WO1997036598A1

WO1997036598A1 - Use of aminoalkylcarboxylic derivatives

Info

Publication number: WO1997036598A1
Application number: PCT/US1997/005114
Authority: WO
Inventors: Richard D. Lippman; A. Glenn Braswell
Original assignee: Lippman Richard D; Braswell A Glenn
Priority date: 1996-03-29
Filing date: 1997-03-28
Publication date: 1997-10-09
Also published as: AU2595297A

Abstract

The present invention is directed to uses of aminoalkylcarboxylic compounds represented by structural formula (I), wherein m is 0, 1, or 2; n is 0 or 1; R1 is hydrogen or methyl; R2 is hydrogen or methyl; R3 is hydrogen or methyl; R4 is hydrogen or methyl; and R5 is selected such that the compound is an acid (R5 = H) or a pharmaceutically acceptable salt or ester thereof. The formula (I) is used with vitamins to make a topical transdermal composition. Aminoalkylcarboxylic compounds of formula (I) comprise 0.001 % to 90 % by weight of the topical solution. Preferably, the aminoalkylcarboxylic compounds of formula (I) comprise 0.5 % to 25 % by weight, and most preferably 2 % to 6 % by weight. In another embodiment, a topical composition for alleviating low normal to abnormal vitamin deficiencies in humans is disclosed wherein said composition comprises: a transdermally effective amount of an amino acid selected from the group consisting of alanine, glycine, dimethylalanine and dimethylglycine; and a vitamin. In a further embodiment the vitamin is selected from the group consisting of vitamin B-12, folate, vitamin C and vitamin A.

Description

USE OF AMINOALKYLCARBOXYLIC DERIVATIVES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from provisional application No. 60/014,751 filed March 29, 1996, which is incorporated herein in its entirety by this reference.

FIELD OF THE INVENTION The present invention generally pertains to topically applied transdermal solutions useful for administering vitamins to humans to achieve sustained release of the vitamins.

BACKGROUND OF THE INVENTION

The accurate and early diagnosis of vitamin deficiencies is especially important since vitamin deficiencies can lead to numerous incapacitating and life threatening abnormalities. Many of these abnormalities can be completely reversed with appropriate vitamin treatment.

Both vitamin B-12 and folate (or folic acid) are known to be essential to human metabolism and are obtained exogenously by foods such as meat and diary products. Exogenous sources of vitamin B-12 and folate can be supplemented with oral (enteral) administration or by injection (parenteral).

Oral and injected vitamin B-12, folate and other vitamins have been used to treat many disorders such as megaloblastic anemia, pernicious anemia, leukopenia, thrombocytopenia, and hypermetabolic states such as hyperthyroidism. Without a diagnosed folate deficiency, vitamin B-12 deficiency by itself is known to cause degeneration of the spinal cord's myelin sheath which in severe cases impairs nerve conduction.

According to current medical practice, vitamin B-12 deficiency should be suspected in individuals with significant anemia, i.e., a decreased level of hemoglobin or a decreased hematocrit, in whom the red blood cells are macrocytic, i.e., mean cell volume is generally greater than 10011., or in individuals who have neurologic abnormalities consisting of peripheral neuropathy and/or ataxia. The administration of exogenous vitamin B-12 almost always stops the progression of neuropsychiatric abnormalities and often leads to their complete correction. Furthermore, U.S. Patent No. 4,940,658 issued 1990 to Allen teaches that the clinical spectrum of vitamin B-12 deficiency is broader than previously recognized and that many vitamin B-12 deficient patients demonstrate normal levels of vitamin B-12 (albeit in the lower range of normal) in a clinical assay. These patients can benefit greatly from the administration of vitamin B-12.

Normal plasma contains approximately 504 to 652 pg of vitamin B-12 per milliliter of serum according to extensive assay investigations of normal populations sponsored by Ciba Corning Diagnostics Corp. All vitamin B-12 in plasma is bound to transport proteins of considerable physiologic complexity which are transcobalamin I (Tel) and transcobalamin II (Tell). When vitamin B-12 enters the bloodstream, 90% of it is initially bound by Tell. The

Tell vitamin B-12 complex is rapidly cleared from the plasma in minutes, and subsequently, Tel takes control in order to form Tel vitamin B-12 complexes. These complexes then carry most of the plasma bound vitamin B-12 which clears the body slowly with a half-life of 9 or 10 days as described in 1 Folates andPterins 9-10, 535-538 (1984). Tel has substantial capacity to bind vitamin B- 12 up to a maximum of about 939 pg/ml of plasma. This suggests that volunteers who ingest large quantities of vitamin B-12 in their diets by eating large quantities of meats, etc., can reach a maximum level of about 939 pg of vitamin B-12 per milliliter plasma. Any excess vitamin B-12 would be rapidly excreted. (See, B-12. Dolphin, D. ed., Vol. 2, Wiley & Sons, N.Y. pp. 18-23.) On the other hand, in the case of folate, no similar phenomena of rapid excretion after reaching a given level is known.

Administering supplemental vitamin B-12 beyond the 939 pg ml level by any method or device will only result in its rapid excretion in a matter of minutes by the previously mentioned Tell vitamin B-12 complex mechanism. Thus, by its intrinsic construction and usage, an ideal vitamin medication should be able to supply a sustained release level of vitamin B-12 which could approach and maintain steady state constant levels near the previously mentioned 939 pg/ml concentration maximum. On the other hand, oral and injected vitamin B-12 do not generally maintain steady state, constant concentrations due to their inherent methods of administration.

