WO2009023122A1

WO2009023122A1 - Cobalamin regimens for the treatment of nitrogen oxide-mediated conditions

Info

Publication number: WO2009023122A1
Application number: PCT/US2008/009479
Authority: WO
Inventors: John A. Mclane; Malcolm Morville
Original assignee: Ariston Pharmaceuticals, Inc.
Priority date: 2007-08-10
Filing date: 2008-08-07
Publication date: 2009-02-19

Abstract

The invention features methods and compositions including cobalamin for the treatment of nitrogen oxide-mediated conditions.

Description

COBALAMIN REGIMENS FOR THE TREATMENT OF NITROGEN OXIDE-MEDIATED CONDITIONS

Back-ground of the Invention

Ever since nitroglycerin was observed to induce violent and migraine- like headaches, nitrogen oxides (e.g., Nitric Oxide (NO), nitrite, and nitrate) have been implicated as early causative agents in migraine headaches (Iversen and Olesen, Cephalalgia 16:412 (1996); Olesen et al., Trends Pharmacol ScL 15:149 (1994)). Nitroglycerin is considered as an exogenous source of NO (Ignarro, Pharm Res. 6:651 (1989); Ignarro, Proc Natl Acad Sci USA 99:7816-7 (2002)). Migraine-like headaches develop shortly after an NO challenge from nitroglycerin or histamines. Endogenous NO maintains vasodilatory tone and can act as a neuronal messenger, so its role in migraine headaches may be a direct effect on cerebral blood vessels or on perivascular sensory nerves (Olesen and Jansen-Olesen, Pathol Biol (Paris) 48:648 (2000)).

Hydroxocobalamin (OHCbI) and it aqueous form, aquacobalamin, can bind directly to nitrogen oxides, such as NO, nitrite, and nitrate, to form a complex (e.g., nitrosylcobalamin or nitrocobalamin; see Roncaroli et al., Inorg Chem. 45:7869 (2006) and Kruszyna et al., J. Pharmacol. Exp. Ther. 285:665 (1998)). Although in vivo complexation has not been shown directly in humans (Brouwer et al., Blood 88:1857 (1996)), in biological systems, OHCbI has been shown to completely or partial antagonize NO-mediated events. In addition, a nitric oxidase synthetase (NOS) inhibitor has also been evaluated in clinical trials and shown to have some efficacy in reducing histamine-induced migraines (Lassen et al., Cephalalgia 23:877 (2003)).

In the previous investigation with OHCbI in migrainers (van der Kuy et al., Cephalalgia 22:265 (2002)) it was observed that after OHCbI was administered intranasally at 1000 μg daily for 3 months, there was a reduction in migraine attacks of greater than 50% in 53% of the patients. New therapies are needed to improve the treatment and prophylaxis of migraine headache and other nitrogen oxide-mediated conditions.

Summary of the Invention The invention provides for cobalamin dosing regimens for the treatment and prophylaxis of nitrogen oxide-mediated conditions. Such regimens can be used to increase the efficacy of cobalamin therapy.

In one aspect, the invention features a method of treating a nitrogen oxide-mediated condition in a subject in need thereof by administering to the subject an initial dose of cobalamin in an amount sufficient to produce a hydroxocobalamin steady state serum concentration of at least 3,000 pg/mL within a period of about 168 hours of administering the initial dose. In certain embodiments the cobalamin is administered in an amount sufficient to produce a hydroxocobalamin steady state serum concentration of at least 3,500; 4,000; 4,500; 5,000; 5,500; 6,000; 6,500; 7,500; 10,000; or 12,500 pg/mL within a period of 28 days, 3 weeks, 2 weeks, 168 hours, 144 hours, 120 hours, 96 hours, 72 hours, 48 hours, or 24 hours. In certain embodiments, the hydroxocobalamin steady state serum concentration is in the range of from 3,000 to 5,000; 5,000 to 7,500; 7,500 to 10,000; 10,000 to 12,500; 12,500 to 15,000; 15,000 to 20,0T)O; or 20,000 to 50,0000 pg/mL.

The invention further features a method of treating a nitrogen oxide- mediated condition in a subject in need thereof by (i) administering intranasally, sublingually, or bucally cobalamin in an amount from 0.5 mg to 10 mg to the subject; (ii) after step (i), measuring the serum concentration of hydroxocobalamin in the blood of the subject; and (iii) if the concentration is less than 3,000 pg/mL, repeating step (i). In certain embodiments, step (i) is repeated if the serum concentration of hydroxocobalamin is less than 3,500; 4,000; 4,500; 5,000; 5,500; 6,000; 6,500; 7,500; 10,000; or 12,500 pg/mL. In still other embodiments, the cobalamin is administered intranasally, sublingually, or bucally in the range of 0.5 to 0.75 mg, 0.75-1.5 mg, 0.5 to 1.5 mg, 1.0-2.0 mg, 1.25-2.25 mg, 1.5-2.5 mg, 1.75-2.75 mg, 1.5-3.5 mg, 2.0-3.5 mg, 2.5-3.5 mg, 3.5 to 10 mg, 5 to 10 mg, or 7.5 to 10 mg.

The invention further features a method of treating a nitrogen oxide- mediated condition in a subject in need thereof by (i) administering subcutanously, intravenously, or intramuscularly cobalamin in an amount from 50 μg to 1,000 μg to the subject; (ii) after step (i), measuring the serum concentration of hydroxocobalamin in the blood of the subject; and (iii) if the concentration is less than 3,000 pg/mL, repeating step (i). In certain embodiments, step (i) is repeated if the serum concentration of hydroxocobalamin is less than 3,500; 4,000; 4,500; 5,000; 5,500; 6,000; 6,500; 7,500; 10,000; or 12,500 pg/mL. In still other embodiments, the cobalamin is administered subcutanously, intravenously, or intramuscularly in the range of 50-400 μg, 100-600 μg, 200-800 μg, 200-1,000 μg, 300-600 μg, 400-800 μg, 500-1,000 μg, or 800-1,000 μg. The invention also features a method of treating a nitrogen oxide- mediated condition in a subject in need thereof by (i) administering orally or enterically cobalamin in an amount from 5 mg to 5,000 mg to the subject; (ii) after step (i), measuring the serum concentration of hydroxocobalamin in the blood of the subject; and (iii) if the concentration is less than 3,000 pg/mL, repeating step (i). In certain embodiments, step (i) is repeated if the serum concentration of hydroxocobalamin is less than 3,500; 4,000; 4,500; 5,000; 5,500; 6,000; 6,500; 7,500; 10,000; or 12,500 pg/mL. In still other embodiments, the cobalamin is administered orally or enterically in the range of 50-5,000 mg, 100-5,000 mg, 500-5,000 mg, 1,000-5,000 mg, 500-1,000 mg, 600-2,000 mg, 250-750 mg, 150-650 mg, or 800-3,000 mg.

