WO1991002535A1

WO1991002535A1 - Cancer prevention composition and method

Info

Publication number: WO1991002535A1
Application number: PCT/US1990/004508
Authority: WO
Inventors: Gerald Shklar
Original assignee: Basil, Inc.
Priority date: 1989-08-11
Filing date: 1990-08-09
Publication date: 1991-03-07

Abstract

Disclosed is a method of preventing cancer in animals involving mixing the animal's food with an exogenous source of vitamin E in admixture with a carotene. The vitamin E and carotene should be in sufficient quantities to prevent tumor growth or occurrence in the animal when administered on a periodic basis. Typically the vitamin E will be dl-alpha tocopherol and will be present in quantities containing 5 to 32 I.U. of vitamin E per kilogram of animal body mass when administered on a daily basis. The carotene will typically be beta carotene and will be present in quantities containing about 140 to 3400 I.U. of carotene per kilogram of animal body mass when administered on a daily basis. In one embodiment, the admixture of vitamin E and carotene is contained within an airtight pouch impermeable to the admixture as a unit dose packet. The invention also includes improvements in animal food. In one embodiment, the animal food will contain exogenous sources of vitamins E and C, glutathione and a carotene.

Description

CANCER PREVENTION COMPOSITION AND METHOD

BACKGROUND OF THE INVENTION Field: This invention generally relates to various compositions and methods for preventing and treating tumors in animals. It specifically relates to the administration of nutritional compositions for the prevention of cancer in animals.

State of the Art: Attempts to prevent or treat various diseases including tumors and cancers with vitamins have been made before. For example, U.S. Patent No. 4,564,686 to Ogata discloses a phosphoric acid diester having a ascorbic acid moiety and tocopherol moiety. The agent of Ogata is used as a proplylactic and therapeutic agent for cataracts.

U.S. Patent No. 4,323,581 to Gander discloses the use of a retinoid to prevent and treat certain forms of epithelial cancer.

U.S. Patent No. 4,665,098 to Gibbs, et al. discloses the use of the same retinoid as Gander in the treatment of bladder cancer, psoriasis, and acne.

U.S. Patent No. 4,320,141 to Komatsu, et al. discloses the use of a ef--tocopherol vitamin A acid ester to treat tumors.

Dosage forms for humans containing vitamin E, . vitamin C and beta carotene are also available. For example, E.R. Squibb & Sons, Inc. of Princeton, New Jersey markets a multivitamin tablet under the name Theragran^® containing these and many other nutrients. Solaray, Inc. of Ogden, Utah has marketed Super Dry ADE¹" vitamin capsule containing vitamin A and D (as fish liver oil) , beta carotene, and vitamin E (d-alpha tocopheryl succinate) . Heretofore, the ability of these agents to act synergistically to prophylactically prevent cancer in animals has not been recognized. - 2 -

SUMMARY OF THE INVENTION

The invention includes a method of preventing cancer in an animal involving mixing the animal's food with an exogenous source of vitamin E in admixture with a carotene. The vitamin E and carotene should be in sufficient quantities to prevent tumor growth in the animal when administered on a periodic (e.g. daily or twice daily) basis. Typically the vitamin E will be dl-alpha tocopherol and will be present in quantities containing about 5 to about 32 international units (I.U.) of vitamin E per kilogram of animal body mass when administered on a daily basis. The carotene will generally be beta-carotene and will be present in quantities containing about 140 to about 3400 I. U. of beta carotene per kilogram of animal body mass when administered on a daily basis.

In one embodiment, the admixture of vitamin E and carotene is contained within an airtight pouch impermeable to the admixture so that the vitamin E or other anti-oxidant do not oxidize too rapidly. The pouch or unit dose packet will typically contain a single dosage of the admixture for mixing with the animal's food.