There are many examples of effective vitamin therapy in the medical literature. For example, in reported studies in "Hyperpigmentation of Skin," Baker et al., Brit. Med. J. (1963)

1713 and in "Hyperpigmentation in Vitamin B-12 Deficiency," 107 Arch, of Dermatology 231 (1973), 21 patients with megaloblastic anemia and generalized hyperpigmentation on the hands and feet and particularly over the joints were completely cleared under vitamin B-12 administration. A study published in 271 New England J. of Medicine 541 (1964) found correction of vitamin B-12 deficiency caused striking and prompt improvement in sense of well being and rise in serum alkaline phosphatase. In 105 Arch, of Internal Medicine 352 (1960), large doses of folic acid (5-10 mg daily) produced sizable reticulocyte responses and suboptimal hemoglobin regeneration in vitamin B-12 and folate deficient patients. Both iii Brit. Med. J. 308 (1975) and ii Brit. J. Oral Surg. 165 (1975), reported a study of 200 patients with recurrent oral ulceration,

36 had vitamin B-12 estimations, 15 had deficiency, and replacement treatment led to resolution in 77% and improvement in 8%. In the case of folate deficiency only, the same study showed improvement or resolution of deficiency in 10 patients out of 13. In I Lancet 899 (1966) a 54 patient study found that all responded to folic acid treatment for megaloblastic anemia. In an earlier issue of the British Medical Journal (Clinical Trails 1969), a study of 14 patients who exhibited clinical and laboratory signs of neurological disease and general vitamin B-12 deficiency showed marked or variable improvement after treatment with vitamin B-12.

Administration of exogenous vitamins can also be used to effectively treat many other clinical conditions. For example, a deficiency in folate can lead to similar hematologic disorders as described above with vitamin B-l 2 deficiencies and these disorders are usually reversible with folate treatment. There are many other examples such as the administration of exogenous vitamin C being employed in the retardation or correction of the following abnormalities or deficiencies: aminoacidaemia, anaemia, thalassemia, microbial action, leprosy, urinary tract infections, atherosclerosis, collagen disorders, common cold, deficiency states, malignant neoplasms, neutrophil dysfunctions, pressure sores, prickly heat, scurvy, and thromboembolism. Another example is the administration of exogenous vitamin D which is employed in the retardation or correction of the following abnormalities or deficiencies: epilepsy, hyperparathyroidism, hypocalcaemia, hypoparathyroidism, hypophosphatemia, osteomalacia, osteoporosis, renal failure, rheumatoid arthritis rickets, and tetany. Administration of exogenous vitamin E is employed to retard or correct the following abnormalities or deficiencies: anaemia, cardiac disease, deficiency syndrome, epidermolysis bullosa dystrophia, muscular dystrophy, neuralgia, porphyria, psychosis, poisoning, and as an antioxidant. A further example is the administration of exogenous vitamin A which is employed in the retardation or correction of the following abnormalities or deficiencies: deficiency syndrome, Hurler's disease, leukoplakia, malignant neoplasms, night blindness, porphyria, sunburn, skin disorders, vaginitis, and wound healing.

Regarding vitamin C, a study by L. Steingold et al., 3 Clinical Trails Journal 459 (1966), showed that anemia could be prevented in pregnancy by treatment with ascorbic acid together with iron rather than iron supplements alone. A study by C. E. Dent in 6 Prescribers' Journal 17 (1966), showed that a deficiency of vitamin D causing rickets could be cured rapidly with a 100 microgram daily vitamin D dose. In a double blind study of 52 infants D. K. Melhorn and S. Gross, 10 International Pharmacology Abstract 31 (1973), showed that anemia caused by inadequate vitamin E could be cured by a 5 mg daily vitamin E dose. A study by G. Venkataswamyin, 51 British Journal of Ophthalogy 854 (1967), showed that vitamin A deficiency in 108 patients and night blindness in 71 patients could be significantly improved by administration of vitamin A supplements.

Topical application of drugs and vitamins are preferred over other methods of administration, i.e., oral (enteral) and by injection (parenteral). However, there are significant limitations and disadvantages to each method which may be summarized as follows: 1. In the case of oral administration, there is a lack of tolerance, especially in the gastrointestinal tract. 2. In the case of oral administration, there is risk of inactivation and/or poor absorption in the gastrointestinal tract.

3. In the case of injection, there is risk of needle injury, infection and emotional trauma.

4. In the case of topical application, few chemical entities can be absorbed through the skin or mucous membranes in efficacious quantities or at an efficacious rate of absorption.

5. Both oral and parental methods risk undesired side effects, especially overdosing and or underdosing the patient. For example, a local dosage for a local effect, i.e., "magic bullet," is often desired, but a larger systemic dose must be given to achieve an effective concentration at the local site where needed. This results in undesired side effects since many substances used in higher doses cause dose related, negative, systemic side effects. In addition, when high dosages are administered, there is risk that the substance's concentration may drop below its efficacious level if a subsequent dosage is omitted or delayed.

6. Intravenous infusion has an advantage of providing a sustained release of a substance, but a significant disadvantage in that it is cumbersome and requires close supervision by trained medical personnel.