In certain embodiments of any of the above methods, if the hydroxocobalamin concentration is greater than 3,000 pg/mL, then cobalamin is administered to the subject in a maintenance dose regimen thereafter. The invention also features a method of treating a nitrogen oxide- mediated condition in a subject by administering cobalamin in a loading-dose regimen. The loading-dose regimen can include: (i) an initial administration of cobalamin at an average daily dose for 2 to 28 days, followed by less than half this average daily dose thereafter; (ii) an average initial daily dose of cobalamin which is at least 200% of the average daily dose over two, three, four, or five subsequent dosing days; or (iii) administering cobalamin at a dose administered on Day 1 that is at least 200% of the dose administered on any of the next four dosing days. In certain embodiments, the loading-dose regimen includes an initial administration of cobalamin at an average daily dose for 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, or 2 to 5 days followed by less than half this average daily dose thereafter.

The loading-dose regimen described above includes the step of administering to the subject an average initial daily dose of cobalamin. Following the initial daily dose, maintenance doses of cobalamin are given to the patient to sustain a desired serum concentration of cobalamin in the subject. The maintenance doses can be administered as a low-dosage or daily regimen, including the maintenance dosing described herein. The maintenance dose can be continued for as long as is needed to treat the nitrogen oxide-mediated condition (e.g., at least 7-14 days, months, or years).

In one embodiment, the loading-dose regimen includes intranasal, buccal, or sublingual administration of cobalamin at an average daily dose of 0.5 to 10 mg for 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, or 1-7 days followed by 0.25 to 5 mg thereafter. In certain embodiments, cobalamin is administered at an average daily loading dose of between 0.75 to 8, 0.5 to 5.0, 0.5 to 3.0, 1.25 to 5, 1.35 to 8, 1.5 to 8, 1.75 to 8, 1.75 to 10, or 2.25 to 3.5 mg for 1-7 days followed by an average daily maintenance dose of between 0.25 to 1.25, 0.25 to 1.0, 0.75 to 1.25, 0.25 to 0.85, or 0.25 to 0.75 mg thereafter.

In one embodiment, the loading-dose regimen includes subcutaneous, intravenous, or intramuscular administration of cobalamin at an average daily dose of 50 to 1000 μg for 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, or 1-7 days followed by 25 to 500 μg thereafter. In certain embodiments, cobalamin is administered at an average daily loading dose of between 50 to 325, 100 to 350, 175 to 350, 175 to 300, 225 to 350, 350 to 500, or or 350 to 1000 μg for 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 1 to 3 days, 1 to 2 days, or 1-7 days followed by an average daily maintenance dose of between 25 to 400, 25 to 300, 25 to 200, 100 to 500, 200 to 500, 300 to 500, 70 to 100, 75 to 100, 50 to 85, or 50 to 75 μg thereafter.

In still another embodiment, the loading-dose regimen includes oral or enteral administration of cobalamin at an average daily dose of 5 to 5,000 mg for 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, or 1-7 days followed by 1 to 5 mg thereafter. In certain embodiments, cobalamin is administered at an average daily loading dose of between 100 to 5,000, 500 to 5,000, 500 to 4,000, 500 to 3,000, 800 to 5,000, 800 to 3,000 or 1,200 to 5,000 mg for 1-7 days followed by an average daily maintenance dose of between 1 to 100, 1 to 75, 2 to 50, 30 to 100, or 4 to 75 mg thereafter.

In yet another embodiment of the methods of the invention, the loading- dose regimen includes administration of cobalamin at least twice daily (e.g., twice daily, three times a day, or four times a day) for a period of 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, or 1-14 days followed by administration daily or less (i.e., once, twice or three times weekly) thereafter.

The invention features a method of treating a nitrogen oxide-mediated condition in a subject in need thereof, the method including (i) intranasal administration of hydroxocobalamin to the subject in a first regimen, the first regimen including an average daily dose of 2.5 to 10 mg of hydroxocobalamin per day for a period of from 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, or 3 to 28 days; and (ii) following step (i), intranasal administration of hydroxocobalamin to the subject in a second regimen, the second regimen including an average daily dose of 0.5 to 5 mg of hydroxocobalamin per day, wherein the average daily dose of the first regimen is greater than the average daily dose of the second regimen.

The invention further features a method of treating a nitrogen oxide- mediated condition in a subject in need thereof, the method including (i) subcutaneous, intravenous, or intramuscular administration of hydroxocobalamin to the subject in a first regimen, the first regimen including an average daily dose of 50 to 5,000 μg of hydroxocobalamin per day for a period of from 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, or 1 to 28 days; and (ii) following step (i), intranasal administration of hydroxocobalamin to the subject in a second regimen, the second regimen including an average daily dose of 0.5 to 5 mg of hydroxocobalamin per day. In certain embodiments, step (i) includes intravenous administration of 1,000 μg to 5,000 μg daily for 1-3 days.

In the above aspects, the invention optionally includes the step of measuring the serum concentration of hydroxocobalamin in the blood of the subject during the first regimen; and, on the basis of the concentration, continuing the first regimen or administering the second regimen.

The invention features a kit including (i) a pharmaceutical composition including cobalamin; and (ii) a label or package insert providing instructions, the instructions describing administration of the pharmaceutical composition as part of a loading-dose regimen for the treatment of a nitrogen oxide-mediated condition. In certain embodiments, the pharmaceutical composition is formulated for oral, intranasal, buccal, sublingual subcutaneous, intravenous, or intramuscular administration. The invention further features a pharmaceutical kit including (i) a pharmaceutical composition formulated for intranasal administration including hydroxocobalamin; and (ii) a label or package insert providing instructions, the instructions describing administration of the pharmaceutical composition for the treatment of migraine headache in a dosing regimen of at least twice daily for a period of 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, or 1-14 days followed by administration daily or less thereafter.

The invention further features a pharmaceutical kit including (i) a first pharmaceutical composition including hydroxocobalamin or cyanocobalamin and formulated for parenteral, sublingual, or oral administration; (ii) a second pharmaceutical composition including hydroxocobalamin and formulated for intranasal administration; and (iii) a label or package insert providing instructions describing administration of the first pharmaceutical composition at least once daily for a period of 7 to 28 days, 14 to 28 days, 7 to 21 days, 2 to 21 days, 2 to 14 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, or 1-14 days followed by administration of the second pharmaceutical composition daily or less thereafter. In certain embodiments, the kit further includes instructions for administering the first pharmaceutical composition and the second pharmaceutical composition to a subject for the treatment of migraine or any other nitric oxide-mediate condition described herein. In other embodiments, the first pharmaceutical composition includes hydroxocobalamin formulated for intravenous administration.

