# Patent

## Title
Method of treating vitamin B12 deficiency

## Abstract

A novel method and composition for treating vitamin B12 deficiency mammals that fail to respond to oral vitamin B12 therapy.

## Background
CROSS-REFERENCE TO PRIOR APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/262,677, filed Oct. 31, 2008, now U.S. Pat. No. 8,022,048, which claims the benefit of U.S. Provisional Application No. 60/984,898, filed Nov. 2, 2007; U.S. Provisional Application No. 61/020,108, filed Jan. 9, 2008; and U.S. Provisional Application No. 61/083,566, filed Jul. 25, 2008, each of which is hereby incorporated by reference.


FIELD OF THE INVENTION
The present invention relates generally to methods of treating vitamin B12 deficiency and pharmaceutical compositions for such treatment.
BACKGROUND OF THE INVENTION
Vitamin B12 is important for the normal functioning of the brain and nervous system and for the formation of blood. It is involved in the metabolism of every cell of the body, especially affecting the DNA synthesis and regulation but also fatty acid synthesis and energy production. Its effects are still not completely known.
Cyanocobalamin is the most stable and widely used form of vitamin B12. It is bound to plasma proteins and stored in the liver. Vitamin B12 is excreted in the bile and undergoes some enterohepatic recycling. Absorbed vitamin B12 is transported via specific B12 binding proteins, transcobalamin I and II, to the various tissues. The liver is the main organ for vitamin B12 storage.
Vitamin B12 deficiency can potentially cause severe and irreversible damage, especially to the brain and nervous system. Oral tablets containing vitamin B12 have been developed to treat vitamin B12 deficiency. However, many patients with vitamin B12 deficiency do not respond to oral vitamin B12 treatment. There is a need to develop a treatment for these patients.
BRIEF SUMMARY OF THE INVENTION
One aspect of the invention is directed to a method for treating vitamin B12 deficiency in a subject, comprising the steps of (a) preparing a pharmaceutical composition for oral administration containing (1) vitamin B12 and (2) at least one substance selected from the group consisting of N-[8-(2-hydroxybenzoyl)amino]caprylic acid and its pharmaceutically acceptable salts; and (b) administering the pharmaceutical composition to the subject to effectively treat said vitamin B12 deficiency.
Another aspect of the invention is directed to a pharmaceutical composition for treating vitamin B12 deficiency in a subject, comprising (1) vitamin B12 and (2) at least one substance selected from the group consisting of N-[8-(2-hydroxybenzoyl)amino]caprylic acid and its pharmaceutically acceptable salts; wherein said subject had failed to respond to existing oral vitamin B12 treatment.
The contents of the patents and publications cited herein and the contents of these documents cited in these patents and publications are hereby incorporated herein by reference to the extent permitted.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of serum vitamin B12 concentration as a function of time.



DETAILED DESCRIPTION
As used herein, the term “SNAC” means Sodium-N-salicyloyl-8-aminocaprylate, Monosodium 8-(N-salicyloylamino) octanoate, N-(salicyloyl)-8-aminooctanoic acid monosodium salt, monosodium N-{8-(2phenoxybenzoyl)amino}octanoate, E414 monosodium salt or sodium 8-[(2-hydroxybenzoyl)amino]octanoate. It has the structure




“N-[8-(2-hydroxybenzoyl)amino]caprylic acid” has an empirical formula C15H21NO4.

