (Hep) activity, or both, in a subject in need thereof.

[0023] In one embodiment, the disease or disorder is associated with COX activity, Hep activity, or both.

[0024] In another aspect, there is a method for inhibiting or reducing enzymatic activity in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention, wherein the enzymatic activity comprises COX activity, Hep activity, or both.

[0025] In one embodiment, reducing enzyme activity comprises preventing or treating a condition selected from inflammation and pain in said subject.

[0026] In one embodiment, the inflammation comprises pancreatitis.

[0027] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions. [Brief description of the drawings]

[0028] [Figure 1]1A-C are bar graphs showing the effect of either PG545, SST0001, aspirin or combination treatment on cerulein-induced pancreatitis as revealed by serum levels of lipase (1A), amylase (1B) and pancreatic index (pancreas weight / body weight ratio) (1C) in WT mice and Hpa-Tg animals. Compared to saline group, *, p<0.05, **, p<0.01 ***, p<0.001; compared to cerulein group, #, p<0.05 ##, p<0.01, ###, p<0.001; compared to combination group, $, p<0.05, $$, p<0.01, $$$, p<0.001. [Diagram 2] Figures 2A-E and 2A'-G' are photomicrographs showing histopathology images of WT and Hpa-Tg mice injected with either saline or caerulein in the presence or absence of PG545, SST0001, Asp or combined pretreatment. Pancreatic tissues were harvested 24 hours thereafter and 5 micron sections from formalin-fixed, paraffin-embedded samples were stained by H&E. Representative photomicrographs at 20x original magnification are shown. Figures 2A-E present hematoxylin stained samples of WT and Hpa-Tg mice injected with either saline (2A) or caerulein (2B) in the presence or absence of Asp (2C), PG545 (2D) or combined pretreatment PG545+Asp (2E). Figures 2A'-G' present eosin-stained samples of WT and Hpa-Tg mice injected with either saline (2A') or caerulein (2B') in the presence or absence of PG545 (2C'), SST0001 (2D'), Asp (2E') or combined pretreatments PG545+Asp (2F') and PG545+Asp+SST0001 (2G'). [Diagram 3]Figures 3A-D are bar graphs and photomicrographs showing the effect of pretreatment with aspirolose (mono- or diester) on cerulein-induced pancreatitis as revealed by serum levels of lipase (3A), amylase (3B), pancreatic index (pancreas weight / body weight ratio) (3C) and histological changes (3D) in WT mice and Hpa-Tg animals. Compared to saline group, *, p<0.05, **p<0.01; compared to cerulein group, #, p<0.05, ##, p<0.01; compared to combination group, $, p<0.05. [Figure 4] Figures 4A-C are bar graphs showing the effect of post-treatment with aspirulose (mono- or diester) on cerulein-induced pancreatitis as revealed by serum levels of lipase (4A), amylase (4B) and pancreatic index (pancreas weight / body weight ratio) (4C) in Hpa-Tg animals. Compared to saline group, *, p<0.05; compared to cerulein group, #, p<0.05; compared to combination group, $, p<0.05. [Diagram 5] 5A-D are bar graphs and photomicrographs showing the effect of pretreatment with Indose (a composite of indomethacin and trehalose) or Diclose (a composite of diclofenac and trehalose) (16 or 32 mg / kg, ip) on caerulein-induced pancreatitis in WT animals as revealed by serum levels of lipase (5A), amylase (5B), pancreatic index (pancreas weight / body weight ratio (P / M)) (5C), and histological analysis (5D). Compared with saline group, *, p<0.05; compared with caerulein group, #, p<0.05; compared with combination group, $, p<0.05. [Figure 6]6A-C are bar graphs and photomicrographs showing the effect of pretreatment with Indose (a complex of indomethacin and trehalose) or Diclose (a complex of diclofenac and trehalose (16 or 32 mg / kg, ip) on caerulein-induced pancreatitis as revealed by serum levels of lipase (6A), amylase (6B) and pancreatic index (pancreas weight / body weight ratio (P / M)) (6C) in Hpa-Tg animals. Compared to saline group, *, p<0.05; compared to caerulein group, #, p<0.05; compared to combination group, $, p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In one aspect of the invention disclosed herein, there is a conjugate comprising a disaccharide covalently attached to a compound selected from the group comprising diclofenac, naproxen, diflunisal, salsalate, paracetamol, ibuprofen, indomethacin, mefenamic acid, meclofenamic acid, clonixin, and licofelone, or any combination thereof.

[0030] In some embodiments, non-limiting examples of disaccharides are sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, kojibiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, turanose, trehalulose, maltulose, leucrose, isomaltulose, gentiobiulose, mannobiose, melibiose, allolactose, melibiulose, lactulose, rutinose, rutinulose, xylobiose, or any combination thereof.

[0031] In some embodiments, the disaccharide comprises two monosaccharide units linked together via a glycosidic bond, and each monosaccharide unit is independently selected from a hexose and a pentose. In some embodiments, each monosaccharide unit is a hexose. In some embodiments, the disaccharide is trehalose or a derivative thereof (e.g., a conformer, diastereomer, enantiomer).

[0032] In some embodiments, the composite comprises trehalose covalently bound to a compound selected from the group comprising diclofenac, naproxen, diflunisal, salsalate, ibuprofen, paracetamol, indomethacin, mefenamic acid, meclofenamic acid, clonixin, and licofelone, or any combination thereof. In some embodiments, the compound is or comprises at least one NSAID selected from diclofenac, naproxen, ibuprofen, indomethacin, and paracetamol, or any combination thereof. In some embodiments, the composite comprises trehalose covalently bound to a plurality of compounds, the compounds being as described herein above. In some embodiments, the compound lacks aspirin.

[0033] In some embodiments, trehalose is covalently attached via a hydrolyzable bond. In some embodiments, trehalose is covalently attached via a biodegradable or biocleavable bond. Such biodegradable or biocleavable bonds are well known in the art and include amide bonds, ester bonds, and disulfide bonds, among others. In some embodiments, the covalent bond is selected from an ester, an amide, a thioester, a carbamate, a carbonate ester, a carbamide, a thiocarbamate, a phosphonate, a phosphodiester, a sulfonate ester, or any combination thereof. In some embodiments, the covalent bond is selected from an ester, an amide, a thioester, or any combination thereof.

[0034] In some embodiments, the conjugates comprise trehalose covalently bound to one or more compounds via ester bonds.