In any of the above methods, kits, and compositions, the administration can be intramuscular, subcutaneous, bronchial, transdermal, intranasal, sublingual, buccal, by vaginal suppository, or by anal suppository.

In any of the above methods, kits, and compositions, the cobalamin can be selected from hydroxocobalamin, cyanocobalamin, methylcobalamin, and adenosylcobalamin. Desirably, the cobalamin is hydroxocobalamin.

In any of the above methods, kits, and compositions, the nitrogen oxide- mediated condition is selected from a condition identified herein. In certain embodiments, the nitrogen oxide-mediated condition is migraine headache.

By "cobalamin" is meant a class of compounds which includes vitamin B 12 and its analogues, derivatives, and conjugates. This class of compounds includes, without limitation, cyanocobalamin (CN-CbI), hydroxocobalamin (OHCbI), aquacobalamin, adenosylcobalamin, methylcobalamin, cyanocobalamin carbanalide, cobinamide, and 5-o-methylbenzylcobalamin [(5- OmeB-za)CN-Cbl] as well as the desdimethyl, monoethylamide and the Diethylamide analogues of all of the above. Also included are the various analogues and homologues of cobinamide such as coenzyme B 12 and 5- deoxydenosylcobalamin. Other analogues include chlorocobalamin, sulfitocobalamin, nitrocobalamin, thiocyanatocobalamin, diaquocobinamide, cyanoaquacobinamide, diaquocobinamide phosphate, 8-epi-aquacobalamin, 13- epi-aquacobalamin, 8-epi-cyanocobalamin, 13-epi-cyanocobalamin or hydroxymethylbenzidazolyl-aquacobamides, hydroxymethylbenzimidazolyl- cyanocobamides, methoxybenzimidazolyl-aquacobamides, methoxybenzimidazolyl-cyanocobamides such as Co-alpha-5 hydroxymethyl-6 methylbenzimidazolyl-Co-beta aquacobamide, or benzimidazole derivatives such as 5,6-dichlorobenzimidazole, 5-hydroxybenzimidazole, trimethylbenzimidazole, as well as adenosylcyanocobalamin [(Adc) CN-CbI], cobalamin lactone, cobalamin lactam and the anilide, ethylamide, monocarboxylic and dicarboxylic acid and salt derivatives of vitamin B 12 or its analogues. Preferred derivatives of vitamin B 12 include the mono-, di- and tricarboxylic acid derivatives or the proprionamide derivatives of vitamin B 12. The singular form, "cobalamin", may mean any one or more compounds from the class of compounds recited above. The dose of non-hydroxocobalamin cobalamins necessary for loading is determined by the physiological efficacy of their being converted to OHCbI and their ability to bind nitric oxide.

The term "administration" or "administering" refers to a method of giving a dosage of a pharmaceutical composition to a mammal, where the method is, for example, intranasal, oral, enteral, buccal, intravenous, intraperitoneal, or intramuscular. The method of administration can vary depending on various factors, such as the components of the pharmaceutical composition, site of the potential or actual disease, and severity of disease.

By "loading-dose regimen" is meant a regimen for the administration of cobalamin that includes at least two administrations of cobalamin in which any of the following criteria are met: i) the average daily dose administered from the first day of treatment to day 28, 21, 14, 7, 5, or 3 is at least 200% of the average daily dose administered over the subsequent following four to fourteen days of administration; ii) the average initial daily dose is at least 200% of the average daily dose over five subsequent dosing days; or iii) the dose administered on Day 1 is at least 200% of the dose administered on any of the next four dosing days. It is recognized that the administration of the cobalamin to attain and to maintain the described serum levels may be by different routes of delivery means or methods or by combinations of different routes of administration and delivery means. Because of the differences in the bioavailability of cobalamin administered by different routes and/ or using different formulations, the amount of cobalamin administered via different dosage forms employed in a regimen must be converted, on the basis of relative bioavailability, for the purpose of assessing whether the regimen is a loading- dose regimen. For example, intramuscular, intravenous and subcutaneous administration are considered herein to be 100% bioavailable. Delivery by intranasal, buccal, sublingual, vaginal or other mucosa membranes administration is considered herein to be 5% bioavailable, or in the range of 1 to 10% bioavailable. Delivery by oral or enteric administration is considered herein to be 1% bioavailable, or in the range of 0.1 to 2% bioavailable. Accordingly, for the purposes of determining whether any given regimen is a loading dose regimen, 1 mg administered by injection is equivalent to 20 mg administered mucosally, and equivalent to 100 mg administered orally or enterically. The exact conversion factor used is determined by the relative bioavailability of the two routes of administration (or two formulations) used in the cobalamin regimen. The bioavailability by any route of administration is dependent on many factors including the formulation characteristics, the health status of the subject and the exact location of the administration or uptake. These biovailabilities can be measured using methods known in the art.

In some embodiments, the loading-dose regimen includes an average daily dose administered from the first day of treatment to 4, 6, 8, 10, 12, or 14 days which is at least 200%, 250%, 300%, 350%, 400%, 500%, 600%, or 700% of the average daily dose administered over the following four to fourteen days of administration; a regimen in which the average initial daily dose is at least 200%, 250%, 300%, 350%, 400%, 500%, 600%, or 700% of the average daily dose over two, three, four, or five subsequent dosing days; or a regimen in which the dose administered on Day 1 is at least 200%, 250%, 300%, 350%, 400%, 500%, 600%, or 700% of the dose administered on any of the next four dosing days.

By "average daily dose" is meant the administered dose, in milligrams, of cobalamin per unit time. The average daily dose is calculated from the instructed regimen. For example, oral dosing of 10 mg twice weekly is an average daily dose of 2.87 mg/day (i.e., 20 mg/7 days); intranasal dosing of 1.25 mg given three times a day for 7 days is 3.75 mg/day (26.25 mg/7 days). For regimens of indefinite intervals, the average daily dose is calculated from the average of the high and low values. For example, a regimen that calls for 15 mg over seven to ten days has an average daily dose of 1.82 mg/day ((15 mg/7 days + 15 mg/10 days)/2).

By "average initial daily dose" is meant the dose of cobalamin administered on Day 1 divided by the time until the next administration. For example, a dosing regimen that calls for administration of 10 mg of cobalamin on Day 1, followed by 1.5 mg on days 8, 10, 14, and 18, has an average initial daily dose of 1.43 mg/day (10 mg/(8-l)).