Trained practitioners in the art of topical and transdermal methods are familiar with only a limited number of techniques and devices that deliver a substance at therapeutic levels through a patient's skin. These techniques and devices usually deliver a substance at a steady state constant rate through the skin. These devices typically include a reservoir enclosed in a membrane through which a drug diffuses at a steady state constant rate. The device is then attached mechanically or adhesively to the skin. Chemical substances will diffuse from the device and permeate epidermal and cutaneous skin layers until they are absorbed by the small blood vessels of the micro vascular system. When these substances enter the bloodstream, they are distributed throughout a patient's body. While such topical or transdermal administration works well for a few substances, i.e., nicotine, nitroglycerin and Dramamine®, such administration is generally unworkable for most chemicals, drugs, vitamins and nutrients. Additionally, very few health practitioners know of or use transdermal administration. This remarkable lack of usage and knowledge of the art is hampered by five problems: 1. The art's devices do not allow an adequate flow rate through the skin.

2. Large molecules such as buprenorphine (see U.S. Patent No. 3,433,791, (Adams, 1969)) do not readily penetrate the skin at a therapeutical ly acceptable and efficacious rate. 3. Common packaging materials do not work well with lipophilic and hydrophilic solvents used in a topical or a transdermal delivery system.

4. Shelf lives of substances contained in topical or transdermal devices are severely limited as a result of the adverse effects of moisture, oxygen and light.

5. Adhesives which secure such devices to a patient's skin are not in isolation from substances and solvents in the topical or transdermal delivery system. This lack of isolation affects adversely the binding of the adhesive to the skin and the transport quantities of substances and solvents.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions useful for sustained release treatment of low normal and abnormal vitamin deficiencies with a topically applied transdermal solution employing aminoalkylcarboxylic acid derivatives and a vitamin. One embodiment of the present invention comprises administering to humans a pharmaceutically acceptable composition, wherein the composition comprises an aminoalkylcarboxylic acid compound or a pharmaceutically acceptable salt or ester of said aminoalkylcarboxylic acid compound or mixtures thereof, wherein said compounds are represented by the following structural formula:

wherein m is 0, 1 , or 2, n is 0 or 1, Ri is hydrogen or methyl, R2 is hydrogen or methyl, R3 is hydrogen or methyl, R4 is hydrogen or methyl, and R5 is selected such that the compound is an acid (R5 = H), or a pharmaceutically acceptable salt or ester thereof; and vitamin B- 12 or folate.

By "pharmaceutically acceptable salts or esters" there are understood to be (a) compounds of the above formula in which the carboxyl group is present in the form of a carboxylic acid salt

— COO^'-VT (where M⁺ is a cation, e.g., alkali metal, alkaline earth metal, cationic amine - such as NH₄ ⁺, etc.); and (b) compounds of the above formula in which the carboxyl group is present in the form of an ester — COOR, (where R is alkyl, aryl, aralkyl, alkaryl, etc.).

Aminoalkylcarboxylic compounds of the above formula comprise 0.001% to 90% by weight of the composition. Preferably, the aminoalkylcarboxylic compounds of the above formula comprise 0.5% to 25% by weight, and most preferably 2% to 6% by weight of the composition. In another embodiment, a topical composition for alleviating low normal to abnormal vitamin deficiencies in humans is disclosed wherein said composition comprises: a transdermally effective amount of an amino acid selected from the group consisting of alanine, glycine, dimethylalanine and dimethylglycine; and a vitamin. In a further embodiment the vitamin is selected from the group consisting of vitamin B-12, folate, biotin, vitamin C, vitamin A, vitamin E and vitamin D.

The topical or transdermal compositions of the present invention described herein permit sustained release of vitamin B-12, folate or other vitamins. This sustained release or steady state constant rate would allow low normal or abnormal deficient patients to obtain and to sustain high systemic levels of vitamins, and therefore, these patients would achieve a more optimal level of health.

DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the following drawings wherein: FIG. 1 is a graph of μmoles of hydrogen peroxide per cm² skin over time after the administration of vitamin C in accordance with the present invention;

FIG. 2a is a graph of serum vitamin B-12 levels over time after the administration of vitamin B-12 in accordance with the present invention;

FIG. 2b is a graph of serum folic acid levels over time after the administration of folate in accordance with the present invention; and

FIG. 3 is a graph of the photomultiplier data collected from glial cells incubated with vitamin E, vitamin C, copper chloride, and control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to compounds which are useful for transdermal administration of vitamins. In particular, the invention relates to topical compositions including vitamins and aminoalkylcarboxylic acid derivatives or a pharmaceutically acceptable salt or ester thereof.

Human skin is wholly permeable to the absorption and transport of some organic molecules. A solution of vitamins and aminoalkylcarboxylic acids is affixed against the skin, and the "uptake" process commences. The term "uptake" as used herein generally means that organic molecules or substances travel through the skin and into the bloodstream of humans.

The uptake must be "significant", and this term used herein would mean substantially that at least a nine percent increase of a substance would enter the bloodstream, i.e., a patient with a blood level of vitamin B-12 at 600 pg/ml serum increasing to 654 pg/ml. On the other hand, insignificant uptake would mean less than a nine percent increase, e.g., an increase in vitamin

B-12 from 600 to 612 pg/ml.