[0035] In some embodiments, trehalose is covalently attached to the compound via at least one hydroxyl group of trehalose. In some embodiments, trehalose is covalently attached to a carboxy group of the compound. In some embodiments, the conjugate comprises trehalose covalently attached to a carboxy group of the compound, the covalent attachment being via a hydroxyl group of trehalose at the 2-position, the 6-position, or both.

[0036] In some embodiments, the compound has Formula 1: [ka] Or formula 1a: [ka] wherein each R is independently or comprises a decarboxylated NSAID selected from decarboxylated diclofenac, decarboxylated naproxen, decarboxylated diflunisal, decarboxylated salsalate, decarboxylated paracetamol, decarboxylated ibuprofen, decarboxylated indomethacin, decarboxylated mefenamic acid, decarboxylated meclofenamic acid, decarboxylated clonixin, or decarboxylated licofelone (including any salt and / or any pharma- ceutically active derivative thereof), and each X or X1 independently comprises O, S, or NH.

[0037] In some embodiments, the conjugate has the formula: [ka] or the formula: [ka] wherein each R, X, and X1 is as disclosed herein above. In some embodiments, each X or X1 is O.

[0038] As used herein, the term "derivative" refers to a small molecule derived from the above-mentioned NSAID and having NSAID functional group or activity. NSAID activity can be determined by well-known methods, such as methods suitable for determining cyclooxygenase inhibitory activity. Furthermore, the NSAID activity of the conjugates of the present invention, including NSAID derivatives, can be evaluated by determining their ability to reduce enhanced Hep activity (e.g., as assessed by reducing elevated lipase / amylase levels).

[0039] Furthermore, the term "derivative" encompasses any structurally similar functional derivative of the above-mentioned NSAIDs, where structurally similar is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% structural similarity, including any range therebetween.

[0040] In some embodiments, the term "structural similarity" refers to fingerprint similarity between two molecules. The term "fingerprint similarity" is well understood by those of skill in the art. In some embodiments, fingerprint similarity is calculated based on circular fingerprints, substructure key-based fingerprints, and / or topological or path-based fingerprints.

[0041] Exemplary circular fingerprints include, but are not limited to, Molprint 2D, ECFP (or Morgan Fingerprint), FCFP, etc. In some embodiments, the term "structural similarity" as used herein is calculated by the Morgan Fingerprint.

[0042] In some embodiments, the term "decarboxylated NSAIDs" includes NSAIDs that lack a carboxy group, i.e., have a bond in place of a carboxy group. In some embodiments, the bond is the point of attachment of R to the -C(=X1)X group of the conjugate molecule represented by Formula 1 or 1a.

[0043] In some embodiments, the compound has Formula 2: [ka] or formula 2a: [ka] where X and R are as described herein above.

[0044] In some embodiments, each R is [ka] where the wavy bond represents the point of attachment of R to the -C(=X1)X or -C(=X)O group of the conjugated molecule.

[0045] In some embodiments, the NSAID comprises a compound that inhibits cyclooxygenase (COX) enzyme activity. In some embodiments, the NSAID comprises a compound that selectively inhibits COX-1 enzyme activity. In some embodiments, the NSAID comprises a compound that selectively inhibits COX-2 enzyme activity. In some embodiments, the NSAID comprises a compound that inhibits COX-1 and COX-2 enzyme activity.

[0046] In some embodiments, non-limiting examples of NSAIDs include, but are not limited to, aspirin, diclofenac, naproxen, indomethacin, diflunisal, salsalate, ibuprofen, mefenamic acid, meclofenamic acid, clonixin, and licofelone (including any pharma- ceutically active derivatives or combinations thereof).

[0047] In some embodiments, the conjugate comprises trehalose covalently bound to at least one of aspirin, diclofenac, indomethacin, and naproxen, including any salt or any combination thereof.

[0048] In some embodiments, the composite of the present invention comprises: [ka] (also used herein as "Diclose"), [ka] (also used herein as "Indose"), [ka] (also used herein as "Asprilose-monoester") and [ka] (also used herein as "asprylose-diester").

[0049] Pharmaceutical Composition (NSAID) In another aspect of the present invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of any one of the compounds of the present invention and a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the compound of the present invention as an active ingredient.

[0050] As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result in a subject. The exact dosage form and regimen will be determined by the physician according to the patient's condition.

[0051] In some embodiments, the pharmaceutical composition comprises a conjugate of the present invention, a pharma- ceutically acceptable salt thereof, or both. In some embodiments, the pharma- ceutically acceptable salt comprises a conjugate of the present invention and a pharma- ceutically acceptable anion.

[0052] In some embodiments, examples of pharma- ceutically acceptable anions include, but are not limited to, acetate, aspartate, benzenesulfonate, benzoate, bicarbonate, carbonate, halides (such as bromide, chloride, iodide, fluoride), bitartrate, citrate, salicylate, stearate, succinate, sulfate, tartrate, decanoate, edetate, fumarate, gluconate, and lactate, or any combination thereof.

[0053] In some embodiments, the pharmaceutical composition comprises a conjugate of the present invention as the only therapeutically active ingredient. In some embodiments, the conjugate of the present invention is the only therapeutically active ingredient in the pharmaceutical composition of the present invention. In some embodiments, the pharmaceutical composition lacks additional therapeutically active ingredients.

[0054] In some embodiments, the pharmaceutical composition comprises trehalose or a pharma- ceutically active derivative thereof covalently bound to an NSAID. In some embodiments, the pharmaceutical composition comprises trehalose or a pharma- ceutically active derivative thereof covalently bound to an NSAID. In some embodiments, the pharmaceutical composition comprises trehalose or a pharma- ceutically active derivative thereof covalently bound to one or more compounds selected from the group comprising aspirin, diclofenac, naproxen, diflunisal, salsalate, paracetamol, ibuprofen, indomethacin, mefenamic acid, meclofenamic acid, clonixin, and licofelone, or any combination thereof. In some embodiments, the pharmaceutical composition comprises trehalose or a pharma-ceutically active derivative thereof covalently bound to one or more compounds selected from the group comprising aspirin, diclofenac, naproxen, diflunisal, salsalate, paracetamol, ibuprofen, indomethacin, mefenamic acid, meclofenamic acid, clonixin, and licofelone, or any combination thereof, including any salt or any combination thereof.