By "average daily dose over N subsequent dosing days" is meant the sum of cobalamin administered in N dosing days subsequent to the day of the initial administration divided by the time over which N+l dosing days subsequent to the initial administration are made. For example, a dosing regimen that calls for administration of 10 mg of cobalamin on Day 1, followed by 2.5 mg on days 8, 10, 14, 18, 23, and 27, has an average daily dose over three subsequent dosing days of 0.75 mg/day ((2.5 + 2.5 + 2.5)/(18-8)); an average daily dose over four subsequent dosing days of 0.66 mg/day ((2.5 + 2.5 + 2.5 + 2.5)/(23-8)); and an average daily dose over five subsequent dosing days of 0.66 mg/day ((2.5 + 2.5 + 2.5 + 2.5 + 2.5)/(27-8)). By "initial administration" is meant administration of cobalamin to a patient to whom cobalamin has not been administered in the previous 15 days. In certain embodiments, the cobalamin has not been administered in the previous 22 days, 1 month, 2 months, or 3 months. By "dose administered on Day 1" is meant the sum total of all cobalamin administered over the first 24 hours of the initial administration.

By "dosing day" is meant a day on which a cobalamin is administered to a patient to whom cobalamin has not been administered in the previous 24 hours, wherein the dose administered on a dosing day is the sum total of all cobalamin administered over a 24 hour period beginning from the first administration on this day.

As used herein, the term "treating" refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. To "prevent disease" refers to prophylactic treatment of a human patient who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disease or condition. To "treat disease" or use for "therapeutic treatment" refers to administering treatment to a patient already suffering from a disease to improve or stabilize the patient's condition. Thus, in the claims and embodiments, treating is the administration to a human patient either for therapeutic or prophylactic purposes. As used herein, "prophylaxis" and "prevention" refer to a reduction in the incidence of nitrogen oxide-mediated conditions, such as a reduction in the number of migraine headaches.

By "nitrogen oxide-mediated conditions" is meant conditions that are associated with the overproduction of nitric oxide, or its metabolites nitrate and nitrite. Examples of such conditions include, without limitation, migraine, inflammatory bowel disease, ulcerative colitis, Crohn's disease, asthma, bronchitis, arthritis, tuberculosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, pre-eclampsia, eclampsia, ischemic heart disease, cardiomyopathy, atherosclerosis, stroke, cerebral ischemia, acute myeloid leukaemia, myelodysplasia, and any other conditions identified herein. A subject in need of the dosing regimens and formulations of the invention is a subject suffering from or predisposed to an NO-mediated condition.

By "effective" amount is meant the amount of cobalamin required to treat a nitrogen oxide-mediated condition. The effective amount of cobalamin used to practice the invention for therapeutic or prophylactic treatment of conditions caused by or contributed to by nitrogen oxides, such as NO, nitrate, and nitrite, varies depending upon the manner of administration, the cobalamin being administered, the age, body weight, and general health of the subject. Ultimately, the attending physician will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

As used herein, "measuring hydroxocobalamin levels" refers to both direct measurement of hydroxocobalamin and indirect measurements made by measuring total cobalamin levels.

Other features and advantages of the invention will be apparent from the following detailed description and the claims.

Detailed Description

The invention provides cobalamin compositions and therapeutic regimens which are useful for the treatment of migraine and other conditions associated with NO, nitrite, and/or nitrate overproduction.

Due to vast differences in physiology of patients and different delivery methods for administering cobalamin (such as hydroxocobalamin) and the differences in physiological concentrations versus pharmacological concentrations needed for differential clinical effects such as treatment of migraine prophylaxis, reduction of asthma or other respiratory conditions, reduction of inflammation in arthritis, inflammatory bowel disease and other conditions where excessive amounts of nitrogen oxides are produced in localized anatomical positions, it is desirable to administer the cobalamin using a loading dose regimen to quickly obtain desirable pharmacological levels, to measure systemic concentrations of cobalamin, and to titrate dosing in order to provide the most benefit for the patients being treated. Contributions to the variability in serum levels following systemic exposure to cobalamin include physiological factors, such as efficiency of transport, excretion, and metabolism (catabolism), as well as variability in cellular and fatty tissue uptake. A hydroxocobalamin serum concentration of at least 3,000 pg/mL is sought in order to selectively promote cellular uptake and possible storage of hydroxocobalamin. Not wishing to be bound by theory, it is believed that saturation of the binding capacity of the haptocorrin and transcobalamin proteins with hydroxocobalamin, or another cobalamin, will result in preferential binding of hydroxocobalamin by haptocorrin or transcobalamin owing to their high affinity in comparison to other cobalamins. This saturation of haptocorrin or transcobalamin will result in a saturated tissue and cellular uptake mechanism and increased transportation of hydroxocobalamin into cells in the body. Once in the serum, tissue, and cellular compartments, the hydroxocobalamin scavenges NO, nitrite, and nitrate at the time of its enzymatic production and, most importantly, prior to activation of adverse downstream physiological events associated with nitrogen oxide-mediated conditions.

An administration of cobalamin to attain levels of at least 3,000 pg/mL (as well as those mentioned above) of hydroxocobalamin can result, for example, from administration of a greater amount of another form of cobalamin, which is converted to hydroxocobalamin in the- serum, tissue and cells. Achieving a serum level of at least 3,000 pg/mL may also increase the total amount of additional serum, tissue and cellular cobalamins (i.e., cyanocobalamin, methylcobalamin, hitrosylcobalamin or adenosylcobalamin) due to interconversion of hydroxocobalamin to these other forms. It is expected that these cobalamins will not be in direct proportion to the hydroxocobalamin and will be inactive, without conversion to hydroxocobalamin, aquacobalamin, or cobinamide, with respect to their ability to treat nitrogen oxide-mediated conditions. However, these cobalamins can be administered to favor cellular and tissue uptake of hydroxocobalamin. A hydroxocobalamin serum concentration of 3000 pg/mL level is significantly greater then the normal levels of cobalamin in the serum which typically fall within the range of about 200 to 900 pg/ml. Furthermore, hydroxocobalamin, or the soluble form aquacobalamin, under physiological conditions is typically a small percentage (e.g., 10-25%) of the total cobalamin measured in the serum.

