L-arginine formulations and methods of managing pain

Abstract

Disclosed herein are compositions and methods for managing pain using L-arginine. In certain embodiments, this disclosure relates to pharmaceutical formulations comprising L-arginine and a biopterin such as tetrahydrobiopterin. In certain embodiments, the pharmaceutical composition further comprises acetaminophen and / or N-acetylcysteine. In certain embodiments, the L-arginine formulation is an aqueous liquid suitable for intravenous injection which increases endogenous formation of kyotorphin.

Description

[0001] L-ARGININE FORMULATIONS AND METHODS OF MANAGING PAIN

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 519,610 filed August 15, 2023 and U.S. Provisional Application No. 63 / 519,938 filed August 16, 2023. The entirety of each of these applications is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under HL122557 and AT009893 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] BACKGROUND

[0007] Painful episodes of vaso-occlusion are the leading cause of hospitalizations and emergency department visits in sickle cell disease patients. Sickle cell disease (SCD) is an autosomal recessive disorder of the P-globin gene in which hemoglobin-S polymerizes in erythrocytes under deoxygenated conditions causing occlusion of the small blood vessels. Pain is the clinical hallmark of SCD with painful vaso-occlusive episodes (VOEs). It is when the pain becomes acutely worse, and unbearable, that they present to the emergency department. Symptomatic relief with analgesics and hydration is a standard treatment. SCD patients often have elevated liver enzymes which prevents the use of aspirin. Extended use of acetaminophen may contribute to “asthma-like” symptoms. Thus, acute vaso-occlusive painful episodes associated with sickle cell disease (SCD) are frequently treated with morphine. SCD often fail to achieve adequate analgesia with standard doses of morphine. Thus, there is a need to find improved therapeutic treatments.

[0008] Morris et al report arginine therapy as a strategy to induce nitric oxide production in sickle cell disease. British J Haematol, 2000, 111, 498-500.

[0009] Morris et al. report a trial of arginine therapy for the treatment of children with sickle cell disease hospitalized with vaso-occlusive pain episodes. Hematol Oncol Clin N Am, 2014, 28 301— 321.

[0010] Rondon et al. report L-arginine supplementation prevents allodynia and hyperalgesia in painful diabetic neuropathic rats. Eur J Nutr, 2018, 57:2353-2363. Onalo et al. report randomized control trial of oral arginine therapy for children with sickle cell anemia hospitalized for pain in Nigeria. Am J Hematol, 2021, 96(l):89-97.

[0011] Ueda reports tyrosine-arginine (kyotorphin) is an opioid analgesic dipeptide that has established the physiological significance as a neuromodulating peptide. Front Med Technol, 2021, 3:662697.

[0012] See also US Patent No. 5,922,332 and WO 1999 / 013717.

[0013] References cited herein are not an admission of prior art.

[0014] SUMMARY

[0015] Disclosed herein are compositions and methods for managing pain generally using L- arginine. In certain embodiments, this disclosure relates to pharmaceutical formulations comprising L-arginine and a biopterin such as tetrahydrobiopterin. In certain embodiments, the pharmaceutical composition further comprises acetaminophen and / or N-acetylcysteine. In certain embodiments, the L-arginine formulation is an aqueous liquid suitable for intravenous injection which increases endogenous formation of kyotorphin.

[0016] In certain embodiments, the L-arginine formulation is an aqueous liquid sufficient for intravenous or oral consumption. In certain embodiments, the L-arginine formulation is in a solid form sufficient for oral consumption.

[0017] In certain embodiments, the L-arginine formulation further comprises a non-steroidal antiinflammatory drug. In certain embodiments, the non-steroidal anti-inflammatory drug is ibuprofen or ketorolac.

[0018] In certain embodiments, the pharmaceutical composition further comprises kyotorphin.

[0019] In certain embodiments, the pharmaceutical composition further comprises morphine.

[0020] In certain embodiments, the pharmaceutical composition further comprises glutathione or liposomal glutathione.

[0021] In certain embodiments, this disclosure relates to methods of treating or preventing pain comprising administering an effective amount of L-arginine in a pharmaceutical formulation as reported herein to a human patient in need thereof. In certain embodiments, the formulation comprises L-arginine and tetrahydrobiopterin.

[0022] In certain embodiments, this disclosure relates to methods of treating or preventing pain, acute or chronic pain, neuropathic pain, traumatic pain, comprising administering an effective amount of L-arginine and a biopterin to a human patient in need thereof wherein the patient is diagnosed with acute or chronic pain, neuropathic pain, traumatic pain, or pain associated with sickle cell disease (SCD).

[0023] In certain embodiments, the patient is diagnosed with sickle cell disease (SCD). In certain embodiments, the patient is diagnosed with homozygous sickle hemoglobin [Hb-SS], thalassemia, hemoglobin S (sickle hemoglobin), or hemoglobin sickle beta zero thalassemia [Hb-SpO- thalassemia]. In certain embodiments, the subject is less than 16 or 21 years old. In certain embodiments, the patient is diagnosed with acute pain. In certain embodiments, the patient is diagnosed with chronic pain.