[0055] In some embodiments, the compounds of the invention present in the pharmaceutical composition are of pharmaceutical grade purity, i.e., characterized by a chemical purity of at least about 90%, at least about 95%, greater than 95% or greater than 99%, and 90-99.999%, 90-95%, 90-97%, 95-99% (including any ranges in between).

[0056] Carrier In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of a conjugate of the present invention and a pharma- ceutically acceptable carrier.

[0057] In some embodiments, the carrier improves the stability of the active ingredient in a living organism. In some embodiments, the carrier improves the stability of the active ingredient within a pharmaceutical composition. In some embodiments, the carrier enhances the bioavailability of the active ingredient.

[0058] In some embodiments, the carrier improves (i) the water solubility of the active ingredient, (ii) the permeability of the active ingredient through lipid membranes, or both.

[0059] As used herein, the term "carrier" refers to any component of a pharmaceutical composition that is not an active agent, such as a diluent, adjuvant, excipient, or vehicle with which an active ingredient is administered (e.g., a material that is in a liquid or solid state at room temperature). Such carriers can be sterile liquids, such as water-based liquids and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents. Suitable pharma- ceutically acceptable carriers, excipients and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entireties.These additional inactive ingredients, as well as effective formulation and administration procedures, are well known in the art and are described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety.

[0060] In some embodiments, the carrier is selected from the group consisting of terpenes from cannabis or total terpene extracts from hemp plants, terpenes from coffee or cocoa, mint extracts, eucalyptus extracts, citrus extracts, tobacco extracts, aniseed extracts, any vegetable oil, peppermint oil, d-limonene, b-myrcene, a-pinene, linalool, anethole, a-bisabolol, camphor, b-caryophyllene and caryophyllene oxide, 1,8-cineole, citrus extracts ... The active ingredient may be or include any of the following: citronella, delta-3-carene, farnesol, geraniol, indomethacin, isopulegol, linalool, unalyl acetate, b-myrcene, myrcenol, l-menthol, menthone, menthol and neomenthol, oridonin, a-pinene, diclofenac, nepafenac, bromfenac, phytol, terpineol, terpinen-4-ol, thymol and thymoquinone. One of skill in the art will recognize that the particular carrier employed within the pharmaceutical composition of the present invention may vary depending on the route of administration.

[0061] In some embodiments, suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, water, ethanol, and the like.

[0062] In some embodiments, non-limiting examples of carriers for pharmaceutical compositions in the form of a cream include, but are not limited to, nonionic surfactants (e.g., glyceryl monolinoleate, glyceryl monooleate, glyceryl monostearate lanolin alcohol, lecithin mono- and diglycerides poloxamer polyoxyethylene 50 stearate and sorbitan trioleate stearate), anionic surfactants (e.g., pharma- ceutically acceptable salts of fatty acids such as, for example, stearic acid, oleic acid, palmitic acid and lauric acid), cationic surfactants (e.g., pharma-ceutically acceptable quaternary ammonium salts such as, for example, benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride), or any combination thereof.

[0063] In some embodiments, the carrier is a liquid carrier. In some embodiments, the carrier is water or an aqueous solution substantially devoid of organic solvents. In some embodiments, water is used when the active agent is water-soluble. In some embodiments, the water solubility of the active agent is sufficient for intravenous administration. As used herein, the term "water-soluble," including any of its grammatical forms, refers to a compound being soluble in water (or in an aqueous solution) to form an aqueous solution substantially devoid of a particular substance (i.e., insoluble aggregates or solid particles of the compound). The presence of particles / aggregates in an aqueous solution can be determined by various methods, such as DLS.

[0064] In some embodiments, the carrier is an aqueous solution. Non-limiting examples of aqueous solutions in some embodiments are saline solution, aqueous dextrose buffers, such as phosphate buffers, and aqueous solutions containing glycerol.

[0065] In some embodiments, the carriers comprise, in total, 0.1%-99.99%, 0.1%-50%, 30-90%, 20-99.99%, 90-99.99%, or 70-90% by weight of the pharmaceutical composition of the invention.

[0066] In some embodiments, the pharmaceutical composition further comprises any one of a wetting agent, an emulsifying agent, a pH buffering agent (such as acetate, citrate or phosphate buffers), an antibacterial agent (such as, for example, benzyl alcohol or methyl parabens), an antioxidant (such as, for example, ascorbic acid or sodium disulfite), an isotonic agent (such as, for example, sodium chloride or dextrose), or any combination thereof.

[0067] In some embodiments, the pharmaceutical composition comprises the incorporation of any one of the active ingredients into or onto microparticle preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar, vesicles, erythrocyte ghosts, or spheroplasts. Such compositions are well known to those skilled in the art and may affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate.

[0068] In some embodiments, liposomes for use in the compositions described herein are generally formed from standard vesicle-forming lipids, including one or more neutral and / or positively and / or negatively charged phospholipids, optionally PEG-lipids and sterols, such as cholesterol. The choice of lipid is generally determined by considerations such as liposome size and stability in blood. Various methods are available for preparing liposomes, as reviewed, for example, by Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0069] In some embodiments, the pharmaceutical composition is in the form of, but not limited to, an emulsion, liquid solution, gel, paste, suspension, dispersion, aerosol, ointment, cream, foam, suppository, patch, pill, capsule, lozenge, wafer, ampoule, vial or pre-filled syringe, pad or gelling stick.

[0070] In some embodiments, the pharmaceutical composition is liquid at a temperature between 15 and 45° C. In some embodiments, the pharmaceutical composition is solid at a temperature between 15 and 45° C. In some embodiments, the pharmaceutical composition is in the form of a suppository.

[0071] In some embodiments, the pharmaceutical composition is semi-liquid at a temperature of 15-45° C. It should be understood that the term “semi-liquid” is intended to mean a material that is flowable under pressure and / or shear stress. In some embodiments, semi-liquid compositions include creams, ointments, gel-like materials, and other similar materials. In some embodiments, the pharmaceutical composition is a semi-liquid composition characterized by a viscosity in the range of 31,000-800,000 cps.

[0072] In some embodiments, the pharmaceutical composition is in the form of a cream. In some embodiments, the cream further comprises a thickening agent.

[0073] In some embodiments, non-limiting examples of thickening agents include, but are not limited to, microcrystalline cellulose, starch, modified starch, tragacanth, gelatin, and polymeric thickeners (eg, polyvinylpyrrolidone), or any combination thereof.