The invention also includes improvements in animal food. Typical animal foods (e.g. dog food) contain a carbohydrate source, a protein source, and a fat mixture. In the invention, the food is supplemented with a vitamin mixture comprising a sufficient quantity of vitamin E and carotene to prevent tumor occurrence or growth in an animal.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The term "vitamin E" as used herein is intended to include vitamin E from natural sources as well as pure alpha tocopherol, alpha tocopherol alkanoates in which the alkanoate has from one to five carbon atoms, and alpha- tocopherol acid malonate, succinate and glutamate. The dextrorotary compound is preferred.

Vitamin E, in its various forms, is available from various sources. For its use in "dry" dosage forms (e.g. tablets, powders, hard gelatin capsules) , a 50 percent dry powder of dl-alpha tocopherol acetate (3,000 I.U. per 6 grams powder) is available from Hoffman LaRoche, Inc. of Nutley, New Jersey. Liquid forms of vitamin E are available from Nature Made Nutritional Products of Los Angeles, California.

Among the carotenes ("carotenoids") useful in the present invention are beta carotene, alpha carotene, ca thaxanthin, and mixtures thereof. Beta carotene (pro vitamin A) and alpha carotene are readily commercially available from Hoffman-LaRoche and other sources.

Canthaxanthin is available under the trademark Orobronze from William H. Rorer (Canada) Ltd. of Bramalea, Ontario.

Vitamin C, as used herein, includes ascorbic acid, sodium ascorbate, and the lipid soluble ascorbyl palmitate. Vitamin C is available from Bronson Pharmaceuticals of LaCanada, California.

Glutathione contains the amino acid cysteine, and is believed to be an anti-oxidant. Glutathione is available from Sigma Chemical Company of St. Louis, Missouri. "Animal food" as used herein is generally intended to refer to commercially prepared animal foods and feeds such as those available from Purina; Kal Kan Foods, Inc. of Vernon, California; and Carnation Company of Los Angeles, California. However, it is to be understood that other sources of animal nutrition are also included, such as table scraps. Absorption of vitamin E and beta carotene is enhanced by admixture with fat containing foods.

To induce tumor regression in an animal, the animal food will generally be mixed to contain dl-alpha tocopherol which will be present in quantities containing from about 5 to about 7 I.U. (3 to 20 mg of dl-alpha tocopherol of vitamin E per kilogram of animal body mass when administered on a daily basis. The animal food will also be mixed to contain beta-carotene which will be present in quantities of about 3 to about 4 mg of beta-carotene per kilogram of animal body mass when administered on a daily basis. In one embodiment, the animal food will further contain vitamin C and glutathione. The vitamin C will then be present in quantities of about 14 to about 28 milligrams per kilogram of animal body mass and the glutathione will be present in quantities of about 4 to about 8 milligrams per kilogram of animal body mass, both on a daily basis.

To prevent tumors (i.e. for use as a prophylactic), the previously described animal food will be mixed with a vitamin source containing a sufficient quantity of vitamin E and carotene to prevent tumor growth in the animal. Typically, the animal food will contain dl-alpha tocopherol present in quantities containing from about 5 to about 32 I.U. of vitamin E per kilogram of animal body mass when administered on a daily basis. The carotene will preferably be beta carotene and will preferably be present in quantities containing about 275 to about 3400 I.U. of beta-carotene per kilogram of animal body mass when administered on a daily basis. The prophylactic animal food may also contain vitamin C and glutathione, with the vitamin C present in quantities of about 14 to about 28 milligrams per kilogram of animal body mass and the glutathione present in quantities of about 4 to about 8 milligrams per kilogram of animal body weight on a daily basis.

The compositions of the present invention are preferably presented for administration to animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, suppositories, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or suspensions, and oral solutions or suspensions and the like, containing suitable quantities of an active ingredient.

For oral administration either solid or fluid unit dosage forms can be prepared.