The aminoalkylcarboxylic acid derivatives useful in practice of principals of this invention may be represented by the following compositional formula:

wherein m is 0, 1 , or 2; n is 0 or 1; Ri is hydrogen or methyl; R2 is hydrogen or methyl; R3 is hydrogen or methyl; R4 is hydrogen or methyl; and R5 is selected such that the compound is an acid (R5 = H) or a pharmaceutically acceptable salt or ester thereof. Pharmaceutically acceptable salts or esters of the compounds of the above formula are useful in practice of the present invention. Furthermore, mixtures of compounds of the above formula including the pharmaceutically acceptable salts or esters may be used in accordance with practice of principals of this invention. Particularly preferred compounds of the above formula are aminoacetic acid (glycine or aminoethanoic acid), dimethylaminoacetic acid (dimethylglycine or dimethylaminoethanoic acid), aminopropionic acid (alanine or 3- aminopropionic acid), and dimethylaminopropionic acid (dimethylalanine or dimethyl-3- aminopropionic acid). The compounds of the above formula are known and may be prepared according to methods disclosed in the literature from known materials. Such references include: U.S. Patent No. 2,663,713 (White et al. 1953); Viscontini, Meier, 33 Helv. Chim. Acta 1773 (1950);

Kenadall, McKi__zie,J C .- Soc. 1399 (1931); the disclosures of which are incorporated herein by reference.

Preferred compositions for topical administration contain from about 0.5% to about 25% by weight of active ingredient (the aminoalkylcarboxylic acid derivative) of the above formula in either acid, ester or salt form. Since the compounds of the above formula are active by themselves as transdermal carriers of vitamins to the bloodstream, they are contemplated as the only "active ingredient" in the compositions along with the vitamins provided in accordance with practice of principles of this invention. More preferably the compositions comprise from about 2% to about 6% by weight of the aminoalkylcarboxylic acid derivative. If the composition contains less than about 0.1%, it will be only marginally effective and greater than about 25%, the economics would not be as favorable as desired, and such high concentrations could possibly produce detrimental side effects. However, compositions for topical application where the aminoalkylcarboxylic acid derivative is in concentrations of from 0.001% to 90% by weight are contemplated. Preferably, the concentration of vitamins in the composition is from about 0.2% to about 8% by weight. It is thought that at less than 0.2% by weight of vitamin, the dosage would not be sufficient to correct vitamin deficiencies, and at more than 8%, there would be a possible overdosage with possible accompanying toxicity problems.

Referring to the above formula, if m is 3 or greater and/or if n is 2 or greater, the compound will not be able to facilitate transport of vitamins as readily as desired through outer skin layers to the bloodstream. It is thought that the use of other transport carriers and penetration enhancers known to practitioners of the art such as ozone, acetamide (DMA), dimethylsulfoxide (DMSO), dimethylformamide (DMF), in place of the compounds of the present invention are not desired. This is because the aforementioned carriers are known to be toxic or have serious side effects as described in Martindale, The Extra Pharmacopeia, Pharm. Press, London, (1992). In contrast with the transport carriers known in the art, the compounds of the present invention are not toxic and do not have serious side effects. Therefore, the compounds of the above formula provided in accordance with the present invention are able to transport vitamins more safely and effectively through the skin of a human than are the aforementioned carriers. Ri in the above formula is a hydrogen or methyl. Without being bound by theory, it is thought that having a hydrogen or methyl at Ri tends to draw electron density to the nitrogen atom of the amine moiety. This increases electron density over the nitrogen, endowing it with a slightly less positive charge. On the other hand, the double bond of the carboxylic moiety spreads its electron density over the entire carboxylic moiety, endowing it with a slightly more positive charge. These positive and negative charges on opposing ends of the compounds of the present invention tend to neutralize charges of membrane molecules of the skin, and thus, they facilitate transport of even large vitamin molecules, e.g., vitamin B-12 (m.w. = 1355.4 daltons), without toxic or other serious side effects.

One important feature of the aminoalkylcarboxylic derivatives set forth as being useful in accordance with practice of the present invention is that they have two highly charged, negative and positive, electron density groups per molecule. A second important feature is that the aminoalkylcarboxylic derivatives are readily metabolized upon completion of their transport function without hamiful toxic or other side effects. None of the other known carrier substances previously disclosed by the literature to be useful in sustained release or as transport carriers have this double feature. A third important feature of the compounds of the present invention is that they are all hydrophilic while all aforementioned prior-known carriers are substantially lipophilic. This hydrophilicity can be modified substantially in the direction of lipophilicity if needed for a desired transport of a specific vitamin. Thus the compounds of the above formula possess unique, surprising, and nonobvious properties since they are nontoxic transport enhancers featuring opposing electron density charges and variable hydrophilicity/lipophilicity structures.