[0024] In certain embodiments, this disclosure relates to methods of treating or preventing traumatic pain, fractures / orthopedic pain, surgical pain / post-op pain. In certain embodiments, this disclosure relates to methods of treating or preventing pain comprising administering an effective amount of a pharmaceutical composition comprising L-arginine as reported herein to a subject in need thereof prior to surgery or during surgery or after surgery.

[0025] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0026] Figure 1A shows data on the impact of intravenous arginine therapy on arginine concentration (pM) after arginine supplementation at different time points. Plasma concentration remains significantly above baseline through 8 hours for arginine.

[0027] Figure IB. shows data on the impact of intravenous arginine therapy on kyotorphin concentrations (pM) after arginine supplementation at different time points. Plasma concentrations remains significantly above baseline through 2 hours for KTP (kyotorphin).

[0028] Figure 2A shows data indicating arginine and kyotorphin levels are closely correlated in SCD patients after arginine therapy.

[0029] Figure 2B shows data indicating arginine levels are elevated in the treatment group at time of discharge suggesting therapy increases kyotorphin levels, improves mitochondrial function, and decreases pain compared to standard of care medications in patients with sickle cell disease.

[0030] Figure 3 shows data on the time-to-crises resolution and total IV opioids in SCD patients after IV arginine therapy. DETAILED DISCUSSION

[0031] In this specification and in the claims that follow reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0032] An “embodiment” of this disclosure refers to an example, and this disclosure is not necessarily limited to such example. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0033] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0034] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0035] "Consisting essentially of' or "consists of' or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps that do not materially affect the basic and novel characteristics of the compositions or methods.

[0036] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or domestic pet. In certain embodiments, the subject is a human subject of any age or a patient 2, 12, 16, or 20 years old or older. In certain embodiments, the subject is a human subject 55 or 65 years old or older. In certain embodiments, the subject is a human subject greater than 65, or 70 years of age.

[0037] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression. As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity is reduced.

[0038] The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on, for example, the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0039] As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

[0040] An “anti-inflammatory agent” refers to a compound generally recognized to be administered to reduce symptoms of inflammatory process such as discolored tissue, tissue swelling, heat, or fever. Suitable anti-inflammatory agents both steroidal and non-steroidal structures such as aspirin and acetaminophen. Additional examples of “nonsteroidal antiinflammatory drugs” or “NSAIDs” include ibuprofen, ketorolac, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, and indomethacin. Examples of “steroidal anti-inflammatory agents” include corticosteroids such as hydrocortisone, cortisol, triamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, desoximetasone, desoxycorticosterone acetate, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadr enol one, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluocinolone acetonide, fluocinonide, fluocortin butyl ester, fluocortolone, fluprednidene acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, fludrocortisone, fludrocortisone, fluocinolone, medrysone, betamethasone and the balance of its esters, chloroprednisone, dichlorisone, flunisolide, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortamate, prednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, and triamcinolone. Mixtures of the above steroidal anti-inflammatory compounds may also be used.

[0041] Formulations of L-arginine

[0042] In certain embodiments, this disclosure relates to pharmaceutical formulations comprising L-arginine and a biopterin such as tetrahydrobiopterin. In certain embodiments, the pharmaceutical composition further comprises acetaminophen and / or N-acetylcysteine. In certain embodiments, the L-arginine formulation is an aqueous liquid suitable for intravenous injection which increases endogenous formation of kyotorphin. In certain embodiments, the L-arginine formulation further comprise kyotorphin.

[0043] In certain embodiments, the L-arginine formulation is an aqueous liquid suitable for intravenous injection which increases endogenous formation of kyotorphin.

[0044] In certain embodiments, the L-arginine formulation is an aqueous liquid sufficient for intravenous or oral consumption. In certain embodiments, the formulation is an isotonic aqueous liquid optionally comprising a saccharide or polysaccharide or optionally comprising salts, e.g., such that the electrolyte content is similar to a solution of 0.9% NaCl or 5% glucose, i.e., solution that has the same osmolarity as blood plasma or approximately 310 mEq / L or between 300 and 320 mEq / L or between 290 and 330 mEq / L.

[0045] In certain embodiments, the L-arginine formulation is in a solid form sufficient for oral consumption.

[0046] In certain embodiments, the L-arginine formulation further comprises an antiinflammatory agent such as a non-steroidal anti-inflammatory drug. In certain embodiments, the non-steroidal anti-inflammatory drug is acetaminophen. In certain embodiments, the non-steroidal anti-inflammatory drug is ibuprofen or ketorolac.

[0047] In certain embodiments, the pharmaceutical composition further comprises kyotorphin.

[0048] In certain embodiments, the pharmaceutical composition further comprises morphine.

[0049] In certain embodiments, the pharmaceutical composition comprises elevated amounts of L-arginine, e.g., 200 mg or more per unit dose.

[0050] In certain embodiments, the pharmaceutical formulation comprises: i) L-arginine, e.g., at 250 mg to 20 grams per unit dose; ii) biopterin e.g., tetrahydrobiopterin, e.g., 10 to 1500 mg per unit dose iii) acetaminophen, e.g., at 50 mg to 650 mg per unit dose; and iv) N-acetyl cysteine, e.g., at 100 mg to 15 grams per unit dose.

[0051] In certain embodiments, the formulation further comprises glutathione.