The term “Vitamin B12” means any member of a group of cobalt-containing compounds known as cobalamins which include, but is not limited to cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5-deoxyadenosylcobalamin.
The term “treatment” or “treating” means any treatment of a disease or disorder in a mammal, including: preventing or protecting against the disease or disorder, that is, causing the clinical symptoms not to develop; inhibiting the disease or disorder, that is, arresting or suppressing the development of clinical symptoms; and/or relieving the disease or disorder, that is, causing the regression of clinical symptoms. The term “mammal” include human subjects.
The terms “carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent, solubilizing agent” are as defined in the Handbook of Pharmaceutical Excipients (fourth edition) by Raymond C. Rowe, Paul J. Sheskey and Paul J. Weller, the content of which is herein incorporated by reference.
The term “intrinsic factor protein” means is a glycoprotein produced by the parietal cells of the stomach. It is necessary for the absorption of vitamin B12 later on in the terminal ileum.
In a preferred embodiment, the treatment is directed to subjects that had failed to respond to existing oral vitamin B12 treatment. Preferably, tablets are used for the treatment. Such tablets contain from about 0.01 mg to about 25 mg of vitamin B12 and from about 1 mg to about 600 mg of SNAC each, preferably from about 0.02 mg to about 25 mg of vitamin B12 and more preferably from about 0.1 mg to about 20 mg of vitamin B12 and the most preferably from about 0.5 mg to 10 mg of vitamin B12 and from about 10 mg to about 200 mg of SNAC in each tablet.
The preferred weight ratio of vitamin B12 and SNAC in the tablet is from about 2:1 to about 1:700, more preferably from about 1:2 to about 1:600 or from about 1:3 to about 1:20 and the most preferably from about 1:4 to about 1:10.
In a preferred embodiment, the pharmaceutical composition is in the form of tablets. Preferrably, each tablet contains from about 0.01 mg to about 25 mg of vitamin B12 and from about 50 mg to about 600 mg of SNAC. More preferably, each tablet contains from about 0.02 mg to about 20 mg of vitamin B12. More preferably, each tablet contains from about 0.1 mg to about 10 mg of vitamin B12. The most preferably, each tablet contains about 15 to 20 mg of vitamin B12 and about 50 to 100 mg of SNAC, or about 0.1 to 1.5 mg of vitamin B12 and about 25 to 150 mg of SNAC.
In another preferred embodiment, the tablet further contains at least one of a carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent, solubilizing agent or combinations thereof.
In another preferred embodiment, the tablet optionally contains from about 1 to 25 mg of Capmul PG-8 and optionally contains from about 0.5 to 10 mg of providone. Preferably, Capmul PG-8 is in an amount from about 2 to 20 mg and Providone is in an amount from about 1 to 8 mg. Preferably, Capmul PG-8 is in an amount from about 5 to 15 mg and the Providone is in an amount from about 1.5 to 5 mg. More preferably, Capmul PG-8 is in an amount from about 5 to 10 mg and Providone is in an amount from about 1.5 to 5 mg.
Without intending to be bound by any particular theory of operation, it is believed that gastrointestinal absorption of vitamin B12 depends on the presence of sufficient intrinsic factor protein, secreted from gastric parietal cells. The average diet supplies about 10 mcg/day of vitamin B12 in a protein-bound form that is available for absorption after normal digestion. Vitamin B12 is bound to intrinsic factor during transit through the stomach; separation occurs in the terminal ileum, and vitamin B12 enters the mucosal cell for absorption via a receptor mediated process. It is then transported by the transcobalamin binding proteins. A small amount (approximately 1% of the total amount ingested) is absorbed by simple diffusion, but this mechanism is adequate only with very large doses. It is also believed that SNAC will allow B12 to bypass its usual receptor mediated process.
The following examples are given as specific illustrations of the invention. It should be understood, however, that the invention is not limited to the specific details set forth in the examples. All parts and percentages in the examples, as well as in the remainder of the specification, are by weight unless otherwise specified.
Further, any range of numbers recited in the specification or paragraphs hereinafter describing or claiming various aspects of the invention, such as that representing a particular set of properties, units of measure, conditions, physical states or percentages, is intended to literally incorporate expressly herein by reference or otherwise, any number falling within such range, including any subset of numbers or ranges subsumed within any range so recited. The term “about” when used as a modifier for, or in conjunction with, a variable, is intended to convey that the numbers and ranges disclosed herein are flexible and that practice of the present invention by those skilled in the art using concentrations, amounts, contents, carbon numbers, and properties that are outside of the range or different from a single value, will achieve the desired result, namely, effective treatment of a subject with vitamin B12 deficiency which failed to respond to existing oral vitamin B12 tablets as well as pharmaceutical compositions for such treatment.
EXAMPLE 1
Preparation of N-[8-(2-hydroxybenzoyl)amino]caprylic acid and SNAC
The preparation method for N-[8-(2-hydroxybenzoyl)amino]caprylic acid and SNAC involves the following steps: The starting material is salicylamide, which is converted to form Carsalam. The second step involves the alkylation of Carsalam. The penultimate step is a hydrolysis to cleave the ethyl protection group at the end of the alkyl chain and spring open the heterocyclic ring forming the free acid of SNAC. In the final step, the sodium salt of the SNAC free acid is formed by reaction with a 1% excess stoichiometric amount of sodium hydroxide base. Upon cooling the precipitated product is isolated by centrifugation and vacuum dried prior to packaging. The in-process controls for the synthetic scheme are given in Table I.







TABLE I







In-process controls for SNAC Manufacturing Process.