The composition and methods of the present invention employ vitamins and aminoalkylcarboxylic acid derivatives or a pharmaceutically acceptable salt or ester thereof. Any pharmaceutically acceptable salt known in the art can be used. Typical salts include alkali metal, alkaline earth metal, ammonia, or organic amine salts as, for example, sodium, potassium, magnesium, calcium, protonated amines such as those derived from ethylamine, triethylamine, ethanolamine, diethylaminoethanol, para-aminobenzoic acid, ethylenediamine, piperidine, morpholine, 2-piperidinoethanol, benzylamine, procaine, and the like. Typical esters include substantially any alkylate such as methylate, acetate, propionate, cholesterate and the like. These esters favor interaction with other desired proteins, nucleic acids and lipid bilayers of the cell. The treatment of vitamin deficiencies or the remission of vitamin deficiency problems in humans is carried out preferably by employing compositions in which effective amounts of vitamins are admixed with the aminoalkylcarboxylic derivatives or pharmaceutically acceptable salts or esters thereof and with acceptable pharmaceutical excipients. Suitable pharmaceutical excipients are those which are typically employed in the topical application of pharmaceuticals and cosmetics. Examples of topically applied pharmaceutical excipients include ointments, creams, gels, solutions, lotions, foams, films, tablets, capsules, pastes, plasters, bandages, pads, tapes and patches, implants, osmotic devices, and transdermal or transmucosal therapeutic systems. The compositions and methods of this invention utilize an effective amount of vitamins and aminoalkylcarboxylic acid derivatives or a pharmaceutically acceptable salt or ester thereof. Typically, an effective amount of the aminoalkylcarboxylic acid derivative is from about 0.01% to about 25.0% by weight of the total composition, while the effective amount of vitamins is from about 0.2% to about 8% by weight of the total composition. The vitamins useful with the present invention may be any vitamin when the aminoalkylcarboxylic derivative is selected from the group consisting of alanine, glycine, dimethylalanine and dimethyl glycine with the following amino acids being preferred: vitamin B-12, folate, biotin, vitamin A, vitamin C, vitamin D, and vitamin E. Other aminoalkylcarboxylic derivatives can be combined with vitamin B-12 and folate.

Topical or transdermal creams and lotions may contain the active ingredients in admixture with conventional cosmetically and pharmaceutically acceptable excipients, e.g., moisturizers and humectants such as uric acid, reticulan, polymucosaccharides, hyaluronic acid, and aloe vera.

Topical or transdermal creams and lotions may all contain various preservatives.

Examples of such preservatives are derivatives of butylated hydroxy-toluene (BHT), butylated hydroxyanisole (BHA), methionine, cystein, ethoxyquin, nordihydroguairetic acid (NDGA), ascorbic acid, tocopherol, catalase, superoxide dismutase, glutatione, glutatione peroxidase, 2- mercaptoethylamine, cystamine, benzoic acid, ethyl-o-hydroxybenzoate, ethylenediamine, tetraacetic acid, and all other mercaptans, thiols and disulfides. The pharmaceutical and cosmetic preparations provided in accordance with practice of principals of this invention may contain up to about 90% by weight of the aminoalkylcarboxylic acid derivatives or pharmaceutically acceptable salts or esters of the present invention in combination with the above described substances or adjuvants.

The foregoing recitation of materials to be used in compositions containing compounds of the present invention are presented for purposes of illustration and not limitation, it being understood that a variety of equivalent materials could also be used if desired.

In the treatment of vitamin deficiencies and remission of vitamin deficiencies, compositions according to the present invention comprising vitamins and aminoalkylcarboxylic acids, or a pharmaceutically acceptable salt or ester thereof should be topically applied to the skin from one to five times daily. The precise regimen in each case will be determined by the physician based upon the exact diagnosis, the severity of the pathology, its responsiveness to treatment, etc. Another aspect of the invention is to have the vitamin and the aminoalkylcarboxylic derivative in a transdermal patch that can be applied to the skin of a patient over extended periods of time. The patch could be as simple as a plastic type material with an absorbent material adhered to one side of the plastic and an adhesive surrounding the absorbent material. The absorbent material would be impregnated with a mixture of a vitamin and an aminoalkylcarboxylic derivative of the present invention. A preferred adhesive and absorbent material is available from 3M company, Minneapolis, MN, and sold commercially as

"Ocupatch." The transdermal patch could also be patches made in accordance with U.S. Patents Nos. 5,008,110, 4,839,174, 4,533,540, 4,943,435, 4,978,531, and 3,71 1,602, the disclosures of which are hereby incorporated by reference. The patches would be constructed identically as described in the patents with the exception of replacing the active transdermal compounds with compounds of the present invention.

The following examples will serve to further typify the nature of the invention but should not be construed as a limitation on the scope thereof, which is defined solely by the appended claims.

Dosage forms suitable for internal use comprise the active compounds of the above formula in either acid, salt or ester form or mixtures thereof in intimate admixture with a solid liquid which are pharmaceutically acceptable diluents. The preferred pharmaceutical compositions from the standpoint of preparation and ease of administration are cream or liquid compositions containing from about 1 to 40 mg of active ingredient in acid, salt or ester form.

Example 1

An ointment is prepared by combining 8.0% (w/w) of vitamin B-12 and 8.0% (w/w) of folate and 84.0% (w/w) of VASELINE® petroleum jelly (Cheeseborough Manuf. Co.) and blending with a suitable mixer until uniform. The ointment is then applied to the skin of a person.

Example 2 Vitamin B-12, 0.2% (w/w), and folate, 0.2% (w/w), are dissolved in a uniform mixture of 6.0%_> (w/w) glycerin and dimethylglycine (DMG) and 93.6% (w/w) of isopropanol in order to form a topical solution. The topical solution is then applied to the skin of a person.

Example 3

Vitamin B-12, 3.0% (w/w), folate 3.0% (w/w), and biotin 0.4% (w/w) are dissolved in a homogenous mixture of 5.6% (w/w) dimethylglycine (DMG) and 88.0% (w/w) of ethanol in order to form a topical solution. The topical solution is then applied to the skin of a person.