[0052] In certain embodiments, the pharmaceutical formulation comprises: i) L-arginine, e.g., at 250 mg to 20 grams per unit dose; ii) biopterin e.g., tetrahydrobiopterin, e.g., at 10 to 1500 mg per unit dose, and iii) ibuprofen.

[0053] In certain embodiments, the pharmaceutical formulation comprises: i) L-arginine, e.g., at 250 mg to 20 grams per unit dose; ii) biopterin, e g., tetrahydrobiopterin, e.g., 10 to 1500 mg per unit dose iii) acetaminophen, e.g., at 50 mg to 650 mg per unit dose; iv) N-acetyl cysteine, e.g., at 100 mg to 15 grams per unit dose; and v) ibuprofen or ketorolac or other nonsteroidal anti-inflammatory drug (NSAID).

[0054] In certain embodiments, the formulation further comprises glutathione.

[0055] In certain embodiments, the pharmaceutical formulation comprises: i) L-arginine, e.g., at 250 mg to 20 grams per unit dose; ii) biopterin, e.g., tetrahydrobiopterin, e.g., 10 to 1500 mg per unit dose iii) acetaminophen, e.g., at 50 mg to 650 mg per unit dose; iv) N-acetyl cysteine, e.g., at 100 mg to 15 grams per unit dose; and v) L-citrulline e.g., 50 mg- 10 grams per unit dose.

[0056] In certain embodiments, the formulation further comprises glutathione.

[0057] In certain embodiments, the formulation comprises 7 grams of L-arginine per unit dose, 1000 mg of tetrahydrobiopterin per unit dose, 650 mg of acetaminophen per unit dose, 900 mg or N-acetyl cysteine, and 2 grams L-citrulline per unit dose.

[0058] In certain embodiments, the formulation further comprises glutathione.

[0059] In certain embodiments, the formulation is used in patients with liver dysfunction that do not tolerate acetaminophen or patients with renal dysfunction or patients that do not tolerate ibuprofen the formulation comprising: i) 250 -20 mg of L-arginine per unit dose, ii) biopterin, e.g., tetrahydrobiopterin, iii) N-acetyl cysteine, iv) L-citrulline, and v) optionally acetaminophen, kyotorphin, ketorolac, or other NSAID.

[0060] In certain embodiments, the formulation further optional comprises ingredients selected from tyrosine and / or other amino acids including glutamine, taurine, glutathione (liposomal glutathione), L-carnitine / acetyl carnitine, curcumin / turmeric, S-adenosyl-methionine, a mitochondrial cocktail (B-vitamins, alpha lipoic acid, vitamin E, vitamin C,) vitamin D, vitamins A,B,C,D,E,K (KI and K2)), alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), zinc, magnesium, calcium, sulforaphane, 5HTP (5 hy droxytryptophan) .

[0061] In certain embodiments, the formulation further optional comprises ingredients selected from vitamins and nutrients to create a nutritional complete medical food or meal replacement such as B-vitamins, methyl-folate, methylcobalamin; thiamine niacinamide, pantothenate, riboflavin, biotin, B6 / P-5-P (pyrodoxal-5-phosphate), naltrexone, choline, phosphatidylcholine, medium chain triglycerides, iron, potassium, iodine, inositol, copper, selenium, resveratrol, long chain fatty acids, esters of long chain fatty acids, alpha-lipoid acid, a carnitine, probiotics, an omega-6 fatty acid, an esters of omega-6 fatty acid, gamma-linolenic acid, ethyl gamma-linolenate, an omega-9 fatty acid, esters of omega-9 fatty acid, oleic acid, ethyl oleate, zinc, calcium, magnesium, selenium, a vitamin A, a vitamin Bi, a vitamin B2, a vitamin B3, a vitamin B5, a vitamin Be, a vitamin B7, a vitamin B9, a vitamin B12, vitamin C, vitamin D, vitamin K, S- adenosylmethionine, a phosphocholine, creatine, a coenzyme Q, taurine, tetrahydrobiopterin, methylcobalamin, betaine, pancreatic enzymes, folinic acid, pancrelipase, a leukotriene inhibitor, an arginine, glutamine, N-acetylcysteine, berberine, an anti-inflammatory agent, anti-bacterial agent, an anti-fungal agent, anti-oxidant, saccharides and polysaccharides or combinations thereof.

[0062] In certain embodiments, the formulation further comprises an omega 3 fatty acid and / or omega 6 fatty acid. In certain embodiments, the composition further comprises fatty acids. In certain embodiments, the composition comprises medium chain fatty acids with no or low amounts of long chain fatty acids e.g., less than 5% or 2% or 1% of total fatty acids by weight.

[0063] In certain embodiments, the formulation further comprises a vitamin E (e.g., alphatocopherol). In certain embodiments, the formulation further comprises one or more or all of the following: coenzyme Q-10 (ubiquinone), B-vitamins (thiamine, riboflavin, niacin, pyridoxine, cobalamin), alpha lipoic acid, vitamin C (L-ascorbic acid), and vitamin E (alpha-tocopherol).

[0064] In certain embodiments, the composition further comprises one or more or all of the following: vitamin KI (phylloquinone), carnitine, and creatine.