OHCbI Measurements

The methods of the invention can include the step of measuring total cobalamin, for the purpose of indirectly measuring hydroxocobalamin levels, or directly measuring hydroxocobalamin concentrations in blood serum. Such measurements can be made using methods know in the art (see, for example, Houeto et al., J. Anal. Toxicol. 18:154 (1994); Astier et al., J. Chromatogr. B Biomed. Appl. 667:129 (1995); Houeto et al., Lancet 346:605 (1995); Demedts et al., Lancet 346:1706 (1995); Yacoub et al., Eur. J. Toxicol. Environ. Hyg. 7:22 (1974); and Ahmad et al., J. Pharm. Biomed. Anal. 10:9 (1992)). Since the soluble form of hydroxocobalamin is aquocobalamin some measurements may express concentration as the amount of aquacobalamin. Measurements of cobalamin bound to proteins, such as the holotranscoblamin assays or the holohaptocorrin assays, will typically under-represent the actual amounts of total cobalamin and so such data may need to be adjusted when used in conjunction with the methods of the invention.

Typically, the serum cobalamin concentration is first measured 24, 48, 72, or 168 hours after the initial cobalamin dosing. This first measurements will assure that serum levels of at least 3000 pg/mL (and the other levels indicated above) of hydroxocobalamin are obtained quickly (e.g., within 1-2 weeks).

Typically subsequent measurements of serum cobalamin can be measured at 1 to 3 months after the initiation of administration and periodically at 6 to 12 month intervals to assure that serum levels are at appropriate levels as mentioned above. Therapy

A number of additional inflammatory and noninflammatory conditions that are associated with NO overproduction or excessive concentration of its metabolites nitrite and nitrate. Exemplary conditions include, without limitation, inflammatory bowel diseases such as ileitis, ulcerative colitis and Crohn's disease; inflammatory lung disorders such as asthma, bronchitis, oxidant-induced lung injury and chronic obstructive airway disease; inflammatory disorders of the eye including corneal dystrophy, ocular hypertension, trachoma, onchocerciasis, retinitis, uveitis, sympathetic ophthalmitis and endophthalmitis; chronic inflammatory disorders of the gum including periodontitis; chronic inflammatory disorders of the joints including arthritis, septic arthritis and osteoarthritis, tuberculosis, leprosy, glomerulonephritis sarcoid, and nephrosis; disorders of the skin including sclerodermatitis, sunburn, psoriasis and eczema; inflammatory diseases of the central nervous system, including amyotrophic lateral sclerosis, chronic demyelinating diseases such as multiple sclerosis, dementia including AIDS- related neurodegeneration and Alzheimer's disease, encephalomyelitis and viral or autoimmune encephalitis; autoimmune diseases including immune- complex vasculitis, systemic lupus and erythematosis; and disease of the heart including ischemic heart disease, heart failure and cardiomyopathy. Acute conditions that may benefit from the use of the methods and compositions of the invention include oncological conditions in which there is excessive production of nitric oxide by nitric oxide synthase enzymes in the cancerous cells including myeloid leukaemia, myelodysplasia. Additional conditions that may benefit from the use of the methods and compositions of the invention include adrenal insufficiency; hypercholesterolemia; atherosclerosis; bone disease associated with increased bone resorption, e.g., osteoporosis, preeclampsia, eclampsia, uremic complications; chronic liver failure, noninflammatory and neurodegenerative diseases of the central nervous system (CNS) including stroke and cerebral ischemia; and other disorders associated with inflammation and undesirable production of nitric oxide or nitrites and/or proinflamatory cytokines such as cystic fibrosis, tuberculosis, cachexia, ischemia/reperfusion, hemodialysis related conditions, glomerulonephritis, restenosis, inflammatory sequelae of viral infections, hypoxia, seasonal and perennial allergic rhinitis, viral or bacteria associated rhinitis, hyperbaric oxygen convulsions and toxicity, dementia, Sydenham's chorea, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, epilepsy, Korsakoff s disease, imbecility related to cerebral vessel disorder, nitrogen oxide-mediated cerebral trauma and related sequelae, ischemic brain edema (stroke), pain, migraine, emesis, immune complex disease, as immunosuppressive agents, acute allograft rejection, infections caused by invasive microorganisms which produce NO and for preventing or reversing tolerance to opiates and diazepines, aging, and various forms of cancer. All these nitric oxide, nitrite, nitrate, and/or proinflammatory cytokine and/or endotoxin induced, mediated, enhanced, and/or aggravated conditions are contemplated as being treatable using the methods and compositions of the invention.

Administration and Formulation

In accordance with the invention, cobalamin is instilled in a carrier matrix, such as controlled release lozenges, pills, troches, tablets, hard or soft capsules, syrups or elixirs, pressed pills, gel caps, chewing gum, gels such as metered gels that can be administered intranasally, creams, lotions, aqueous or oily suspensions, dispersible powders or granules, emulsions, sprays or aerosols using flowing propellants, like liposomal sprays, nasal drops, nasal sprays etc., douches and suppositories, transdermal patches etc., all for patient- friendly, self-administration of effective amounts of cobalamin. The cobalamin in formulations such as lozenges, troches, tablets, hard or soft capsules, gum etc. are preferably absorbed directly via the mucosa, such as buccal, nasal mucosa, into the blood stream. Preferred cobalamin formulations include nasal sprays, sublingual lozenges, nasal drops, nasal or pulmonary or other mucosal sprays, fast absorbing capsules or tablets. Therapeutic formulations may, for example, be in the form of liquid solutions for injection, and for intranasal formulations, in the form of powders, intranasal drops, droplets, or aerosols. Suitable intranasal formulations include, for example, the hydroxocobalamin formulation described in Example 1 and those described by van der Kuy et al., Cephalalgia 22:265 (2002). Powders and solutions can also be formulated for buccal administration and for sublingual administration. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a cobalamin.

The tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the drugs used in this invention may be incorporated for administration orally include liquid-filled gelatin capsules. Liquid forms include aqueous solutions, oil solutions, aqueous or oil suspensions, alcohol solutions, and similar pharmaceutical vehicles.

Methods well known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A.R. Gennaro, 2000, Lippincott Williams & Wilkins). Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycholate and deoxycholate, or may be oily solutions for administration in the form of intranasal drops, or as a gel. The concentration of cobalamin in the formulation will vary depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.

Cobalamin may optionally be formulated as a pharmaceutically acceptable salt, such as a non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Formulations for oral use may also be provided as chewable tablets. Cobalamin may optionally be formulated for controlled release. Many strategies can be pursued to obtain controlled release in which the rate of release outweighs the rate of metabolism of the therapeutic compound. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.