[0074] In some embodiments, the pharmaceutical compositions of the present invention are stable for a period ranging from at least 10 hours, at least 24 hours, at least 2 days, at least 10 days, at least 20 days, at least 1 month, at least 6 months, at least 1 year, including any values ​​and ranges therebetween. As used herein, the term "stable" means that the pharmaceutical composition retains at least 80%, at least 85%, at least 90%, at least 95%, at least 99% by weight of the initial conjugate loading after the aforementioned period, including any ranges therebetween. The term stability, as used herein, encompasses the chemical stability of the composition (i.e., at least 90%, at least 95%, at least 99% or 90-95%, 90-99% by dry weight of each of the components of the composition retain their chemical identity as determined by well-known analytical methods such as HPLC, GC / LC-MS, etc.), as well as physical stability (e.g., appearance, mechanical / physical integrity, homogeneity, etc.).

[0075] In some embodiments, the pharmaceutical compositions described herein are topical compositions. In some embodiments, the pharmaceutical compositions are oral compositions. In some embodiments, the pharmaceutical compositions are injectable compositions. In some embodiments, the pharmaceutical compositions are for systemic use.

[0076] In some embodiments, the pharmaceutical composition is for use in the prevention and / or treatment of a condition in a subject. In some embodiments, the condition comprises a disease or disorder selected from inflammation, pain, or both. In some embodiments, the condition comprises a disease or disorder associated with inflammation and / or pain. In some embodiments, the pharmaceutical composition comprises a disaccharide covalently attached to a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, the pharmaceutical composition comprises a disaccharide covalently attached to multiple NSAIDs, wherein the NSAIDs are as described herein above.

[0077] In some embodiments, the pharmaceutical composition is for use in increasing internalization of the NSAID into cells when compared to a similar pharmaceutical composition comprising an unconjugated NSAID, or when compared to a conjugate of the invention lacking a pharma- ceutically acceptable carrier, i.e., not formulated in a pharmaceutical composition (also referred to herein as a "control"). In some embodiments, the conjugate of the invention has an increased ability to internalize the NSAID into cells when compared to an unconjugated NSAID. In some embodiments, the conjugate of the invention has an increased bioavailability when compared to an unconjugated NSAID. In some embodiments, the conjugate of the invention has an increased specificity or therapeutic effect of treating a disease or condition disclosed herein when compared to a control. In some embodiments, the pharmaceutical composition is for increasing any one of the bioavailability, specificity and / or therapeutic effect of the NSAID when compared to a similar pharmaceutical composition comprising an unconjugated NSAID, or when compared to a conjugate of the invention lacking a pharma- ceutically acceptable carrier, i.e., not formulated in a pharmaceutical composition. In some embodiments, the cell is a human cell. In some embodiments, the cell is a pancreatic tissue cell. In some embodiments, the cell is or comprises an endocrine cell. In some embodiments, the cells are or comprise alpha cells, beta cells, delta cells, epsilon cells, gamma cells, or any combination thereof.

[0078] In some embodiments, the terms "increased" and "enhanced" are used interchangeably herein and refer to an enhancement of at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 2-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, including any ranges therebetween, when compared to a control.

[0079] In some embodiments, the pharmaceutical compositions and / or conjugates of the invention are for use in preventing or treating a disease or disorder in a subject in need thereof. In some embodiments, the disease or disorder comprises inflammation. In some embodiments, the pharmaceutical composition is for use in preventing or treating a disease or disorder associated with inflammation. In some embodiments, the inflammation comprises neuroinflammation. In some embodiments, the pharmaceutical composition is for use in preventing or treating a neuroinflammatory disorder. In some embodiments, the inflammation comprises a peripheral inflammatory disease.

[0080] In some embodiments, non-limiting examples of peripheral inflammatory diseases include, but are not limited to, pancreatitis (such as acute pancreatitis, chronic pancreatitis, and autoimmune pancreatitis), arthritis, atherosclerosis, asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS) (such as viral and / or bacterial infection-associated ARDS, toxin-associated ARDS, sepsis-associated ARDS, etc.), thromboembolic diseases, chronic dermatitis, chronic hepatitis, liver cirrhosis, intestinal inflammatory disorders, inflammatory bowel disease (IBD), colitis, and Crohn's disease, or any combination thereof.

[0081] In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of pancreatitis. In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of a disease or disorder associated with pancreatitis. In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of a disease or disorder selected from the group including acute pancreatitis, chronic pancreatitis, autoimmune pancreatitis, and post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis, or any combination thereof.

[0082] In some embodiments, the disease or disorder is associated with abnormal (e.g., enhanced) enzyme activity in at least one cell or in a subject when compared to the same enzyme activity in a healthy cell or in a healthy subject. In some embodiments, the abnormal enzyme activity comprises enhanced cyclooxygenase (COX) activity, enhanced heparinase (Hep) activity, or both. In some embodiments, the conjugates of the invention are capable of inducing inhibition or reduction of COX activity and / or Hep activity in a cell or in a subject (e.g., in a peripheral organ of a subject). In some embodiments, the abnormal (e.g., enhanced) Hep activity is associated with enhanced lipase and / or amylase levels in a sample from a subject.

[0083] In some embodiments, the disease or disorder is selected from brain inflammation, abnormal (e.g., increased) blood clotting, pain, and fever, or any combination thereof. In some embodiments, the pharmaceutical composition is for use in preventing or treating brain inflammation. In some embodiments, the pharmaceutical composition is for use in preventing or treating a disease or disorder associated with brain inflammation. In some embodiments, the pharmaceutical composition is for use in preventing or treating pain (e.g., headache, migraine, etc.). In some embodiments, the pharmaceutical composition is for use in preventing or treating fever. In some embodiments, the pharmaceutical composition is for use in preventing or treating abnormal blood clotting.

[0084] In some embodiments, the pharmaceutical composition is for use in inhibiting or reducing enzyme activity in a subject in need thereof. In some embodiments, the enzyme activity comprises cyclooxygenase (COX) activity, heparinase (Hep) activity, or both. In some embodiments, the pharmaceutical composition is for use in inhibiting or reducing COX activity and / or Hep activity in a peripheral organ of the subject.