[0065] In certain embodiments, the formulation further comprises one or more all of the essential amino acids, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

[0066] In certain embodiments, the formulation further comprises zinc. In certain embodiments, zinc is in amount of greater than 8, 11, 15, or 20 mg. In certain embodiments, zinc is in an about of greater than 15, 20, or 30 mg. In certain embodiments, zinc is in an amount of between 30 - 50 mg.

[0067] In certain embodiments, the formulation further comprises S-adenosyl-L-methionine (SAMe).

[0068] In certain embodiments, the formulation further comprises melatonin.

[0069] In certain embodiments, the formulation further comprises glutathione.

[0070] In certain embodiments, the formulation further comprises calcium.

[0071] In certain embodiments, the formulation further comprises magnesium.

[0072] In certain embodiments, the formulation further comprises selenium.

[0073] In certain embodiments, the formulation further comprises vitamin A.

[0074] In certain embodiments, the formulation further comprises vitamin Bl.

[0075] In certain embodiments, the formulation further comprises vitamin B2.

[0076] In certain embodiments, the formulation further comprises vitamin B3.

[0077] In certain embodiments, the formulation further comprises vitamin B5.

[0078] In certain embodiments, the formulation further comprises vitamin B6.

[0079] In certain embodiments, the formulation further comprises vitamin B7.

[0080] In certain embodiments, the formulation further comprises vitamin B9.

[0081] In certain embodiments, the formulation further comprises vitamin B 12

[0082] In certain embodiments, the formulation further comprises vitamin C.

[0083] In certain embodiments, the formulation further comprises a vitamin D.

[0084] In certain embodiments, the formulation further comprises a vitamin K. In certain embodiments, the formulation further comprises glutamine.

[0085] In certain embodiments, the formulation further comprises pyridoxal-5-phosphate.

[0086] In certain embodiments, the formulation further comprises methylcobalamin.

[0087] In certain embodiments, the formulation further comprises L-methylfolate (5-MTHF).

[0088] In certain embodiments, the formulation further comprises citrulline.

[0089] In certain embodiments, the formulation further comprises citrulline and glycine.

[0090] In certain embodiments, the additional components are selected from long or medium chain fatty acids, esters of long chain fatty acids, alpha-lipoic acid, carnitine, an omega-6 fatty acid, esters of omega-6 fatty acid, gamma-linolenic acid, ethyl gamma-linolenate, an omega-9 fatty acid, esters of omega-9 fatty acid, oleic acid, ethyl oleate, zinc, calcium, magnesium, selenium, a vitamin A, a vitamin Bi, a vitamin B2, a vitamin B3, a vitamin B5, a vitamin Be, a vitamin B7, a vitamin B9, a vitamin B12, vitamin C, vitamin D, vitamin K, S-adenosylmethionine, a phosphocholine, creatine, a coenzyme Q, taurine, tetrahydrobiopterin, methylcobalamin, betaine, pancreatic enzymes, folinic acid, pancrelipase, a leukotriene inhibitor, glutamine, N- acetylcysteine, an anti-fungal agent, berberine, an anti-inflammatory agent, anti-bacterial agent, anti-oxidant, saccharides and polysaccharides or combinations thereof.

[0091] “Fatty acids” refers to a family of carboxylic acids having a saturated or unsaturated hydrocarbon chain of about 4 to about 28 carbons in length and is intended to include carboxylic acid salt forms. Medium chain fatty acids (MCFAs) refer to a family of carboxylic acids having a saturated or unsaturated hydrocarbon chain of from about 6 to 10 carbons in length and is intended to include carboxylic acid salt forms. Examples include capric acid, caprylic acid, and hexanoic acid. Medium-chain triglycerides (MCTs) are esters (tri-, di-, monoglyceride esters) of medium chain fatty acids and glycerol. Natural sources of MCFAs and MCTs include coconut, palm kernel oil, and bovine milk. Long chain fatty acids are typically between 12 and 28 carbons in length. Unsaturated fatty acids have at least one carbon-carbon double bond in the hydrocarbon chain. Unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs). Unsaturated fatty acids are designated by the position of the first double bond from the methyl end of the hydrocarbon chain. Omega-3 fatty acids have a first double bond at the third carbon from the methyl end of the chain; and include, e.g., alpha-linolenic acid (ALA) (octadeca- 9,12,15-trienoic acid), stearidonic acid (octadeca-6,9,12,15-tetraenoic acid), eicosapentaenoic acid (eicosa-5,8,11,14,17-pentaenoic acid; "EPA"), docosapentaenoic acid (docosa-7, 10,13,16,19- pentaenoic acid), eicosatetraenoic acid (eicosa-8,l l,14,17-tetraenoic acid), and docosahexaenoic acid (docosa-4,7,10,13,16,19-hexaenoic acid; "DHA"). Ethyl eicosapentaenoate, icosapent ethyl (Vascepa™) is an omega-3 polyunsaturated fatty acid (PUFA) ester composition FDA approved for the treatment of hypertriglyceridemia. Omega-6 fatty acids have a first double bond at the sixth carbon from the methyl end of the chain; and include, e.g., linoleic acid (9,12-octadecadienoic acid), gamma-linolenic acid (6,9,12-octadecatrienoic acid; GLA), eicosadienoic acid (11,14- eicosadienoic acid), dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid), arachidonic acid (5,8,11,14-eicosatetraenoic acid), docosadienoic acid (13,16-docosadienoic acid), adrenic acid (7,10,13,16-docosatetraenoic acid), docosapentaenoic acid (4,7,10,13,16-docosapentaenoic acid), and calendic acid (8E,10E,12Z-octadecatrienoic acid), and the like. Omega-9 fatty acids have a first double bond at the ninth carbon from the methyl end of the chain; and include, e.g., oleic acid (cis-9-octadecenoic acid); eicosenoic acid (cis-ll-eicosenoic acid); mead acid (all-cis-5,8, 11 - eicosatrienoic acid); erucic acid (cis-13-docosenoic acid); and nervonic acid (cis-15-tetracosenoic acid).