Packaging and Instructions

The methods and compositions of the present invention can be disclosed in the form of instructions for the administration of cobalamin in a loading- dose regimen. Typically, the method is disclosed to a patient along with the sale or distribution of cobalamin. In some instances, instructions may be included on a label or on a package insert accompanying a pharmaceutical formulation containing cobalamin. The method of the present invention can be incorporated into a prepackaged therapeutic regimen designed to deliver a loading-dose regimen of cobalamin to a patient using the prepackaged regimen. For example, cobalamin can be packaged as a kit in dosage units containing varying quantities of cobalamin along with instructions to the patient to administer the larger quantities followed by the smaller quantities over a particular time period. Such doses may be delivered via a nasal pump for intranasal administration; a vial for injection; a suspension, powder, or lozenge for buccal or sublingual administration; or one or more tablets, pills, capsules, or caplets for oral administration. The kit can include instructions for administration via a loading dose regimen, achieved, for example, by varying the dosing frequency. Kits of the invention can include instructions to the patient, caregiver, physician, and/or a nurse practitioner directing the cobalamin to be administered in any regimen of the invention described herein. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

Example 1. Intranasal Formulation of Hydroxocobalamin.

A kit is supplied in which hydroxocobalamin is formulated for intranasal administration for use in a loading dose regimen. Hydroxocobalamin is formulated as an isotonic, aqueous solution of OHCbI acetate in a formulation with antibacterial preservatives at a concentration of 1.07% (0.750 mg/70 μL) and 1.79% (1.25 mg/70 μL) packaged in a container-closure system (nasal pump actuator) designed to deliver 70 μL of solution (less than 1 drop in volume). These can be administered according to the methods of the invention. For example, a loading regimen of 1.25 mg can be administered 4 times daily (twice daily in each nostril) for 4-28 days, followed by a maintenance regimen of 0.75 mg once daily. The average daily dose of hydroxocobalamin given during the loading period is 5.0 mg/day and the average daily dose of hydroxocobalamin given during the maintenance period is 0.75 mg/day.

Example 2. Oral Cobalamin Loading Dose Followed by Twice Weekly Intranasal Hydroxocobalamin Thereafter.

A kit is supplied in which hydroxocobalamin is formulated for intranasal administration and cobalamin is formulated for oral administration, both for use in a loading dose regimen. Cobalamin is orally administered to a human subject in a loading doses of 20 mg each on Days 1, 2, 3, 4, 5, 6, and 7. Cobalamin serum levels are measured on Day 7 to confirm that hydroxocobalamin serum levels at least 3,000 pg/mL. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated (e.g., up to 28 days). After the first week, hydroxocobalamin is intranasally administered to the subject in doses of 1.25 mg (1.79% solution, 70 μL per dose) daily. The average daily dose of cobalamin given during the loading period (week 1) of the regimen is 20 mg/day. The average daily dose of cobalamin (as hydroxocobalamin) given during the maintenance period (after week 1) of the regimen is 1.25 mg/day. The portion of the regimen that is administered nasally is scaled to account for the difference in bioavailability (e.g., 1.25 mg/day x 5 = 6.25 mg/day). Comparing the doses scaled to account for differences in bioavailability, the average daily dose during the loading period is 5 mg/day and the average daily dose in the maintenance period is 1.785 mg/day.

Example 3. Subcutaneous Cobalamin Loading Dose Followed by Subcutaneous Cobalamin Once Weekly Thereafter.

A kit is supplied in which hydroxocobalamin is formulated for subcutaneous administration for use in a loading dose regimen. Cobalamin is subcutaneously administered to a human subject in doses of 350 μg each on Days 1, 2, 3, 4, 5, 6, and 7. Cobalamin serum levels are measured on Day 7 to confirm that hydroxocobalamin serum levels at least 3,000 pg/mL. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated. After the first week, cobalamin is subcutaneously administered to the subject in doses of 700 μg once weekly thereafter. The average daily dose of cobalamin given during the loading period (week 1) of the regimen is 350 μg/day . The average daily dose of cobalamin given during the maintenance period (after week 1) of the regimen is 100 μg/day.

Example 4. Intranasal Hydroxocobalamin Loading Dose Followed by Four

Times a Week Intranasal Hydroxocobalamin Thereafter. A kit is supplied in which hydroxocobalamin is formulated for intranasal administration for use in a loading dose regimen. Hydroxocobalamin is intranasally administered to a human subject in loading doses of 1.25 mg

(1.79% solution, 70 μL per dose) twice daily on Days 1, 2, 3, 4, 5, 6, and 7.

Cobalamin serum levels are measured on Day 7 to confirm that serum levels of hydroxocobalamin of at least 3,000 pg/mL. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated (e.g., up to 28 days). After the first week, hydroxocobalamin is intranasally administered to the subject in maintenance doses of 1.25 mg (1.79% solution, 70 μL per dose) four times a week. The average daily dose of hydroxocobalamin given during the loading period (week 1) of the regimen is 2.5 mg/day. The average daily dose of hydroxocobalamin given during the maintenance period (after week 1) is 0.714 mg/day.

Example 5. Intramuscular Cobalamin Loading Dose Followed by Twice Weekly Intranasal Hydroxocobalamin Thereafter. A kit is supplied in which hydroxocobalamin is formulated for intranasal administration and hydroxocobalamin is formulated for injection, both for use in a loading dose regimen. Cobalamin is intramuscularly administered to a human subject in doses of 100 μg each on Days 1, 2, 3, 4, 5, 6, and 7. Cobalamin serum levels are measured on Day 7 to confirm that hydroxocobalamin serum levels of at least 3,000 pg/mL. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated (e.g., up to 28 days). After the first week, hydroxocobalamin is intranasally administered to the subject in doses of 1.25 mg (1.79% solution, 70 μL per dose) four times a week. The average daily dose of cobalamin given during the loading period (week 1) of the regimen is 100 μg/day. The average daily dose of cobalamin (as hydroxocobalamin) given during the maintenance period (after week 1) of the regimen is 0. 714 mg/day. The portion of the regimen that is administered intramuscularly is scaled to account for the difference in bioavailability (e.g., 100 μg/day x 20 = 2 mg/day). Comparing the doses scaled to account for differences in bioavailability, the average daily dose during the loading period is 2 mg/day and the average daily dose in the maintenance period is 0. 714 mg/day. Example 6. Intramuscular Cyanocobalamin Loading Dose Followed by Twice Weekly Intranasal Hydroxocobalamin Thereafter.