In-Process


Step
Reaction
Desired Product
Specification
Control





1
Carsalam
Carsalam
<10%
HPLC





salicylamide


2
Alkylation
Alkylated
<8% Carsalam
HPLC




Carsalam


3
Hydrolysis
SNAC Free
 <0.5%
LOD




acid


4
Sodium Salt
SNAC Sodium
95-105%
HPLC




salt









EXAMPLE 2
Preparation of Vitamin B12 Tablets
The tablet die and punches are checked to ensure that they are clean and that their surfaces are dusted with magnesium stearate powder. Vitamin B12, SNAC, carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent, solubilizing agent are screened through a #35 sieve and transferred into a sealed containers. 50 mg of Vitamin B12 is weighed and mixed thoroughly with 11 grams of a carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent and/or solubilizing agent. 100 vitamin B12 tablets are made, with each tablet containing 0.5 mg of Vitamin B12 and 110 mg of a carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent and/or solubilizing agent. These tablets are used as a control.
EXAMPLE 3
Preparation of Vitamin B12 and SNAC Tablets
50 mg of Vitamin B12, 1 gram of SNAC are weighed and thoroughly mixed with 10 grams of a carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent and/or solubilizing agent. 100 vitamin B12 tablets are made, with each tablet containing 0.5 mg of Vitamin B12. 10 mg of SNAC and 100 mg of a carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent and/or solubilizing agent. The process is repeated to make tablet batches containing 1.0 mg, 0.8 mg, 0.6 mg, 0.4 mg and 0.2 of Vitamin B12, respectively. These tablets have the following specifications for release of SNAC component:












Tests
Specification
Analytical Method







Appearance
White to light-tan powder with
AM001



pink hue


Identification


Test for Sodium
Confirms presence of Sodium
USP <191>


FTIR
Conforms to reference standard
USP <197K>


Melting Range/Temperature
193-203° C. with a range not to
USP <741>



exceed 5° C.


Water Content
NMT 3.0%
USP <921> Method I


Heavy Metals
<20 ppm
USP <231> Method II


Sodium Content
6.9 to 8.4%
AM017


Residual Solvents


Ethanol
Less than 4000 ppm
AM008


Heptane
Less than 500 ppm
AM008


Assay as SNAC Sodium salt
90.0-110.0% w/w
AM016


(As Is)









EXAMPLE 4
Preparation of Tablets for Testing on Rats
Tablets with four types of different ingredients were made as follows: (1) 8.8 mg of vitamin B12, 35 mg of SNAC were weighed, thoroughly mixed and made into a tablet for dosing on rat; (2) 8.8 mg of vitamin B12, 35 mg of SNAC and 5 mg of Capmul PG-8 were weighed, thoroughly mixed and made into a tablet; (3) 8.8 mg of vitamin B12, 35 mg of SNAC and 0.9 mg of Providone were weighed, thoroughly mixed and made into a tablet. Each of the four processes was repeated to produce more tablets.
EXAMPLE 5
Dosing Sprague-Dawley Rats
Male Sprague-Dawley rats (325-350 g) were dosed with vitamin B12 intravenously (0.5 mg/kg) alone, or orally with the tablets made in Example 4 at a dose of 50 mg/kg vitamin B12 alone or in combination with SNAC at 200 mg/kg. Blood samples were collected at 0, 3, 10, 20, 30, 60, 120, 240 and 360 minutes post dosing. Plasma samples were analyzed for B12 by RIA. The model independent PK metrics obtained following B12-SNAC combination were compared to those obtained following B12 alone. The testing results are shown in Table 1.







TABLE 1







Comparative Testing Results for Vitamin B12 Absorption














AUC
Mean



Cmax
Tmax
(ug * min/
Bio-


Group
(ug/mL)
(min)
mL)
availability














(N = 5)
Mean
S.D
Mean
S.D
Mean
S.D
%

















0.5 mg/kg
2.15
0.64
4.4
3.13
65.84
11



Vitamin


B12 (IV)


50 mg/kg
0.14
0.07
52
17.9
28.72
13
0.42


Vitamin


B12


alone (PO)


50 mg/kg
7.99
2.41
24
5.48
522.37
179
7.93


Vitamin


B12 +


200 mg/kg


SNAC (PO)









EXAMPLE 6
Preparation of Tablets for Testing on Human Subjects
Tablets were made from Cyanocobalamin, SNAC, Kollidon 90F, Anhydrous Emcompress USP/EP and Magnesium Stearate, NF/BP/EP/JP. Each tablet contains the followings:














Ingredients
mg/tablet



















Cyanocobalamin, USP (Intragranular)
5.00



SNAC (Intragranular)
100.00



Kollidon 90F, NF/EP/JP
2.00



(Providone K90; Intragranular)