[0092] In certain embodiments, formulations may contain an additional pharmaceutically acceptable carrier. The composition can be in dosage unit form such as liquid, gel, tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, particles, or the like. An acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the disclosure. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.

[0093] The phrase "carrier" as used herein means a material, composition, or vehicle, such as a liquid or solid fdler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the recipient. Some examples of materials which can serve as acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) ethyl alcohol; and (17) other non-toxic compatible substances employed in formulations.

[0094] Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and the like, each containing a predetermined amount of compounds. Compositions or compounds may also be administered as a bolus, electuary, or paste.

[0095] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, powders, granules and the like), the active ingredient(s) are mixed with one or more acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fdlers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills. Compositions may also comprise buffering agents, e.g., such that the solution is around a pH of 7 or between a pH of 6.5 to 8.0. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0096] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0097] The tablets, and other solid dosage forms of the compositions, such as capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient(s) can also be in micro-encapsulated form, if appropriate, with one or more of the abovedescribed excipients.

[0098] Compositions suitable for parenteral administration may comprise one or more active compounds in combination with one or more sterile isotonic solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation aqueous pH buffered and isotonic with the blood of the intended recipient or suspending or thickening agents.

[0099] Examples of suitable nonaqueous carriers that may be employed in the compositions of the disclosure include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0100] If desired, the effective daily doses of the compounds may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present disclosure, the compounds may be administered two or three times daily. In preferred embodiments, the compounds will be administered once daily.

[0101] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the formulations.

[0102] Methods of managing pain

[0103] In certain embodiments, this disclosure relates to treating or preventing general pain such as acute or chronic pain, neuropathic pain, traumatic pain, or pain associated with sickle cell disease (SCD) or associated side effects comprising administering an effective amount of arginine, a biopterin such as tetrahydrobiopterin, and to a subject or human patient in need thereof. It is contemplated that methods of treating pain are not limited to sickle cell disease i.e., SCD is one example of a group experiencing pain who would benefit. In certain embodiments, the biopterin is biopterin, dihydrobiopterin, tetrahydrobiopterin, or combinations thereof. In certain embodiments, the patient is diagnosed with general acute or chronic pain, neuropathic pain, traumatic pain, or pain associated with sickle cell disease (SCD). In certain embodiments, the patient is diagnosed with acute pain. In certain embodiments, the patient is diagnosed with chronic pain. In certain embodiments, the subject is a human child diagnosed with SCD having vasoocclusive pain episodes (VOE). In certain embodiments, the patient is diagnosed with sickle cell disease (SCD). In certain embodiments, the patient is diagnosed with homozygous sickle hemoglobin [Hb-SS] or hemoglobin sickle beta zero thalassemia [Hb-SpO-thalassemia], In certain embodiments, this disclosure relates to methods of treating or preventing traumatic pain, fractures / orthopedic pain, or surgical pain / post-op pain. In certain embodiments, this disclosure relates to methods of treating or preventing pain comprising administering an effective amount of a L-arginine and a biopterin and optionally other agents reported herein prior to surgery or during surgery or after surgery.

[0104] In certain embodiments, this disclosure relates to treating or preventing pain associated with sickle cell disease comprising administering an effective amount of L-arginine and a biopterin to a human patient in need thereof.

[0105] In certain embodiments, L-arginine is administered to the subject intravenously at 200 mg / kg or greater. In certain embodiments, L-arginine is administered intravenously at 300 mg / kg or greater. In certain embodiments, L-arginine is administered intravenously at 400 mg / kg or greater. In certain embodiments, L-arginine is administered intravenously at 500 mg / kg or greater.

[0106] In certain embodiments, the biopterin is tetrahydrobiopterin. In certain embodiments, the formulation further comprises glutathione or / and liposomal glutathione.

[0107] In certain embodiments, methods further comprise administering L-arginine and the biopterin in combination with acetaminophen or N-acetylcysteine to the subject.

[0108] In certain embodiments, methods further comprise administering L-arginine and the biopterin in combination with morphine to the subject.

[0109] In certain embodiments, methods include administration of L-arginine and the biopterin in a single pharmaceutical composition wherein the pharmaceutical composition further optionally comprises acetaminophen and / or N-acetylcysteine. In certain embodiments, the formulation further comprises glutathione.