A kit is supplied in which hydroxocobalamin is formulated for intranasal administration and cyanocobalamin is formulated for injection, both for use in a loading dose regimen. Cyanocobalamin is intramuscularly administered to a human subject in doses of 500 μg each on Days 1, 2, 3, 4, 5, 6, and 7. Cobalamin serum levels are measured on Day 7 to confirm that hydroxocobalamin serum levels of at least 3,000 pg/mL. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated (e.g., up to 28 days). After the first week, hydroxocobalamin is intranasally administered to the subject in doses of 1.25 mg (1.79% solution, 70 μL per dose) four times a week. The average daily dose of cobalamin given during the loading period (week 1) of the regimen is 500 μg/day. The average daily dose of cobalamin (as hydroxocobalamin) given during the maintenance period (after week 1) of the regimen is 0. 714 mg/day. The portion of the regimen that is administered intramuscularly is scaled to account for the difference in bioavailability (e.g., 500 μg/day x 20 = 10 mg/day). Comparing the doses scaled to account for differences in bioavailability, the average daily dose during the loading period is 10 mg/day and the average daily dose in the maintenance period is 0. 714 mg/day.

Example 7. Intranasal and Sublingual Hydroxocobalamin Loading Dose Followed by Daily Intranasal Hydroxocobalamin Thereafter.

A kit is supplied in which hydroxocobalamin is formulated for intranasal administration and hydroxocobalam is formulated for sublingual administration, both for use in a loading dose regimen. Hydroxocobalamin is intranasally administered to a human subject in doses of 1.25 mg (1.79% solution, 70 μL per dose) four times daily (twice daily in each nostril) on Days 1, 2, 3, 4, 5, 6, and 7; and sublingually in doses of 2 mg daily on Days 1, 2, 3, 4, 5, 6, and 7. Hydroxocobalamin serum levels are measured on Day 7 to confirm that serum levels at least 3,000 pg/mL. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated (e.g., up to 28 days). After the first week, hydroxocobalamin is intranasally administered to the subject in doses of 1.25 mg (1.79% solution, 70 μL per dose) daily without further sublingual administration. The average daily dose of hydroxocobalamin given during the loading period (week 1) of the regimen is 7.0 mg/day. The average daily dose of hydroxocobalamin given during the maintenance period (after week 1) is 1.25 mg/day. The portion of the regimen that is administered sublingually need not be scaled to account for the difference in bioavailability because sublingual and nasal routes of administration have comparable bioavailabilities.

Example 8. Intramuscular Cobalamin Loading Dose Followed by Twice Weekly Intranasal Hydroxocobalamin Thereafter.

A kit is supplied in which hydroxocobalamin is formulated for intramuscular administration and hydroxocobalamin is formulated for intranasal administration, both for use in a loading dose regimen. Hydroxocobalamin is intramuscularly administered to a human subject in doses of 200 μg each on Days 1, 2, 3, 4, 5, 6, and 7. Cobalamin serum levels are measured on Day 7 to confirm that hydroxocobalamin serum levels at least 3,000 pg/mE. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated (e.g., up to 28 days). After the first week, the kit contains a formulation so that hydroxocobalamin is intranasally administered to the subject in doses of 1.25 mg (1.79% solution, 70 μL per dose) daily. The average daily dose of cobalamin given during the loading period (week 1) of the regimen is 200 μg/day. The average daily dose of cobalamin (as hydroxocobalamin) given during the maintenance period (after week 1) of the regimen is 1.25 mg/day. The portion of the regimen that is administered intramuscularly is scaled to account for the difference in bioavailability (e.g., 200 μg/day x 20 = 4 mg/day). Comparing the doses scaled to account for differences in bioavailability, the average daily dose during the loading period is 4 mg/day and the average daily dose in the maintenance period is 1.25 mg/day.

Example 9. Sublingual Cobalamin Loading Dose Followed by Daily Intranasal Hydroxocobalamin Thereafter.

A kit is supplied in which methylcobalamin is formulated for sublingual administration and hydroxocobalamin is formulated for intranasal administration, both for use in a loading dose regimen. Methylcobalamin is sublingually administered to a human subject in doses of 1000 μg twice daily on Days 1, 2, 3, 4, 5, 6, and 7. Cobalamin serum levels are measured on Day 7 to confirm that hydroxocobalamin serum levels at least 3,000 pg/mL. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated (e.g., up to 28 days). After the first week, the kit contains a formulation so that hydroxocobalamin is intranasally administered to the subject in doses of 1.25 mg (1.79% solution, 70 μL per dose) every other day. The average daily dose of cobalamin given during the loading period (week 1) of the regimen is 2 mg/day. The average daily dose of cobalamin (as hydroxocobalamin) given during the maintenance period (after week 1) of the regimen is 0.625 mg/day. The portion of the regimen that is administered sublingually need not be scaled to account for the difference in bioavailability because sublingual and nasal routes of administration have comparable bioavailabilities .

Example 10. Oral Cobalamin Loading Dose Followed by Daily Intranasal Hydroxocobalamin Thereafter.

A kit is supplied in which cyanocobalamin is formulated for oral administration and hydroxocobalamin is formulated for intranasal administration, both for use in a loading dose regimen. Cyanocobalamin is orally administered to a human subject in doses of 5,000 mg daily on Days 1, 2, 3, 4, 5, 6, and 7. Cobalamin serum levels are measured on Day 7 to confirm that hydroxocobalamin serum levels at least 3,000 pg/mL. If a serum level of 3,000 pg/mL has not been reached, then the loading period can be repeated (e.g., up to 28 days). After the first week, the kit contains a formulation so that hydroxocobalamin is intranasally administered to the subject in doses of 1.25 mg (1.79% solution, 70 μL per dose) daily. The average daily dose of cobalamin given during the loading period (week 1) of the regimen is 5 g/day. The average daily dose of cobalamin (as hydroxocobalamin) given during the maintenance period (after week 1) of the regimen is 1.25 mg/day. The portion of the regimen that is administered nasally is scaled to account for the difference in bioavailability (e.g., 1.25 mg/day x 5 = 6.25 mg/day). Comparing the doses scaled to account for differences in bioavailability, the average daily dose during the loading period is 5 g/day and the average daily dose in the maintenance period is 6.25 mg/day.

Other Embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims. What is claimed is:

Claims

1. A method of treating a nitrogen oxide-mediated condition in a subject in need thereof, said method comprising administering to said subject an initial dose of cobalamin in an amount sufficient to produce a hydroxocobalamin steady state serum concentration of at least 3,000 pg/mL within a period of approximately 28 days of administering said initial dose.

2. The method of claim 1, wherein said hydroxocobalamin steady state serum concentration of at least 3,000 pg/mL is obtained within a period of 24 hours of administering said initial dose.

3. A method of treating a nitrogen oxide-mediated condition in a subject in need thereof, said method comprising:

(i) Administering cobalamin in an intranasal, sublingual or buccal amount from 0.5 mg to 10 mg to said subject;

(ii) After step (i), measuring the serum concentration of hydroxocobalamin in the blood of said subject; and

(iii) if said concentration is less than 3,000 pg/mL, repeating step (i).