Example 4

A solution is prepared from the following components:

In the first container, alcohol is warmed on a steam bath and all ingredients except vitamin B-12, folate, and ascorbic acid are added to the alcohol and dispersed with mixing. In a second container, vitamin B-12 and folate is dissolved in the propylene glycol. The alcohol mixture in the first container is added to the second container, mixing until uniform. Ascorbic acid is then slowly added to the mixture to achieve a pH of about 5.5. Finally more alcohol is added to bring the mixture to final weight. The mixture is then applied to the skin of a person.

Example 5 In Example 5 the composition of Part a is formulated separately from the composition of Part b and then equal amounts of Parts a and b are mixed together.

The ingredients listed under Parts a and b of Example 5 are distributed and manufactured under their respective trademarks by Croda, Inc., New York, N.Y., except for "GG Germabern II" which is manufactured and distributed by Sutton Laboratories, Inc., Chatham, N.J., and "Volatile Silicone 03314" which is manufactured and distributed by SWS Silicones Corp., Adrian, Michigan. The mixtures of Parts a and b are brought to 100 % w/w by adding a carrier such as petroleum jelly, alcohol and the like. The mixture is then applied to the skin of a person.

Example 6 Referring now to FIG. 1 , results of a double blind time study using a composition containing 5% by weight vitamin C can be seen. This composition was the same as the composition set forth in Example 4 with the exception that the percentage of alcohol was reduced to approximately 3.9%, vitamins A, D, and E were excluded, and vitamin C was added to a total of 5% by weight. Starting at time zero, 15 milliliters of this solution was vigorously applied topically onto the skin of the wrist of a volunteer. Measurements were taken at different intervals using noninvasive, reflective, near infrared spectroscopy. The methods of noninvasive, reflective, near infrared spectroscopy used in this example are set forth in detail in R. Lippman, "Rapid In Vivo Quantification and Comparison of Hydroperoxides and Oxidized Collagen in Aging Mice, Rabbits and Man", 20 Experimental Gerontology 1 -5, (1985), which is incorporated herein by reference. Results showed that lipid hydroperoxides in the other skin layers and microvascularization decreased from about 151 to 130 micromole/cm² after about one and a half hours. The pharmacokinetics of topical vitamin C application showed a gradual return to a normal level of 151 micromole/cm² level after approximately 6 hours. This experiment showed that vitamin C uptake did occur and cellular damage due to any substantial deficiency was prevented according to the procedure of this example.

Example 7 This composition and experimental procedure was the same as the composition and experimental procedure in Example 6 with the exception that a 5% by weight concentration of vitamin E was substituted for vitamin C. Table I below is a table of the micromoles/cm² of lipid hydroperoxides as measured in Example 6. Results showed that lipid hydroperoxides in the other skin layers and micrOvascularization decreased from about 158 to 142 micromole/cm² after about one and a half hours. This experiment showed that vitamin E uptake did occur and cellular damage due to any substantial deficiency was prevented according to the procedure of this example.

Table I

Example 8

This composition and experimental procedure was the same as the composition and experimental procedure in Example 6 with the exception that a 5% by weight concentration of vitamin A was substituted for vitamin C. Table II illustrates the results. The results showed that lipid hydroperoxides in the other skin layers and microvascularization decreased from about 155 to 136 micromole/cm² after about one and a half hours. This experiment showed that vitamin A uptake did occur and cellular damage due to any substantial deficiency was prevented according to the procedure of this example. Table II

Example 9

This composition and experimental procedure was the same as the composition and experimental procedure in Example 6 with the exception that a 5% by weight concentration of vitamin D was substituted for vitamin C. Results showed in Table III below demonstrate that lipid hydroperoxides in the other skin layers and microvascularization decreased from about 153 to 135 micromole/cm² after about one and a half hours. This experiment showed that vitamin D uptake did occur and cellular damage due to any substantial deficiency was prevented according to the procedure of this example.

Table III

Example 10

Glial cells from rat and human brain, respectively, were cultivated by monolayering on Petri dishes. Antioxidant versus prooxidant activity was monitored as an indicator of health status, i.e., deficiency, of these brain cells. These cells received 50 micromole supplements each of vitamin E and orotic acid during experiments mixed ultrasonically with 1 micromole of the carrier substance, glycine. Control cell cultures received only 50 micromoles of vitamin E and orotic acid without the carrier glycine. The health status was evaluated using special chemical probes, i.e., luminol-carnitine derivatives, which were pipetted into the glial cell culture media. Uptake of the probes occurred in the mitochondrial inner membrane where oxygen metabolism and toxic oxygen byproducts are produced. The probes emitted chemiluminescent light in proportion to superoxide (0₂ ) and hydroxyl radicals (»OH). This light was measured by a photomultiplier, and the results quantified and recorded. Vitamin E and orotic acid added to the glial cells with glycine significantly reduced the steady state light to 30.8% and 32.9% of controls, respectively. Therefore, vitamin E and orotic acid given with glycine significantly reduced toxic oxygen byproducts and increased glial cell metabolic efficiency and health status compared to giving vitamin E and orotic acid without glycine.