[0110] In certain embodiments, methods include administration of L-arginine and the biopterin in combination with morphine (co-admini strati on) at lower doses. In certain embodiments, the subject is administered less than 20 mg of morphine every 12 hours. In certain embodiments, the subject is administered less than 15 mg of morphine every 12 hours. In certain embodiments, the subject is administered less than 10 mg of morphine every 12 hours. In certain embodiments, the subject is administered less than 5 mg of morphine every 12 hours. Sickle cell disease (SCD)

[0111] Low L-arginine bioavailability is associated with sickle cell disease (SCD) mortality and morbidity, including acute pain severity. Multiple phase 2 trials support the safety and efficacy of arginine therapy in children with SCD experiencing vaso-occlusive pain. Nitric oxide (NO) is a potent vasodilator depleted in SCD due to hemolysis. As the obligate substrate for NO production, mechanism-of-action of arginine is unknown but thought to be related in part to NO production. Kyotorphin (KTP) is an endogenous opioid analgesic peptide composed of two amino acids: tyrosine and arginine. Its ability to reduce nociceptive pain are believed to be due to the induction of Met-enkephalins release in the brain. Oral administration of arginine in wild-type mice increases the levels of KTP centrally. Arginine supplementation increases NO metabolites (NOx), improves mitochondrial function, decreases oxidative stress, and has opioid sparing effects in children with SCD. The association between IV arginine supplementation as a precursor to KTP in SCD is unknown and may represent a mechanism by which arginine decreases pain in patients with SCD. Safe and effective non-opioid pain medications are needed. Current standard is acetaminophen, ibuprofen, or opioids for more severe pain. Effective treatments with opioid sparing, i.e., eliminating or reducing the amounts, are needed. Thus, contemplated herein is a combination pain cocktail.

[0112] Experiments reported herein indicate arginine therapy increases kyotorphin levels in the plasma of patients with sickle cell disease experiencing pain. Arginine alone or in combination with other ingredients provides a non-toxic treatment of pain in sickle cell disease that can be applied to other acute and chronic pain syndromes. The addition of BH4 (tetrahydrobiopterin) or other biopterins supports the arginine pathway that is sometimes dysfunctional in pain syndromes. Additional ingredients contemplated in the analgesic formulations include acetaminophen and N- acetylcysteine (which may protect against the adverse effects of acetaminophen on the liver and other organs), and / or glutathione (liposomal glutathione may be added to provide stability and bioavailability), curcumin, ibuprofen, and / or S-adenosyl-methionine (which supports reduced glutathione production) and optionally exogenous kyotorphin.

[0113] Interventions that improve mitochondrial function may have added benefit to treat pain. Contemplated mitochondrial cocktails include agents such as B-vitamins, alpha lipoic acid, co- Q10, vitamin E, vitamin C, an omega 3 fatty acid, a vitamin E, other essential amino acids, SAMe, melatonin, and GSH. Examples of ingredients in a mitochondrial cocktail include active B- vitamins including methylcobalamin, L-methylfolate (5-MTHF), Bl (thiamine), B2 (riboflavin), niacin, B6 (5-P-5), biotin, pantothenic acid, alpha lipoic acid, Co-QlO, and phosphatidylcholine. Arginine (or kyotorphin) and acetaminophen / N-acetyl cysteine and ibuprofen also represents a contemplated analgesic.

[0114] Acetaminophen and Glutathione

[0115] Data indicates patients with SCD have elevated liver enzymes and have unique risks of developing asthma like vascular effects from acetaminophen. Arginine is hepatoprotective in SCD providing a benefit to using acetaminophen combination with arginine. There is evidence of asthma as an unanticipated adverse effect associated with acetaminophen. Patients with SCD and “asthma-like” symptoms, may be due to acetaminophen treatment, e.g., in acute and chronic vasoocclusive pain.

[0116] The addition of N-acetyl cysteine (NAC) to acetaminophen / Tylenol® antidote is contemplated to increase the safety of acetaminophen. Using NAC and glutathione as a pain reliever targets an unmet need and makes acetaminophen safer for patients with SCD who are vulnerable to the effects of low glutathione bioavailability. Low plasma and erythrocyte glutathione levels make sickle-red blood cells more vulnerable to hemolysis. Given acetaminophen depletes glutathione levels, the addition of NAC to an acetaminophen pain reliever creates a safer analgesic.

[0117] Although the formulations provided herein are in the context of opioid-sparing analgesics for SCD, the formulations and methods disclosed herein may be applied in the treatment of acute and chronic pain generally in the context of other diseases and conditions.

[0118] Pharmacokinetics / pharmacodynamics studies

[0119] Low arginine bioavailability is associated with vaso-occlusive pain episode (VOE) severity in sickle cell disease (SCD), with opioid-sparing effects reported in children hospitalized with SCD-VOE in national / international phase-2 randomized placebo-controlled trials (RCT). Experiments were performed to determine the role of intravenous arginine therapy for SCD-pain. Prospective double-blind RCT of intravenous arginine was conducted. Participants were between 3-21 years of age with SCD-VOE hospitalized for pain requiring parenteral opioids. Of 1,548 patients screened, 108 were randomized, with 36 in each study-arm. Participants were randomized into l-of-3 intravenous arms: Standard dose (SD): 100 mg / kg / dose L-arginine every 8 hours, loading dose: 200 mg / kg followed by SD or placebo, until discharge. Total parenteral opioid use (TPO, intravenous morphine equivalents, mg / kg) and secondary outcomes: Time-to-cri sisresolution, pain scores, patient-reported outcomes (PROs), arginine bioavailability and biomarkers of oxidative stress / mitochondrial function were evaluated.