4. A method of treating a nitrogen oxide-mediated condition in a subject in need thereof, said method comprising:

(i) Administering cobalamin in a subcutaneous, intravenous or intramuscular amount from 50 μg to 1,000 μg to said subject;

(iii) if said concentration is less than 3,000 pg/mL, repeating step (i).

5. A method of treating a nitrogen oxide-mediated condition in a subject in need thereof, said method comprising:

(i) Administering cobalamin in an oral or enteric amount from 5 to 5,000 mg to said subject; (ii) After step (i), measuring the serum concentration of hydroxocobalamin in the blood of said subject; and

(iii) if said concentration is less than 3,000 pg/mL, repeating step (i).

6. The method of any of claims 1-5, wherein said hydroxocobalamin steady state serum concentration of at least 5,000 pg/mL.

7. The method of claim 6, wherein said hydroxocobalamin steady state serum concentration of at least 7,500 pg/mL.

8. The method of any of claims 1-5, wherein if said concentration is greater than 3,000 pg/mL, administering to said subject cobalamin in a maintenance dose regimen thereafter.

9. A method of treating a nitrogen oxide-mediated condition in a subject in need thereof, said method comprising administering a loading-dose regimen of cobalamin to said subject.

10. The method of claim 9, wherein said loading-dose regimen comprises: a) an initial administration of an average daily dose of cobalamin for 2 to 28 days; b) following said initial administration, administration of less than half said average daily dose thereafter.

11. The method of claim 9, wherein said loading-dose regimen comprises an average initial daily dose of cobalamin which is at least 200% of the average daily dose over any of the next two, three, four, or five subsequent dosing days.

12. The method of claim 9, wherein said loading-dose regimen comprises a dose of cobalamin administered on Day 1, said dose administered on Day 1 being at least 200% of the dose administered on any one of the next four dosing days.

13. The method of any of claims 1-12, wherein said administration is intramuscular, subcutaneous, bronchial, transdermal, intranasal, sublingual, buccal, by vaginal suppository, or by anal suppository.

14. The method of claim 9, wherein said loading-dose regimen comprises intranasal, buccal, or sublingual administration of cobalamin at an average daily dose of 0.5 to 10 mg for 1-28 days followed by 0.25 to 5 mg thereafter.

15. The method of claim 9, wherein said loading-dose regimen comprises subcutaneous, intravenous, or intramuscular administration of cobalamin at an average daily dose of 50 to 1000 μg for 1-28 days followed by 25 to 500 μg thereafter.

16. The method of claim 9, wherein said loading-dose regimen comprises oral administration of cobalamin at an average daily dose of 5 to 5,000 mg for 1-28 days followed by 1 to 5 mg thereafter.

17. The method of claim 9, wherein said loading-dose regimen comprises administration of cobalamin at least twice daily for a period of 1-28 days followed by administration daily or less thereafter.

18. The method of any of claims 1-17, wherein said cobalamin is selected from hydroxocobalamin, cobinamide, aquacobalamin, cyanocobalamin, methylcobalamin, and adenosylcobalamin.

19. The method of claim 18, wherein said cobalamin is hydroxocobalamin.

20. The method of any of claims 1-19, wherein said nitrogen oxide- mediated condition is migraine headache.

21. A pharmaceutical kit comprising:

(i) a pharmaceutical composition comprising cobalamin; and (ii) a label or package insert providing instructions, said instructions describing administration of said pharmaceutical composition as part of a loading-dose regimen for the treatment of a nitrogen oxide-mediated condition.

22. The kit of claim 21, wherein said cobalamin is selected from hydroxocobalamin, cyanocobalamin, cobinamide, aquacobalamin , methylcobalamin, and adenosylcobalamin.

23. The kit of claim 21, wherein said pharmaceutical composition is formulated for intranasal administration.

24. The kit of claim 21, wherein said nitrogen oxide-mediated condition is migraine headache.

25. A pharmaceutical kit comprising:

(i) a pharmaceutical composition comprising hydroxocobalamin and formulated for intranasal administration; and

(ii) a label or package insert providing instructions, said instructions describing administration of said pharmaceutical composition in a dosing regimen of at least twice daily for a period of 1-28 days followed by administration once daily or less thereafter.

26. A pharmaceutical kit comprising:

(i) a first pharmaceutical composition comprising hydroxocobalamin or cyanocobalamin and formulated for parenteral, sublingual, or oral administration;

(ii) a second pharmaceutical composition comprising hydroxocobalamin and formulated for intranasal administration; and

(iii) a label or package insert providing instructions, said instructions describing administration of said first pharmaceutical composition at least once daily for a period of 1-28 days followed by administration of said second pharmaceutical composition daily or less thereafter.

27. The kit of claim 25 or 26, further comprising instructions for administering said first pharmaceutical composition and said second pharmaceutical composition to a subject for the treatment of a nitric oxide- mediated condition.

28. The kit of claim 27, wherein said nitric oxide-mediated condition is migraine.

29. The kit of claim 26, wherein said first pharmaceutical composition comprises hydroxocobalamin formulated for intravenous administration.

30. A method of treating a nitrogen oxide-mediated condition in a subject in need thereof, said method comprising (i) intranasal administration of hydroxocobalamin to said subject in a first regimen, said first regimen comprising an average daily dose of 2.5 to 10 mg of hydroxocobalamin per day for a period of from 3 to 28 days; and (ii) following step (i), intranasal administration of hydroxocobalamin to said subject in a second regimen, said second regimen comprising an average daily dose of 0.5 to 5 mg of hydroxocobalamin per day, wherein the average daily dose of said first regimen is greater than the average daily dose of said second regimen.

31. A method of treating a nitrogen oxide-mediated condition in a subject in need thereof, said method comprising (i) subcutaneous, intravenous, or intramuscular administration of hydroxocobalamin to said subject in a first regimen, said first regimen comprising an average daily dose of 50 to 5,000 μg of hydroxocobalamin per day for a period of from 1 to 28 days; and (ii) following step (i), intranasal administration of hydroxocobalamin to said subject in a second regimen, said second regimen comprising an average daily dose of 0.5 to 5 mg of hydroxocobalamin per day.

32. The method of claim 30 or 31, further comprising measuring the serum concentration of hydroxocobalamin in the blood of said subject during said first regimen; and, on the basis of said concentration, continuing said first regimen or administering said second regimen.

33. The method of claim 31, wherein step (i) comprises intravenous administration of 1,000 μg to 5,000 μg daily for 1-3 days.