Powders are prepared quite simply by comminuting the active ingredient (e.g. powdered vitamin E) to a suitably fine size and mixing with a similarly comminuted diluent. The diluent can be an edible carbohydrate material such as lactose or starch. Advantageously, a sweetening agent or sugar is present as well as a flavoring oil Capsules are produced by preparing a powder mixture as hereinbefore described and filling onto formed gelatin sheaths. Advantageously, as an adjuvant to the filling operation, a lubricant such as talc, magnesium stearate, calcium stearate and the like is added to the powder mixture before the filling operation.

Soft gelatin capsules are prepared by machine encapsulation of a slurry of active ingredients with an acceptable vegetable oil, light liquid petrolatum or other inert oil or triglyceride.

Tablets are made by preparing a powder mixture, granulating or slugging, adding a lubricant and pressing into tablets. The powder mixture is prepared by mixing an active ingredient, suitably comminuted, with a diluent or base such as starch, lactose, kaolin, dicalcium phosphate and the like. The powder mixture can be granulated by wetting with a binder such as corn syrup, gelatin solution, methylcellulose solution or acacia mucilage and forcing through a screen. As an alternative to granulating, the powder mixture can be slugged, i.e., run through the tablet machine and the resulting imperfectly formed tablets broken into pieces (slugs) . The slugs can be lubricated to prevent sticking to the tablet-forming dies by means of the addition of stearic acid, a stearic salt, talc or mineral oil. The lubricated mixture is then compressed into tablets.

Advantageously the tablet can be provided with a protective coating consisting of a sealing coat or enteric coat of shellac, a coating of sugar and methylcellulose and polish coating of carnauba wax. Fluid unit dosage forms for oral administration such as syrups, elixirs and suspensions can be prepared wherein each teaspoonful of composition contains predetermined amounts of active ingredients for administration. The water- soluble forms can be dissolved in an aqueous vehicle together with sugar, flavoring agents and preservatives to form a syrup. An elixir is prepared by using a hydroalcoholic vehicle with suitable sweeteners together with a flavoring agent. Suspensions can be prepared of the insoluble forms with a suitable vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like. For parenteral administration, fluid unit dosage forms are prepared utilizing active ingredients and a sterile vehicle, ethyl oleate being preferred especially for* intramuscular injection. The active ingredients, depending on the form and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions water- soluble active ingredients can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampule and sealing. Advantageously, adjuvants such as local anesthetic, preservative and buffering agents can be dissolved in the vehicle. Parenteral suspensions are prepared in substantially the same manner except that active ingredient is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. Active ingredients can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active ingredients.

In addition to oral and parenteral administration, the rectal and vaginal routes can be utilized. An active ingredient can be administered by means of a suppository. A vehicle which has a melting point at about body temperature or one that is readily soluble can be utilized. For example, cocoa butter and various polyethylene glycols (Carbowaxes) can serve as the vehicle.

The active ingredients can also be admixed in animal feed. The active ingredients can conveniently be prepared in the form of a food premix. The food premix can comprise active ingredients in admixture with an edible pharmaceutical diluent such as starch, oatmeal, flour, calcium carbonate, talc, dried fish meal and the like nontoxic, orally acceptable pharmaceutical diluents. The prepared premix is then conveniently added to the regular feed.

The term "unit dosage form" as used in the specification and claims refers to physically discrete units suitable as unitary dosages for animal subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitation inherent in the art of compounding such an active material for therapeutic use in humans, as disclosed in this specification, these being features of the present invention. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, troches, suppositories, powder packets, wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampules, vials, segregated multiples of any of the foregoing, and other forms as herein described.

The active ingredients to be employed as anti-tumor agents can be easily prepared in unit dosage form with the employment of pharmaceutical materials which themselves are available in the art and can be prepared by established procedures. The following formulations are illustrative of the formulation of the unit dosage forms of the present invention, but are not intended to be limiting.

FORMULATIONS Formulation 1

6.5 grams of dl-alpha tocopherol (sigma chemical of St. Louis, Missouri) are admixed with 1.625 grams of beta carotene (Hoffman-LaRoche) under sterile conditions in an inert (e.g. nitrogen) atmosphere with a suitable carrier (e.g. vegetable oil) . The resulting admixture is then divided into 100 separate dosage units each containing 21 I.U. of dl-alpha tocopherol and 1837.5 I.U. of beta carotene. The dosage units are separately packaged into individual vacuum sealed packets for use as unit dosage forms.