[0085] In some embodiments, the pharmaceutical compositions / compounds of the invention are for use in inhibiting or reducing cyclooxygenase (COX) activity and / or Hep activity in the brain of a subject in need thereof. In some embodiments, reducing COX activity includes reducing COX-1, COX-2 or both activity. In some embodiments, the pharmaceutical compositions / compounds are for selective inhibition or reduction of COX-1 or COX-2 activity. In some embodiments, the pharmaceutical compositions / compounds are for selective inhibition or reduction of Hep activity. In some embodiments, selective is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% selective.

[0086] In some embodiments, the pharmaceutical composition is for use in preventing or treating a disease or disorder associated with abnormal COX and / or Hep activity in a subject. In some embodiments, the pharmaceutical composition is for use in preventing or treating inflammation and / or pain associated with abnormal COX and / or Hep activity in a subject. In some embodiments, the abnormal activity comprises an increase in enzyme activity, where the increase is a % increase of at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 200%, at least 1000% or more, including any ranges therebetween.

[0087] In some embodiments, the pharmaceutical composition is for delivery of a conjugate of the present invention to the brain of a subject in need thereof. In some embodiments, the pharmaceutical composition is for delivery of an NSAID to the brain of a subject in need thereof. Without being limited to any theory or mechanism, it is hypothesized that trehalose induces or enhances the permeability of the NSAID across the blood-brain barrier (BBB).

[0088] In some embodiments, trehalose induces or increases BBB permeability and / or accumulation of NSAIDs.

[0089] In some embodiments, the pharmaceutical composition is for increasing BBB permeability of the NSAID. In some embodiments, the pharmaceutical composition is for increasing the concentration of the NSAID in the brain of a subject.

[0090] In some embodiments, the pharmaceutical compositions described herein are topical compositions. In some embodiments, the pharmaceutical compositions are oral compositions. In some embodiments, the pharmaceutical compositions are injectable compositions. In some embodiments, the pharmaceutical compositions are for systemic use.

[0091] In some embodiments, the pharmaceutical composition comprising the compound of the present invention is in unit dosage form. As used herein, the term "unit dosage" means that each unit contains the compound in an amount equivalent to a dose in the range of 0.5-5 mg / kg, 0.5-1.5, 1.2-2.6, 1-5, 1-3, 2-4, 1-5, 1.25-3.75 mg / kg body weight, including any value therebetween.

[0092] In some embodiments, the unit dosage form is in the form of a tablet, capsule, lozenge, wafer, patch, ampoule, vial, or pre-filled syringe. In some embodiments, the pharmaceutical compositions are prepared by any of the methods well known in the art of pharmacy.

[0093] In some embodiments, in vitro assays can optionally be used to help identify optimal dosage ranges. The precise dose to be used in the formulation will depend on the route of administration and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves from in vitro or in vivo animal model test bioassays or systems. In some embodiments, the effective dose is determined as described herein above.

[0094] In another embodiment, the pharmaceutical compositions of this invention are administered in any conventional oral, parenteral, or transdermal dosage form.

[0095] As used herein, the terms "administering," "administration," and the like refer to any method of delivering a composition containing an active agent to a subject in sound medical practice in a manner that produces a therapeutic effect.

[0096] In some embodiments, the pharmaceutical compositions are administered via oral (i.e. enteral), rectal, vaginal, topical, nasal, ocular, transdermal, subcutaneous, intramuscular, intraperitoneal, or intravenous routes of administration. In some embodiments, the route of administration of the pharmaceutical composition depends on the disease or condition being treated.

[0097] In some embodiments, suitable administration routes include, but are not limited to, parenteral injection, such as intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other injection methods known in the art.In addition, it may be desirable to introduce the pharmaceutical composition of the present invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated, for example, by an intraventricular catheter connected to a reservoir.Pulmonary administration may also be used, for example, by using an inhaler or nebulizer.

[0098] In some embodiments, for oral application, the pharmaceutical composition is in the form of a tablet or capsule, which may contain any of the following ingredients or compounds of a similar nature: binders, such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate; or glidants, such as colloidal silicon dioxide. When the unit dosage form is a capsule, it may contain a liquid carrier, such as fatty oil, in addition to the above types of materials. In addition, the unit dosage form may contain various other materials that modify the physical form of the dosage form, such as sugar coating, shellac or other enteric agents. In some embodiments, the tablet of the present invention is further film coated. In some embodiments, the oral application of the pharmaceutical composition or kit is in the form of a drinkable liquid. In some embodiments, the oral application of the pharmaceutical composition or kit is in the form of an edible product.

[0099] For the purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be used, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent may first be rendered isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.

[0100] In some embodiments, the method of the present invention includes a preliminary step of selecting a subject suitable for treatment with the inventive compound, the preliminary step being performed before the administration step. In some embodiments, the preliminary step includes determining whether the subject has abnormal (e.g., elevated) cyclooxygenase (COX) activity, abnormal (e.g., increased) heparinase (Hep) activity, or both, where abnormal is compared to a healthy subject. In some embodiments, the subject suitable for treatment has abnormal (e.g., enhanced) COX and / or Hep activity, and / or COX and / or Hep expression. In some embodiments, the abnormal activity / expression can be determined by standard detection methods, for example, by measuring COX and / or Hep expression / activity in a sample from the subject (e.g., a blood sample, a tissue sample, etc.). In some embodiments, the abnormal Hep activity / expression is associated with enhanced lipase and / or amylase levels in a sample from the subject. In some embodiments, aberrant Hep activity / expression is determined by determining lipase and / or amylase concentrations in a sample from the subject, where increased lipase and / or amylase concentrations compared to healthy subjects are indicative of aberrant Hep activity / expression.

[0101] In some embodiments, the subject suffers from an inflammatory disease. In some embodiments, the inflammatory disease comprises brain inflammation. In some embodiments, the subject suffers from pain (such as a headache or migraine). In some embodiments, the subject suffers from a neurological disorder (such as epilepsy). In some embodiments, the subject suffers from epilepsy.

[0102] In some embodiments, the subject is a mammal. In some embodiments, the subject is a laboratory animal. In some embodiments, the subject is a pet. In some embodiments, the subject is a rodent. In some embodiments, the subject is a livestock animal. In some embodiments, the subject is a human subject.

[0103] In another aspect, there is a method comprising administering an effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In some embodiments, the method is for inhibiting or reducing COX activity and / or Hep activity in the brain in a subject in need thereof. In some embodiments, the COX / Hep activity is as described herein.