Example 11 The experimental conditions of Example 10 were repeated with different tissue and with the help of the special chemical probes. Mitrochondria from rat and human liver were freshly isolated and maintained in Petri dishes in order that normal respiration and ATP production occurred. 15 micromoles of Vitamins C and E were ultrasonically mixed with 0.3 micromoles of glycine and were added to the mitochondria in a ratio of 1 micromole vitamin C and E to 2 milligrams mitochondria in respective experiments. The control cultures were identically prepared except for the glycine was omitted. Low concentrations, i.e., 1 micromole per 2 mg mitochondria, of vitamins C and E were shown to reduce free radical activity to approximately one-third versus controls. Vitamins C and E are potent antioxidants and scavengers of free radicals, and therefore, the health status of these mitochondria was improved. Referring to FIG. 3, human liver mitochondria were probed with the CML probe and subsequently incubated with (A) 15 μmole vitamin E with 0.3 micromoles glycine, (B) 10 μmole vitamin C with 0.3 micromoles glycine, (C) 30 μmole copper chloride, and (D) control mitochondria only. Rapid injection of superoxide free radicals was quenched by all compounds studied versus controls.

Example 12

Turning to FIGs. 2a and 2b, graphs of results of a pharmacokinetic time course (double blind) study of single topical doses of vitamin B-12 combined with folate and glycine and administered to human volunteers at time zero is shown. Topically applied single doses of vitamin B- 12 of 18 mg and folate of 18 mg were given to volunteers who had fasted at least 12 hours (overnight). Placebos (patches without vitamin B-12 and folate) were given to several other volunteers. Using the above referenced technique of radioimmunoassay according to Ciba Corning Diagnostics Corp., changes in serum concentrations of vitamin B-12 and folate were monitored during 4 hour test periods. The graphs of FIGs. 2a and 2b plot concentrations of vitamin B-12 and folate in pg/ml and ng/ml serum, respectively, on the vertical axis (as measured by the radioimmunoassay technique) versus time in hours on the horizontal axis. Subjects received topically applied, single doses of vitamin B-12 combined with folate, and placebos, respectively. The topical solution was placed using a patch. The patch comprised an absorbent material impregnated with the vitamin B-12, folate, and glycine and covered with a plastic outer layer. On the edges of the plastic outer layer surrounding the absorbent material was an adhesive for securing the patch to the skin. The preferred adhesive is an ocular adhesive purchased from 3M company, Minneapolis, MN. The results, as can be seen in FIGs. 2a and 2b, show that both vitamin B-12 and folate were absorbed into the blood stream after about 1 to 2 hours upon topical application on the underside of either wrist. Vitamin B-12 increased 134% from a baseline level of 265 pg/ml serum to a four-hour level of 620 pg/ml. Folate increased 187% from a baseline level of 4.0 ng/ml serum to a four hour level of 11.5 ng/ml serum. Two nearly flat lines were recorded for several volunteers who received placebos. It is concluded that vitamin B-12 and folate are effectively absorbed into the bloodstream with glycine in the patch and effectively raise serum concentration levels thereof, when taken at regular intervals of at least 4 hours, 3 times daily.

Example 13

Fifty-four volunteers were chosen of which five received a placebo unknown to the administrating doctor. All were of adult age (21 to 76 years of age), male or female, in average health, non-smokers and vegetarians. This meant that they did not eat meat in any form, and therefore, extraneous sources of vitamin B-12 and folic acid from meat were not allowed to interfere with this study's test results. All fifty-four volunteers fasted 12 hours before testing and refrained from eating vitamin supplements a week before testing since extraneous foods and vitamins may interfere with this example's test results. Samples of blood were drawn at time zero in order to establish a baseline for folic acid and vitamin B-12. Five of the fifty-four volunteers received an identical placebo, i.e., red food coloring was substituted for the regular vitamin B-12 and folic acid solution. The remaining 49 volunteers received a topical solution containing 18 mg vitamin B-12, 18 mg folic acid, and all other substances except vitamins according to Example 4. The topical solution was administered on the underside of the wrist of each volunteer at time zero. Further blood samples were drawn after 2 and 4 hours, respectively, while volunteers read, walked or listened to music.

Blood concentrations of only vitamin B-12 and folic acid were determined by known methods of radioimmunoassay as referenced herein (Ciba Corning Diagnostics Corp. Bulletin, Medfield, MA (1993)). Also, hemoglobin (HGB) and erythrocyte sedimentation rates (ESR) were determined separately in order to correlate any possible relationships between them and the transdermal uptake of vitamin B-12 and folic acid.

Radioimmunoassay results showed that 77.6% and 59.2% of the non-placebo volunteers experienced uptake of vitamin B-12 and folic acid, respectively. Of those who experienced uptake, the mean level of vitamin B-12 increased from a normal mean of 614.5 (baseline) to a transdermal mean of 856.6 pg ml serum. This was a 39.4 ± 10.8% increase. Most non-placebo volunteers, i.e., the 77.6%, showed a minimum of a 15.5% increase of vitamin B-12, which was statistically significant for this sampling. Also, most non-placebo volunteers, i.e., the 77.6%, raised thei' vitamin B-12 to clinically significant levels, namely up to a mean of 856.6 pg/ml serum. Th iame group increased their folic acid from a normal mean of 15.6 to a transdermal mean of 21.6 ng ml serum. This was a 38.5 ± 6.9% increase. Again, most non-placebo volunteers, i.e., the 59.2%, showed a minimum of a 9.7% increase of folic acid, which was statistically significant for this sampling. All five volunteers receiving placebos showed no changes in their vitamin B-12 and folic acid levels. Exceptionally healthy volunteers who registered low sedimentation (ESR) rates, i.e., under 5, and high hemoglobin (HGB) rates, i.e., over 14, did not show transdermal uptake of vitamin B-12 nor folic acid. The detailed reasons for this phenomena are unknown, but one hard fact is that their sera was already saturated at maximum levels, i.e., 939 pg of Tel bound vitamin B-12 per milliliter, and any supplemental vitamin B-12 would be excreted in minutes as described in the book, B-12, D. Dolphin, ed., 1, Wiley & Sons, New York, N.Y., pp. 18-23 (1982). The majority of the volunteers were in average health with mean ESR and HGB rates of 13.1 and 13.2, respectively. These rates correspond to an "average" health condition of normal, smoking and non-smoking, American adults. All results were statistically accurate within a 90% confidence level of a normal bell shaped curve.