Anhydrous Emcompress USP/EP (Diabasic
70.00



Calcium Phosphate, Anhydrous; Intragranular)



Anhydrous Emcompress USP/EP (Diabasic
21.00



Calcium Phosphate, Anhydrous; Extragranular)



Magnesium Stearate, NF/BP/EP/JP
2.00



(extragranular)




Total Weight
200.0










EXAMPLE 7
Dosing Human Subjects
Sixteen healthy male subjects were randomized to receive one of the following treatments:

    
    
        (1) Treatment B: a single oral dose of cyanocobalamin/SNAC (5 mg cyanocobalamin/100 mg SNAC) administered in the fasted state as a tablet. (6 subjects);
        (2) Treatment C: a single oral dose of cyanocobalamin alone (5 mg cyanocobalamin, VitaLabs, commercial) administered in the fasted state as a tablet. (6 subjects).
        (3) Treatment D: a single intravenous dose of cyanocobalamin (1 mg cyanocobalamin) administered in the fasted state. (4 subjects). Each subject received a 1 mL intravenous injection of a 1 mg/mL (1000 μg/mL) solution resulting in a total dose of 1 mg cyanocobalamin.
    
    


The subjects were fasted overnight prior to dosing and had no liquids (including water) consumption for at least one hour before and after dosing. The oral forms of cyanocobalamin/SNAC tablets were administered in a single dose as tablets with 50 mL of plain water. Twenty-five blood samples were drawn for cyanocobalamin analyses at the following time points: within 30 minutes pre-dose and at Minutes 2, 5, 10, 20, 30, 40, 50, and at Hours 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 20 and 24 post-dose.
Pharmacokinetic metrics was obtained following a model independent pharmacokinetic analysis of individual cyanocobalamin concentrations. Descriptive statistics was used to summarize the results.
Following 1 tablet of 5 mg B12/100 mg SNAC mean B12 peak concentration is 12847±6613 pg/mL and occur within 1 hour post dose (mean tmax of 0.50±0.21 hours). Mean AUClast (0-24) value is 54618±16392 hr*pg/mL. The percent coefficient of variation (% CV) is 51.5% for Cmax and 30.0% for AUC.
Following a single oral dose of cyanocobalamin alone (5 mg cyanocobalamin, VitaLabs, commercial) mean B12 peak concentration is 1239±450 pg/mL and occur between 3 to 10 hours post-dose (mean tmax of 6.8±3.2 hours). Mean AUClast (0-24) value is 23131±8343 hr*pg/mL. The percent coefficient of variation (% CV) is 36.3% for Cmax and 36.1% for AUC.
Following a single intravenous dose of cyanocobalamin (1 mg cyanocobalamin) administered in the fasted state (4 subjects). Mean B12 peak concentration is 221287±80248 pg/mL and mean AUClast (0-24) value is 215391±44602 hr*pg/mL. The percent coefficient of variation (% CV) is 36.3% for Cmax and 20.7% for AUC.
The mean bioavailability of 1 tablet of 5 mg vitamin B12 alone, 1 tablet of 5 mg vitamin B12/100 mg SNAC, and 2 tablets of 5 mg vitamin B12/100 mg SNAC are 2.15±0.77%, 5.07±1.52, and 5.92±3.05%, respectively. (Note: 2 tablets of 5 mg vitamin B12/100 mg SNAC were dosed previously in a pilot arm are designated Treatment A).
The mean tmax of 1 tablet of 5 mg vitamin B12 alone, 1 tablet of 5 mg vitamin B12/100 mg SNAC, and 2 tablets of 5 mg vitamin B12/100 mg SNAC are 6.8±3.2 hours, 0.50±0.21 hours, and 0.54±0.32 hours, respectively.
No adverse events were observed during the given treatments. All formulations appear to be safe and well tolerated.
It was found surprisingly that the extent of B12 absorption, measured as Cmax and AUC, was significantly enhanced by the administration of the cyanocobalamin/SNAC combination. Vitamin B12 bioavailability was  ̃240% greater for the 1 tablet of 5 mg B12/100 mg SNAC compared to 5 mg B12 commercial formulation. Mean peak B12 concentrations following B12 commercial oral formulation occurred significantly later compared to that following the B12/SNAC combinations likely due to a different site of absorption between the two oral formulations. This is consistent with literature data describing intestinal absorption of B12 occurring in the distal section of the gastrointestinal tract in the absence of the carrier.
The principles, preferred embodiments, and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art, without departing from the spirit of the invention.