[0120] The mean age of participants was 12.6, 52% were female, and 65% had hemoglobin-SS. TPO, time-to-crisis-resolution, pain scores and PROs at discharge were similar across arms. However, an ad hoc sensitively analysis of children 5-16 years old was performed in response to residual confounding of younger children 3-4 randomized to placebo only, while 15 / 18 older children >16 years were randomized to arginine; TPO utilization was significantly reduced in those receiving arginine vs. placebo (1.54 vs. 2.85 mg / kg, p = 0.02, n = 87). Time-to-crisis-resolution strongly correlated to TPO (r = 0.79). Plasma arginine was low at presentation in 74% of patients (mean 50 ± 28 pM) and increased significantly in those treated with arginine. Arginine bioavailability at VOE-presentation inversely correlated to time-to-crisis-resolution in the placebo arm only, an association lost after arginine therapy. A dose-dependent increase in platelet- mitochondrial activity was noted after arginine versus no change after placebo; plasma proteincarbonyl levels decreased, suggesting a decrease in oxidative stress with arginine that worsened in the placebo-group.

[0121] SCD-VOE is associated with an acquired arginine deficiency that correlates with worse clinical outcomes. Intravenous arginine improves mitochondrial function and decreases oxidative stress in patients with SCD-VOE compared to placebo. Significant opioid-sparing is noted in children of ages 5-16 years.

[0122] Impact of arginine therapy on kyotorphin in children with sickle cell disease and vasoocclusive pain

[0123] A single-center, institutional review board-approved, prospective, randomized, open-label pharmacokinetics (pK) / pharmacodynamics study of IV arginine was conducted to assess the impact of arginine therapy on plasma arginine and NOx concentrations over time. Kyotorphin assessment was a post hoc analysis. Patients with SCD (homozygous sickle hemoglobin [Hb-SS] or hemoglobin sickle beta zero thalassemia [Hb-SpO-thalassemia]) aged 7 to 21 years hospitalized for VOE requiring parenteral opioids were eligible. Exclusion criteria included hemoglobin < 5 gm / dL, hepatic / renal dysfunction, acute stroke, allergy to arginine, pregnancy, emergency department discharge, hospital discharge within the past 7 days, or previous enrollment into the study.

[0124] The research pharmacist performed blocked randomization using lists prepared by the biostatistician to randomize the patients into 1 of 3 IV arginine dosing arms: (1) 100 mg / kg every 8 hours (standard dose, n = 4); (2) loading dose (200 mg / kg) followed by standard dose (n = 5); or (3) loading dose (200 mg / kg) followed by continuous infusion (300 mg / kg per day) (n = 4). Arginine was administered over 30 minutes. Blood was obtained at 6 time points: preinfusion (time 0) and at 1, 1.5, 2, 4, and 8 hours after the initiation of the first arginine infusion, and then at about 8 AM daily until discharge or for 7 days, whichever came first. Plasma arginine, kyotorphin, and NOx levels were measured, pK / pharmacodynamics analyses were performed, including determination of arginine maximum concentration (Cmax), time to reach Cmax (Tmax), area under the curve (AUC; calculated using the trapezoid rule), rate of clearance, and half-life. Numeric pain scores were extracted from the electronic medical records. The daily highest / worst, lowest, and mean pain scores were assessed for correlations with the peak kyotorphin concentration, arginine Cmax, change in arginine concentration from baseline to discharge (pM), and peak NOx.

[0125] Sixteen patients were consented and 13 patients were randomized. Three patients were excluded: 1 with elevated creatinine and 2 with emergency department discharge. Participant demographics, clinical characteristics, and laboratory values at initial presentation (pre-dose) were recorded. Although no statistically significant differences between randomized study arms were identified, participants randomized to the standard dose arm (100 mg / kg IV every 8 hours) trended to be younger in age and had clinically relevant lower hemoglobin levels and blood biomarkers, suggesting an increased hemolytic rate that could impact arginine bioavailability. Plasma arginine and kyotorphin levels (Figure 1A) were significantly higher after arginine infusion, peaking at 1 hour, with no significant differences in the peak concentrations across the study arms. The mean plasma arginine peak for all participants was 331.6 ± 95.4 pM, 30 minutes after infusion completion (Tmax). All participant except 1 achieved peak plasma arginine levels above the Km (100-150 pM) of the cationic amino acid transporter-1 after arginine infusion. The AUC was the highest in the loading dose + continuous infusion arm. Kyotorphin levels were strongly correlated with plasma arginine concentration (Figure 2A). Plasma NOx also significantly increased from pre-dose to Tmax, returning to baseline by 8 hours. Although NOx increased primarily in those receiving an argi nine-loading dose, no correlation was found between arginine and NOx concentration, arginine Cmax and peak NOx levels, or the mean change / percent change in NOx. No significant changes in the kyotorphin-precursor tyrosine were observed. Significant inverse correlations were identified between daily pain scores and changes in plasma arginine concentration (pM) from baseline to discharge and peak kyotorphin levels on day 1 when the arginine-loading dose arms were combined. Nonsignificant inverse correlations between daily pain scores and day 1 peak kyotorphin, arginine Cmax, and peak NOx levels for all participants were also noted when standard dose was included.