[0104] In some embodiments, the effective amount is equivalent to a body weight dose of a compound of the invention in the range of 0.5-5 mg / kg, 0.5-1.5, 1.2-2.6, 1-5, 1-3, 2-4, 1-5, 1.25-3.75 mg / kg body weight, including any value therebetween.

[0105] In some embodiments, the method is for increasing the BBB permeability of NSAID.In some embodiments, the method is for increasing the concentration of NSAID in the brain of a subject.In some embodiments, the method is for increasing the cellular internalization of NSAID.In some embodiments, the method is for increasing the bioavailability of NSAID.

[0106] In some embodiments, the method is for preventing or treating brain inflammation. In some embodiments, the method is for preventing or treating a disease associated with brain inflammation. In some embodiments, the method is for preventing or treating pain (e.g., headache, migraine, etc.). In some embodiments, the method is for preventing or treating fever. In some embodiments, the method is for preventing or treating platelet adhesion (preventing thrombus formation). In some embodiments, the method further comprises the primary step of determining the suitability of a subject for treatment with the complex / pharmaceutical composition of the present invention, the preliminary steps being as described herein above.

[0107] General As used herein, the term "treatment" or "treating" of a disease, disorder, or condition includes alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, a composition useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement to the quality of life of the patient or subject.

[0108] As used herein, the term "prevention" of disease, disorder or condition includes delay, prevention, suppression or inhibition of the onset of disease, disorder or condition. When used according to the subject matter described herein, the term "prevention" refers to a prophylactic process in which a subject is exposed to the active ingredient described herein before the induction or onset of the disease / disorder process. This can be done when an individual has a genetic lineage that shows a predisposition to the occurrence of the disease / disorder to be prevented. For example, this can be the case for an individual whose ancestors show a predisposition to a certain type of inflammatory disorder.

[0109] The term "suppression" is used to describe a state in which the disease / disorder process has already begun, but overt symptoms of the condition have not yet been noticed. Thus, an individual's cells may have the disease / disorder, but the outward signs of the disease / disorder have not yet been clinically recognized. In either case, the term prophylaxis may be applied to encompass both prevention and suppression.

[0110] Conversely, the term "treatment" refers to the clinical application of an active agent to treat an already existing condition, whose clinical symptoms are already noticeable in the patient.

[0111] In the discussion, unless otherwise indicated, it is understood that adjectives such as "substantially" and "about" modifying a condition or relationship characteristic of one or more properties of an embodiment of the invention are meant to define that the condition or characteristic is within an acceptable tolerance for the operation of the embodiment for its intended application. Unless otherwise indicated, the term "or" in this specification and claims is deemed to be an inclusive "or" rather than an exclusive "or" or to indicate at least one or any combination of the items it conjugates.

[0112] The terms "a" and "an," as used above or elsewhere herein, should be understood to refer to "one or more" of the listed components. It will be apparent to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Thus, the terms "a," "an," and "at least one" are used interchangeably in this application.

[0113] For a better understanding of the present teachings, and without in any way limiting the scope of the present teachings, all numbers expressing quantities, percentages or proportions and other numerical values ​​used in the specification and claims, unless otherwise indicated, should be understood to be modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, each numerical parameter should be construed, at least in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0114] In the description and claims of this application, the verbs "comprise," "include," and "have," and their conjugations, are used to indicate that the object(s) of the verb is not necessarily an exhaustive list of components, elements, or parts of the object(s) of the verb.

[0115] Other terms, as used herein, are meant to be defined by their well-known meaning in the art.

[0116] Unless specifically stated or clear from the context, as used herein, the term "or" is understood to be inclusive.

[0117] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" refer to the inclusion of any recited integer or group of integers but not to the exclusion of any other integer or group of integers.

[0118] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of" as used throughout this specification and claims indicates the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that does not materially change the basic or novel characteristics of the specified method, structure or composition.

[0119] As used herein, the terms "comprises," "comprising," "containing," "having," and the like can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially of," and the like have the meaning set forth in U.S. Patent Law, which terms are open-ended and allow for the presence of more than what is recited, but exclude prior art embodiments, so long as the basic or novel characteristics of the recited are not altered by the presence of more than what is recited. In one embodiment, the terms "comprises," "comprising," and "having" are interchangeable with "consisting."

[0120] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0121] Working Example Materials and Methods Animals. Studies utilized wild-type (WT) BALB / c mice and heparanase transgenic (Hpa-Tg) mice in which the human heparanase gene is driven by a constitutive β-actin promoter (19) in a BALB / c genetic background. Animals were fed standard mouse chow and tap water ad libitum. All experiments were approved and performed in accordance with the Technion's guidelines of the Committee for the Supervision of Animal Experiments.

[0122] Induction of acute pancreatitis. Mice were injected with either caerulein (intraperitoneally, 50 mg / kg, 5 times with 1 hour intervals) (Sigma-Aldrich) or saline (0.9% NaCl) (control group). Additional groups of mice were pretreated with aspirin (250 mg / kg, SC) and trehalose (2000 mg / kg, ip), either alone or in combination. Additional groups of animals were pre- and post-treated with aspirin (10 mg / mouse). In the next step, we tested the pancreatic protective effect of indomethacin and diclofenac (16 and 32 mg / kg ip), alone or in combination with trehalose. Finally, we tested the efficacy of "Indose" and "Diclose" at various concentrations against AP. Mice were sacrificed 24 hours later, and serum samples and pancreatic tissues were collected for measurement of blood amylase and lipase levels, pancreatic index (pancreas weight / body weight ratio), and for histological analysis. Portions of pancreatic tissue were also homogenized, and lysate samples were subjected to immunoblotting.

[0123] Light microscopy. Pancreatic tissue samples were fixed in 10% neutral buffered formalin, dehydrated in graded alcohols, and embedded in paraffin. Five-micron sections were stained with hematoxylin and eosin (H&E).

[0124] Electron Microscopy Tissue samples were stabilized in glutaraldehyde 2% and osmium tetroxide (OsO4) 4%, subsequently dehydrated in graded ethanol, embedded in a commercial resin medium, and stained with uracil acetate and lead nitrate to achieve better image contrast for further examination in a JEOL 1011 transmission electron microscope.

[0125] Statistical analysis. Data are presented as the mean ± standard error of the mean (SEM) of repeated measures. After testing for equal variance, comparisons between two parameter groups were performed using an unpaired Student's t-test. A repeated measures one-way analysis of variance (ANOVA) test was used to test more than two paired groups, followed by Tukey's post-hoc test for multiple comparisons.