## Claims

1. A method for administering vitamin B12 to a subject to treat vitamin B12 deficiency, comprising orally administering a pharmaceutical composition comprising (1) from about 0.01 mg to about 25 mg of vitamin B12 and (2) from about 1 mg to about 600 mg of at least one substance selected from N-[8-(2-hydroxybenzoyl) amino]caprylic acid and its pharmaceutically acceptable salts to said subject.

2. The method of claim 1, wherein said subject had failed to respond to existing oral vitamin B12 treatment.

3. The method of claim 1, wherein said pharmaceutical composition is a tablet.

4. The method of claim 1, wherein said pharmaceutical composition has a weight ratio of vitamin B12 to monosodium N-[8-(2-hydroxybenzoyl) amino]caprylate of from about 2:1 to about 1:700.

5. The method of claim 4, wherein said weight ratio is from about 1:2 to about 1:600.

6. The method of claim 4, wherein said weight ratio is from about 1:3 to about 1:20.

7. The method of claim 1, wherein said further comprises at least one of a carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent, solubilizing agent or combinations thereof.

8. An oral pharmaceutical composition comprising (1) from about 0.01 mg to about 25 mg of vitamin B12 and (2) from about 1 mg to about 600 mg of at least one substance selected from N-[8(2-hydroxybenzoyl) amino]caprylic acid and its pharmaceutically acceptable salts.

9. The pharmaceutical composition of claim 8, wherein said pharmaceutical composition comprises a tablet.

10. The pharmaceutical composition of claim 9, wherein said tablet comprises from about 0.01 to about 25 mg of vitamin B12 and from about 50 to about 600 mg of monosodium N-[8-(2-hydroxybenzoyl) amino]caprylate.

11. The pharmaceutical composition of claim 9, wherein said tablet comprises from about 1 to about 15 mg of vitamin B12 and from about 50 to about 200 mg of monosodium N-[8-(2-hydroxybenzoyl) amino]caprylate.

12. The pharmaceutical composition of claim 8, further comprising at least one of a carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent, solubilizing agent or combinations thereof.

13. The method of claim 1, wherein said pharmaceutical composition comprises about 0.1 to 1.5 mg of vitamin B12 and from 25 to 100 mg of monosodium N-[8-(2-hydroxybenzoyl) amino]caprylate.

14. The method of claim 1, wherein the vitamin B12 is a cobalamin.

15. The method of claim 1, wherein the vitamin B12 is cyanocobalamin.

16. The method of claim 1, wherein the vitamin B12 is hydroxocobalamin.

17. The method of claim 1, wherein the vitamin B12 is methylcobalamin.

18. The method of claim 1, wherein the vitamin B12 is 5-deoxyadenosylcobalamin.

19. The method of claim 13, wherein the vitamin B12 is a cobalamin.

20. The method of claim 13, wherein the vitamin B12 is cyanocobalamin.

21. The method of claim 13, wherein the vitamin B12 is hydroxocobalamin.

22. The method of claim 13, wherein the vitamin B12 is methylcobalamin.

23. The method of claim 13, wherein the vitamin B12 is 5-deoxyadenosylcobalamin.

24. The oral pharmaceutical composition of claim 8, wherein said pharmaceutical composition comprises about 0.1 to 1.5 mg of vitamin B12 and from 25 to 100 mg of monosodium N-[8-(2-hydroxybenzoyl) amino]caprylate.

25. The oral pharmaceutical composition of claim 8, wherein the vitamin B12 is a cobalamin.

26. The oral pharmaceutical composition of claim 8, wherein the vitamin B12 is cyanocobalamin.

27. The oral pharmaceutical composition of claim 8, wherein the vitamin B12 is hydroxocobalamin.

28. The oral pharmaceutical composition of claim 8, wherein the vitamin B12 is methylcobalamin.

29. The oral pharmaceutical composition of claim 8, wherein the vitamin B12 is 5-deoxyadenosylcobalamin.

30. The oral pharmaceutical composition of claim 24, wherein the vitamin B12 is a cobalamin.

31. The oral pharmaceutical composition of claim 24, wherein the vitamin B12 is cyanocobalamin.

32. The oral pharmaceutical composition of claim 24, wherein the vitamin B12 is hydroxocobalamin.

33. The oral pharmaceutical composition of claim 24, wherein the vitamin B12 is methylcobalamin.

34. The oral pharmaceutical composition of claim 24, wherein the vitamin B12 is 5-deoxyadenosylcobalamin.

35. The method of claim 1, wherein the substance in component (2) is monosodium N-[8-(2-hydroxybenzoyl)amino]caprylate.

36. The oral pharmaceutical of claim 8, wherein the substance in component (2) is monosodium N-[8-(2-hydroxybenzoyl)amino]caprylate.