[0126] There was acute increase in the plasma concentration of the opioid-like analgesic kyotorphin in patients with SCD-VOE after IV arginine infusion. Kyotorphin concentrations remained elevated for 2 hours before returning to the pre-dose baseline level by 4 hours, strongly correlating with arginine concentration. Low arginine bioavailability is associated with SCD mortality and morbidity, including acute pain and the severity of pain. Arginine is contemplated to be the rate-limiting amino acid for kyotorphin production, potentially contributing to the efficacy of arginine therapy for pain reduction. In particular, the opioid-sparing effect of arginine supplementation is not fully understood in SCD; induction of an endogenous opioid-like dipeptide, such as kyotorphin, represents a potential mechanism of analgesia that would decrease opioid utilization during VOE.

[0127] Arginine administered IV rapidly increased plasma arginine concentration 2 to 5 times above baseline at presentation for VOE, reaching a maximum concentration within 1 hour of infusion initiation, regardless of the study-dose administered. Although there was intersubject variability in the peak arginine levels achieved, the loading dose, which was double the standard dose, interestingly, did not result in a significantly higher Cmax. Although greater renal excretion or elevated metabolism of arginine are potential explanations, this observation may potentially reflect higher arginine intracellular transport in the loading dose arms, ultimately leading to changes in pharmacodynamic outcomes, such as mitochondrial function and oxidative stress, which favor the utilization of higher doses.

[0128] Kyotorphin levels correlated with arginine concentration and rapidly peaked within 1 hour of arginine infusion initiation. Although there were no significant changes in plasma tyrosine concentration after arginine infusion, there were no significant differences in peak kyotorphin levels between our loading (200 mg / kg) and the standard dose (100 mg / kg). The administration of higher doses of IV arginine may have a greater impact on kyotorphin production and, ultimately, pain relief. Studies evaluating higher doses for SCD-VOE are indicated to potentially maximize pain management, particularly in the acute care setting.

[0129] Arginine supplementation significantly increased plasma NOx levels, supporting the role of vasodilation as an additional potential mechanism of action during SCD-VOE. Hemolysis depletes tetrahydrobiopterin (BH4), an essential cofactor for both tyrosine synthesis and NO production from arginine. BH4 converts phenylalanine to tyrosine and is also a cofactor for NO synthase in the production of NO from arginine. Because BH4 is unstable, it becomes non enzymatically oxidized to dihydrobiopterin under oxidative stress, disrupting both metabolic pathways and compromising tyrosine synthesis and NO production. Particularly relevant to SCD, lack or BH4 is contemplated to disrupt both metabolic pathways compromising tyrosine synthesis and NO production, adversely impacting kyotorphin production, and potentially contributing to pain. Thus, the addition use of BH4 is contemplated.

Claims

CLAIMS1. A pharmaceutical formulation comprising L-arginine and a biopterin.

2. The pharmaceutical formulation of claim 1 further comprising acetaminophen or N- acetylcysteine.

3. The pharmaceutical formulation of claim 1 further comprising acetaminophen and N- acetylcysteine.

4. The pharmaceutical formulation of claim 1, wherein the biopterin is tetrahydrobiopterin.

5. The pharmaceutical formulation of any of claims 1-4 which is a pH buffered isotonic aqueous liquid.

6. The pharmaceutical formulation of claim 5, wherein the pH buffered isotonic aqueous liquid is sufficient for intravenous injection or oral consumption.

7. The pharmaceutical formulation of any of claims 1-4 which is in a solid form sufficient for oral consumption.

8. The pharmaceutical formulation of any of claims 1-7 further comprising a non-steroidal anti-inflammatory drug.

9. The pharmaceutical formulation of claim 8, wherein the non-steroidal anti-inflammatory drug is ibuprofen or ketorolac.

10. The pharmaceutical composition of any of claims 1-9 further comprising kyotorphin.

11. The pharmaceutical composition of any of claims 1-10 further comprising morphine.

12. The pharmaceutical composition of any of claims 1-11 further comprising glutathione or liposomal glutathione.

13. A method of treating or preventing pain comprising administering an effective amount of L-arginine and a biopterin to a human patient in need thereof as in any of claims 1-12.

14. The method of claim 13, wherein the patient is diagnosed with acute or chronic pain, neuropathic pain, traumatic pain, and / or sickle cell disease (SCD).

15. The method of claim 13, wherein L-arginine is administered intravenously at 200 mg / kg or greater.

16. The method of claim 13, wherein L-arginine is administered intravenously at 500 mg / kg or greater.

17. The method of claim 13, wherein the biopterin is tetrahydrobiopterin.

18. The method of claim 13, further comprising administering L-arginine and the biopterin in combination with acetaminophen or N-acetylcysteine to the subject.

19. The method of claim 13, further comprising administering L-arginine and the biopterin in combination with morphine to the subject.

20. A method of treating or preventing pain comprising administering an effective amount of a pharmaceutical composition as in any of claims 1-12 to a subject in need thereof prior to surgery or during surgery or after surgery.

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