These unit dosage forms can be opened and the ingredients mixed with the food of an animal to be treated immediately before service of the food to the animal. For tumor prophylaxis, one unit dosage form per 5 kilograms of animal body mass on a daily basis is preferably used. Formulation 2 The unit dosage forms of Formulation 1 may be further admixed with vitamin C especially ascorbyl palmitate, in amounts of 21 milligrams (mg) per unit dosage form. These unit dosage forms too may be admixed with the animal's food prior to service. Formulation 3 and 4

The unit dosage forms of Formulation 1 and Formulation 2 respectively, are further admixed with the compound glutathione in amounts of 6 mg per unit dosage form. These unit dosage forms too may be admixed with the animal's food prior to service.

The invention is further explained by reference to the following Examples and appended claims.

EXAMPLES

Example A

One hundred young male adult Syrian hamsters (Mesocricetus auratus) are randomly divided in to five equal groups of 20 animals. The hamsters would be randomly bred (e.g. Lakeview Strain LVG available from Charles River Breeding Laboratories) . The animals are housed five to a cage and maintained in a controlled environment under standardized conditions of temperature and humidity with an alternative twelve hour light-dark cycle. The animals would be 60-90 days of age and would weigh 95-125 grams (g) at the beginning of the test.

The animals are fed standard Purina Laboratory - ~ -

pellets and water as desired. The five groups are prophylactically treated as follows:

Group 1 - no treatment Group 2 - beta carotene 200 ug and dl-alpha tocopherol 200 ug in 0.2 ml vegetable oil daily by mouth. Group 3 - beta carotene 400 ug in 0.2 ml vegetable oil daily by mouth. Group 4 - dl-alpha tocopherol 400 ug in 0.2 ml vegetable oil daily by mouth. Group 5 - 0.2 ml vegetable oil vehicle only.

After 14 weeks of the aforementioned prophylactic treatment, all of the animals would have their right buccal pouches "painted" three times per week with a 0.5 weight % solution of 7,12 dimethyl-benz (a) anthracene (DMBA) in heavy mineral oil USP using a number 3 sable brush. DMBA is a known carcinogen in this concentration when applied in this manner. Each painting with DMBA leaves approximately 0.6 mg of the carcinogen on the ucosal surface of the buccal pouch of the animals. The DMBA was applied in such a manner for 14 weeks. Animals in all groups are examined weekly. They are weighed and the presence or absence and size of any tumor noted.

After 14 weeks of DMBA application (week 28) , moderate size tumors are present in Groups 1 and 5. Smaller tumors are present in Groups 3 and 4. No tumor or only very small tumors are present in Groups 2 animals. Prophylactic treatment continues. DMBA application is stopped after Week 28.

Eight weeks after the DMBA application is stopped. Groups 1 and 5 animals have large and extensive tumor growth. Some of the animals in these groups will have died. The animals in Groups 3 and 4 should appear more healthy, and the tumors are smaller than those in Groups 1 and 5. The Group 2 animals have even smaller, or no, tumors. The animals may be subsequently sacrificed for analysis of the tumors. Such sacrifice may be accomplished through the use of carbon dioxide chamber. Pouches may be excised and sections fixed in 10 percent formalin, sectioned in paraffin and stained with hematoxylin and eosin. Cervical lymph nodes may also be dissected with adjacent tissue and also prepared for histologic study.

The tumors should be epidermoid carcinomas, consistent with the application of DMBA. Groups 1 and 3-5 have animals displaying metastatic spread to cervical lymph nodes, while no such tumor activity is present in the Group 2 animals.

Example B

The test of Example A may be duplicated except that 500 I.U. of alpha carotene on a kilogram basis may be substituted for the beta-carotene. Similar results are obtained.