The present invention is not to be limited to the specific compositions and examples detailed above which are merely illustrative. Various and numerous other compositions and examples may be devised by one skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A topical composition for alleviating low normal to abnormal vitamin deficiencies in humans, said composition comprising:

a transdermally effective amount of an amino acid selected from the group consisting of alanine, glycine, dimethylalanine and dimethylglycine; and

a vitamin.

2. The topical composition of claim 1 wherein the vitamin is selected from the group consisting of vitamin B-12, folate, vitamin C and vitamin A, vitamin E and vitamin D.

3. The topical composition of claim 1 wherein the concentration of amino acid ranges from about 0.5% to about 25% by weight.

4. The topical composition of claim 3 wherein the concentration of amino acid ranges from about 2% to about 6% by weight.

5. The topical composition of claim 2 wherein the concentration of the vitamin ranges from about 0.2% to about 8% by weight.

6. The topical composition of claim 1 wherein three vitamins are selected, vitamin

B-12, folate and biotin.

7. a topical composition for alleviating vitamin deficiencies in humans, said composition comprising:

a transdermally effective amount of an aminoalkylcarboxylic acid compound or a pharmaceutically acceptable salt or ester of said aminoalkylcarboxylic acid compound or mixtures thereof, wherein said compound is represented by the following structural formula:

wherein

m is 0, 1, or 2,

n is 0 or 1,

R₁ is hydrogen or methyl,

R₂ is hydrogen or methyl,

R₃ is hydrogen or methyl,

R₄ is hydrogen or methyl, and

R₅ is selected such that the compound is an acid or a pharmaceutically acceptable salt or ester thereof; and

a vitamin selected from the group consisting of vitamin B-12 and folate.

8. The topical composition of claim 7 wherein the concentration of the aminoalkylcarboxylic acid compound ranges from about 0.5% to about 25% by weight.

9. The topical composition of claim 8 wherein the concentration of the aminoalkylcarboxylic acid compound ranges from about 2% to about 6% by weight.

10. The topical composition of claim 7 wherein the concentration of vitamin B-12 or folate ranges from about 0.2% to about 8% by weight.

11. A transdermal patch comprising:

an outer layer;

an adhesive suitable for use on skin; and

a layer of compounds wherein the compounds are the topical composition of claim 1.

12. A transdermal patch comprising:

an outer layer;

an adhesive suitable for use on skin; and

a layer of compounds wherein the compounds are the topical composition of claim 7.

AMENDED CLAIMS

[received by the International Bureau on 23 September 1997 (23.09.97) ; new claims 13-26 added ; remaining claims unchanged (3 pages ) ]

11. A transdermal patch comprising,

an outer layer,

an adhesive suitable for use on skin; and

12. A transdermai patch comprising:

an outer layer.

an adhesive suitable for use on skin; and

13. A method for treating a human who has a low normal or abnormal vitamin deficiency, comprising topically administering to said human a composition which comprises:

a transdermally effective amount of an ammo acid selected from the group consisting of alanine, glycine, dimethylalanine and dimethylglycine: and

at least one vitamin.

14. The method of claim 13 wherein the vitamin is selected from the group consisting of vitamin B-12, folate, vitamin C and vitamin A, vitamin E and vitamin D.

15. The method of claim 13 wherein the concentration of amino acid ranges from about 0-5% to about 25% by weight.

16. The method of claim 15 wherein the concentration of amino acid ranges from about 2% to about 6% by weight.

17. The method of claim 14 wherein the concentration of said vitamin ranges from about 0.2% to about 8% by weight.

18. The method of claim 13 wherein three vitamins are included, wherein said vitamins are vitamin B-12, folate and biotin.

19. The method of claim 13 wherein said topical administration of said composition is by means of a transdermal patch.

20. The method of claim 13 wherein said composition is in a form selected from a cream and a lotion.

21. A method for treating a human who has a low normal or abnormal vitamin deficiency comprising topically administering to said human a composition which comprises:

wherein

m is 0, 1, or 2,

n is 0 or 1,

R₁ is hydrogen or methyl,

R₂ is hydrogen or methyl,

R₃ is hydrogen or mathyl,

R₄ is hydrogen or methyl, and

a vitamin selected from the group consisting of vitamin B-12 and folate.

22. The method of claim 21 wherein the concentration of the aminoalkylcarboxylic acid compound ranges from about 0.5% to about 23% by weight.

23. The method of claim 22 wherein the concentration of the aminoalkylcarboxylic acid compound ranges from about 2% to about 6% by weight.

24. The method of claim 21 wherein the concentration of vitamin B-12 or folate ranges from about 0.2% to about 8% by weight.

25. The method of claim 21 wherein said topical administration of said composition is by means of a transdermal patch.

26. The method of claim 21 wherein said composition is in a form selected from a cream and a lotion.