[0126] Example 1 The present inventors have synthesized various NSAID-trehalose conjugates according to the synthetic procedures disclosed herein below.

[0127] Aspirin, diclofenac sodium salt, D+trehalose dihydrate, indomethacin, N,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, hydroxybenotriazole, dimethylformamide and silica gel 230-400 mesh were purchased from Sigma Aldrich.

[0128] Asprilose The inventors have tested various trehalose:aspirin ratios to obtain the desired mono- and / or diesters in acceptable yields. Based on these experiments, a 2:1 trehalose:aspirin ratio was selected. The synthesis of asprylose has been carried out as follows:

[0129] Aspirin (acetylsalicylic acid) (1.8 gr (10 mmole)), N,N-dicyclohexylcarbodiimide (2.06 gr (10 mmole)), 4-dimethylaminoprylidine (1.22 gr (10 mmole)), hydroxybenotriazole (1.35 gr (50 mmole)) and trehalose (6.84 gr (20 mmole)) were dissolved in dimethylformamide (20 ml) and stirred at room temperature for 1-10 hours. The precipitated dicyclohexylurea was removed by filtration. The solvent was removed by evaporation (Rotavap). The resulting asprylose (mixture of mono- and diesters) was purified on a reversed phase silica gel C-18 chromatography column using a gradient of the mobile phase composed of a mixture of water and water-miscible organic solvents (e.g. methanol, ethanol, acetonitrile, ethyl acetate).

[0130] The chemical structures of the purified mono- and diesters were verified by LC-MS and NMR.

[0131] Diclose and Indose Diclose and Indose have been synthesized following procedures similar to those described herein below.

[0132] The reaction was carried out in a 100 ml three-neck flask equipped with a thermometer, magnetic stirrer and a condenser with a nitrogen bubbler.

[0133] Indomethacin (3.57 gr (10 mmole) or 2.95 g Diclofena (10 mmole) was added to the reaction flask followed by DCC (2.06 gr (10 mmole), 4-DIMAP (1.22 gr (10 mmole)), HOBT (1.35 gr (10 mmole)) and Trehalose (6.84 gr (20 mmole). 25 ml DMF was added and stirred at room temperature for 24 hours. Precipitated Dicyclohexylurea was removed by filtration and washed with 5 ml DMF. Solvent was removed by evaporation (Rotavap) using high vacuum at 45° C. and 1 mm Hg.

[0134] The oily residue was dissolved in methanol and purified using silica gel 230-400 mesh column chromatography using a gradient of 5 to 25% methanol in ethyl acetate as the eluent.

[0135] Trehalose-indomethacin conjugate (Indose) and trehalose-dichlorofenac conjugate (Diclose) were obtained as pale yellow solids in 32% and 15% yields, respectively.

[0136] Analytical and structural analysis Trehalose-Indomethacin (Indose): HRMS: (m / z:): C 13 H 36 NO 14 M+Na calculated for ClNa, 704.1722, found: 704.1727. 1HNMR(400MHz,CD3OD),δ,ppm7.71(dd,J1=8Hz,J2=4Hz,2H Ar),7.58(dd,J1=8Hz,J2=4Hz,2H,Ar),7.01(dd,J1=4Hz,1H Ar),6.90(d,J=8Hz,1H Ar),6.67(dd,J1=8Hz,J2=4Hz,1H,Ar),5.0(d,J=4Hz,1H),4.42(dd,j1=12Hz,J2=4Hz,1H),4. 23(dd,J1=8Hz,J2=4Hz,1H),4.0-3.9(M,1H),3.82(S,3H),3.77(t,J=4Hz,1H),3.76(S,2H),3. 72(dd,J1=8Hz,J2=4Hz,1H),3.65(dd,J1=12Hz,J2=8Hz,1H),3.38(dd,J1=12Hz,J2=4Hz,1H),3 .27(dd,J1=8Hz,J2=4Hz,1H),3.27(dd,J1=8Hz,J2=4Hz,1H),3.21(t,J=8Hz,1H),2.33(S,3H).

[0137] 13C NMR:(400MHz,CD3OD),δ ppm:172.9,170,157.7,140.3,137.2,135.8,132.6,132.4,132.2,130.4,116.1,114.3,112.7,105 .8,102.9,95.3,95.15,74.8,74.5,74,73.2,72.0,71.9,71.5,56.3,30.95,17.7,14.6,13.8,13.6.

[0138] Trehalose-Dichlorofenac (Diclose): HRMS: (m / z): C 26 H 31 NO 12 Calculated for Cl2Na: 642.1121, measured: 642.1143 1HNMR(400MHz,DMSO-d6),δ,ppm:7.70-7.76(m,4H,Ar),7.02(d,J=4Hz,1H,Ar),6. 90(d,J=8Hz,1H,Ar),6.71(dd,J1=12Hz,J2=4Hz,Ar),5.06(d,J=8Hz,1H),4.95(br S 1H),4.85(d,J=8Hz,1H),4.86(t,J=8Hz,3H),4.68(dd,J1=12Hz,J2=8Hz,2H),4.35(t,J=4Hz,1H),4.27(d,J=12H) z,1H),4.08(dd,J1=12Hz,J2=4Hz,1H),3.94-3.91(m,1H),3.66-3.63(m,1H),3.6-3.4(m,5H),3.25-3.15(m,4H).

[0139] The reaction was followed by TLC using 25% methanol in ethyl acetate. The ester has an aromatic ring with strong absorption in the UV range, therefore it was detected using a UV lamp. The trehalose derivatives of diclofenac and indomethacin were also detected using an anisaldehyde-ethanolic sulfuric acid spray. A green spot was obtained on the TLC plate at 120°C.

[0140] Example 2 Previous studies have highlighted the strong involvement of heparanase in the pathogenesis of inflammatory diseases, including AP. Specifically, the present inventors demonstrated that pancreatic heparanase expression and activity were significantly increased after caerulein-induced AP. Furthermore, pancreatic edema and inflammation, as well as the induction of cytokines and signaling molecules after caerulein administration, were significantly attenuated by selective heparanase inhibitors PG545 and SST0001, suggesting that heparanase plays an important role in AP.

[0141] Notably, all of the above characteristics appear to be even more evident in transgenic mice overexpressing heparanase, suggesting that these mice may serve as a sensitive model system to elucidate the molecular mechanisms by which heparanase functions in AP.