Examples C and D The tests of Examples A and B may be repeated except that the prophylactic treatment admixture is administered in a suitable vehicle (e.g. ethyl oleate) by intramuscular injection. Similar results are obtained.

Example E

One hundred tumor inflicted dogs are randomly divided into five equal groups of 20 animals. The animals would be of various breeds. The animals are housed in a kennel, and maintained in a controlled environment under standardized conditions of temperature and humidity with an alternative twelve hour light-dark cycle. Each of the animals would be weighed. The size of each animal's tumor would be determined along with a preliminary classification as to tumor type.

The dogs are fed standard Purina Dog Chow and water - li ¬

as desired. The five groups are treated as follows: Group 1 - no treatment Group 2 - One packet per 5 kg of animal body mass of

Formulation 1 is mixed with the animal's food daily immediately prior to service.

Group 3 - Formulation 2 is mixed with the animal's food daily immediately prior to service. Group 4 - One packet of Formulation 3 per 5 kg of animal body mass is mixed with the animal's food daily immediately prior to service.

Group 5 - One packet of Formulation 4 per 5 kg of animal body mass is mixed with the animal's food daily immediately prior to service. The animals are monitored on a daily basis, and tumor size, body mass, and survival times are recorded. Group 1 animals die quickly. The animals in Groups 2-5 retain body mass longer, exhibit a decreased tumor size and growth, and the animals survive longer.

Reference herein to specific embodiments and formulations is not intended to limit the scope of the appended claims.

Claims

What is claimed is: 1. A method of preventing tumor occurrence in an animal comprising: administering daily to the animal a prophylactic treatment composition, said composition comprising vitamin E - in admixture with a carotene, said components being in a sufficient quantity to prevent the occurrence on growth of a tumor in said animal.

2. The method according to Claim 1, wherein said prophylactic treatment composition further composes glutathione.

3. The method according to Claim 2 further comprising administering to the animal vitamin C at regular intervals throughout the day.

4. The method according to Claim 3, wherein said regular intervals are twelve hours apart.

5. The method according to Claim 4, wherein said vitamin E is present in quantities of about 10 I.U. to about

32 I.U. per kilogram of animal body mass, wherein said carotene is present in quantities of about 275 I.U. to about 3400 I.U. per kilogram of animal body mass; said glutathione is present in quantities of about 4 milligrams to about 8 milligrams per kilogram of body mass, and said vitamin C is present in quantities of about 14 milligrams to about 28 milligrams per kilogram of body mass.

6. The method according to Claim 5, wherein said vitamin E is alpha-tocopherol and said carotene is beta carotene.

7. An improvement in animal food of the type containing an admixture of a carbohydrate source, a protein source, and a fat source, wherein the improvement comprises mixing with said admixture a vitamin source comprising a sufficient quantity of vitamin E and carotene to prevent tumor growth in an animal.

8. The animal food of claim 7 wherein said vitamin E is dl-alpha tocopherol and is present in quantities containing about 10 to about 32 international units of vitamin E per kilogram of said animal's body mass when administered on a daily basis.

9. The method of claim 8 wherein said carotene is beta carotene and is present in quantities containing about 275 to about 3400 international units of beta carotene per kilogram of said animal's body mass when administered on a daily basis.

10. The animal food of claim 7 wherein said vitamin source consists essentially of vitamin E and carotene.

11. The animal food of Claim 10 wherein said vitamin E is dl-alpha tocopherol present in quantities containing about 5 to about 32 international units of vitamin E on a daily basis and said carotene is beta- carotene and is present in quantities containing about 275 to about 3400 international units of beta carotene per kilogram of said animal's body mass when administered on a daily basis.

12. The animal food of claim 7 wherein said vitamin source further comprises vitamin C and glutathione, and said vitamin C is present in quantities of about 14 to about 28 milligrams per kilogram of animal body mass and said glutathione is present in quantities of about 4 to about 8 milligrams per kilogram of animal body mass on a daily basis.