[0142] Heparanase expression and activity in induced acute pancreatitis (AP). Serum levels of amylase and lipase typical of AP were substantially (4-fold) increased in WT mice treated with caerulein (Figure 1), and an additional approximately 7-fold higher induction of lipase and amylase levels was observed in Hpa-Tg mice after caerulein treatment (Figure 1). Furthermore, AP was accompanied by pancreatic edema, as evidenced by an increase in pancreatic index (% pancreas weight / body weight) (Figure 1). Notably, the elevation of biochemical parameters and the increase in pancreatic edema were significantly reduced by the heparanase inhibitors PG545 and Roneparstat in WT and Hpa-Tg mice (Figure 1), together suggesting that heparanase plays a substantial role in AP.

[0143] Histological analysis. To further investigate the involvement of heparanase in the pathogenesis of AP, pancreatic morphology was evaluated in WT and Hpa-Tg mice by histopathological and electron microscopic analysis. Treatment with caerulein resulted in typical edema in WT mice, and even more severe edema was observed in Hpa-Tg mice exposed to caerulein (Figure 2). Notably, tissue edema was significantly reversed in mice treated with PG545 and SST0001 (Figure 2), further supporting the idea that heparanase is involved in AP.

[0144] In the following steps, we investigate the effect of Asprilose as pre-treatment and post-treatment in AP, and the results are presented in Figures 3 and 4. As can be seen, administration of Asprilose (monomer or dimer) as either early or post-treatment exerts pancreatic protective effect against AP. The beneficial effect of Asprilose was also evident at the histological level (Figure 3). It is assumed that this novel compound may be effective as a novel therapeutic agent for the treatment of AP.

[0145] Furthermore, we investigated the pancreatic protective effects of Indose and Diclose in AP. The newly synthesized compounds, Indose and Diclose, were well tolerated and effective against AP in WT and Hpa-Tg mice, reducing the severity of AP at doses of 16 and 32 mg / kg (see Figures 5 and 6, respectively).

[0146] Furthermore, the inventors investigated the acute toxicity of the compounds Indose and Diclose. A series of doses: 8 mg / kg, 32 mg / kg, 64 mg / kg, 128 mg / kg, 256 mg / kg, 512 mg / kg, 768 mg / kg, 1024 mg / kg, 1100 mg / kg and 1200 mg / kg were injected by intraperitoneal injection in the presence of sterile water as a carrier. Groups of 5 wild-type mice were injected with each dose. Indose showed an LD50 of 900 mg / kg and Diclose had an LD50 of 780 mg / kg. At a dose of 500 mg / kg, no mortality was observed for both Indose and Diclose, indicating low toxicity of both compounds.

[0147] To this end, the novel pharmaceutical compositions of the present invention have shown significant efficacy in vivo with negligible toxicity. It is therefore envisioned that the pharmaceutical compositions or compounds of the present invention may provide novel therapeutic strategies for the treatment of the diseases / conditions disclosed herein.

[0148] Although the present invention has been described in detail, those skilled in the art will recognize that many variations and modifications may be made. Accordingly, the present invention should not be construed as being limited to the specifically described embodiments, and the scope and spirit of the present invention will be more readily understood by reference to the following claims.

Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of a complex and a pharmaceutically acceptable carrier, wherein the complex comprises a disaccharide covalently bonded to a nonsteroidal anti-inflammatory drug (NSAID), Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the covalent bond is mediated by an ester bond between the carboxyl group of the NSAID and the hydroxyl group of the disaccharide.

3. The pharmaceutical composition according to claim 1 or 2, wherein the NSAID comprises one or more compounds selected from aspirin, diclofenac, indomethacin, naproxen, diflunisal, sarsalate, ibuprofen, mefenamic acid, meclofenamic acid, clonixin, lycopherone, and COX-2 inhibitors, the NSAID comprising any pharmaceutically acceptable salt, pharmaceutically active derivative, or combination thereof.

4. The pharmaceutical composition according to claim 1, wherein the disaccharide is trehalose.

5. The pharmaceutical composition according to claim 1, wherein the compound is the compound described in claim 1; or the NSAID is selected from aspirin, diclofenac, and indomethacin.

6. The pharmaceutical composition according to claim 1, for use in preventing or treating a disease or disorder in a subject requiring it.

7. The pharmaceutical composition according to claim 6, wherein the disease or disorder is inflammation, pain, or both, or comprises inflammation, pain, or both.

8. The pharmaceutical composition according to claim 6, wherein the disease or disorder is pancreatitis or includes pancreatitis.

9. The pharmaceutical composition according to claim 1 for use in inhibiting and / or reducing cyclooxygenase (COX) activity, heparinase (Hep) activity, or both, in a subject requiring such inhibition.

10. The pharmaceutical composition according to claim 6, wherein the disease or disorder is related to COX activity, Hep activity, or both.

11. This includes trehalose covalently bound to a nonsteroidal anti-inflammatory drug (NSAID) selected from the group including diclofenac, naproxen, diflunisal, sarsalat, ibuprofen, indomethacin, mefenamic acid, meclofenamic acid, clonixin, lycopherone, or combinations thereof. Composite.

12. The composite according to claim 11, wherein the trehalose is covalently bonded to the carboxyl group of the NSAID.

13. The composite according to claim 12, wherein the covalent bond is mediated by a bond selected from esters, amides, thioesters, carbamates, carbonates, carbamides, thiocarbamates, phosphonates, phosphodiesters, sulfonic acid esters, or any combination thereof.

14. The composite according to claim 11, wherein the trehalose is covalently bonded via a hydroxyl group at the 2-position, the 6-position, or both.

15. Formula 1: 【Chemistry 1】 Or formula 1a: 【Chemistry 2】 A compound according to claim 11, represented by the formula, wherein each R independently comprises decarboxylated diclofenac, decarboxylated indomethacin, decarboxylated naproxen, decarboxylated diflunisal, decarboxylated sarsalate, decarboxylated paracetamol, decarboxylated ibuprofen, decarboxylated mefenamic acid, decarboxylated meclofenamic acid, decarboxylated clonixin, or decarboxylated lycopherone, each X or X １ A composite substance containing O, S, or NH independently. 【Request Item 16】 【Chemistry 3】 【Chemistry 4】 The composite material according to claim 11, comprising any of the following:

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