WO1994006413A1

WO1994006413A1 - Therapeutic elevated levels of trimethylamine oxide

Info

Publication number: WO1994006413A1
Application number: PCT/US1993/008513
Authority: WO
Inventors: Eric T. Fossel; Tenley A. Albright
Original assignee: The Beth Israel Hospital Association; Vital Sciences, Inc.
Priority date: 1992-09-21
Filing date: 1993-09-10
Publication date: 1994-03-31
Also published as: AU4854493A

Abstract

This invention comprises a method of anticancer prophylaxis comprising chronic maintenance administration of trimethylamine oxide, analogs and derivatives thereof and precursors (collectively 'trimethylamine oxide-class compounds'), and unit dosage forms for such method. In particular embodiments, the method comprises administration of trimethylamine oxide-class compound in the range of at least about 35 mg/kg at least about every 1 to 5 days, or, alternatively, maintained blood levels of about 100 mM.

Description

THERAPEUTIC ELEVATED LEVELS OF TRIMETHYLAMINE OXIDE

Field of the Invention

This invention comprises a method of anticancer prophylaxis comprising chronic maintenance administration of trimethylamine oxide ("TMAO"), analogs and derivatives thereof and precursors (collectively

"trimethylamine oxide-class compound"), and unit dosage forms for such

method. In particular embodiments, the method comprises administration of

trimethylamine oxide-class compound in the range of at least about 35 mg/kg/ at least about every 1 to 5 days, or, alternatively, maintained blood levels of about 100 mM, or in increments of the "no effect" dosage.

Statement Regarding Federally Sponsored Research The funding for work described may have been provided by the Federal

Government, under a grant from the Department of Health and Human Services. Thus the Government may have certain rights in this invention.

Background of the Invention The role of trimethylamine oxide has been widely studied in animal systems over the last century -- without result. The high levels of trimethylamine oxide in certain elasmobranch fish has been noted. In particular, trimethylamine oxide has been studied as a component that contributes to loss of quality in marine fish for food uses. One review of this topic is "The Chemistry and Metabolism of the Nitrogenous Extractives in Fish," J.M. Shewan, Biochemical Society Symposia, R.J. Williams, Ed., 8:28- 48 (1 951 ). More recent studies are Food Chemistry, pp. 614, 61 5, 759,

772, 778 (Marcel Decker, 1 985); and, Chemistry & Biochemistry of Marine

Food Products, Roy E. Martin, Ed., pp. 1 55-1 69 (AVI Publishing). Empirical

determination of trimethylamine oxide is provided in "The Automated

Determination of Volatile Bases (trimethylamine, dimethylamine and ammonia)

in Fish and Shrimp," A. Ruiter, et al., J. Fd. Techno/. , 1 1 :59-68 (1 976).

Other methodology is presented in "Trimethylamine Oxidase of Nurse Shark

Liver and Its Relation to Mammalian Mixed Function Oxidase," Leon Goldstein

et al., Comp. Biochem. Physio/, 45B:895-903 (1 973). The teachings of

these references are incorporated herein by reference. Determination of

trimethylamine and trimethylamine oxide levels as well as tertiary amines are well known in the art.

Physiologically, trimethylamine oxide has also been the subject of

inquiry. Reference is made to "A Comparative Study of Trimethylamine-oxide

Biosynthesis," John R. Baker et al., Biochimica et Biophysica Acta, 71 : 58-64

( 1 963); "Studies on Trimethylamine Oxide," Earl R. Norris et al., J. Bio/.

Chem, 1 58:433-438 (1 945); "Studies on Trimethylamine Oxide," Earl R.

Norris et al., J. Bio/. Chem, 1 58:443-448 (1 945). Metabolism has been

studied in "Urinary Excretion of Choline and Trimethylamines after

Intravenous Administration of Choline in Liver Diseases," J. de la Huerga et

al., J. Lab. Clin. Med., 39:904-910 (1 951 ); and, "Trimethylamine Oxidase of

Nurse Shark Liver and Its Relation to Mammalian Mixed Function Oxidase," Leon Goldstein et al., Comp. Biochem. Physio/, 45B:895-903 (1973). In particular the de la Huerga article points out intestinal conversion of choline

to trimethylamine oxide. Excretion of administered trimethylamine oxide in humans is studied in "The Fate of Trimethylamine Oxide and Trimethylamine

in Man," H.L. Tarr, J. Fish. Res. Bd. Can., 5:21 1 -216 (1941 ). In addition,

intraperitoneal trimethylamine oxide administration to cats and dogs at 200mg/kg and 500mg/kg is disclosed in "Pharmacologically Effective Substances Occurring in Fish," Malorny, G. and N. Rietenbrock, The

Technology of Fish Utilization, FAO Symp., 79-83 (1965) as resulting in

increased urinary excretion of formaldehyde and formic acid. Similarly, Malorny, G. and N. Rietenbrock "Nahrungsabhangige Ausscheidung von Formaldehyd und Ameisensaure im Urin bei Katzen," Naturwiss, 49:520. The teachings of these references are incorporated herein by reference.

In a separate field of study, marine organisms have been investigated

as a potential source of antineoplastic drugs. For example, "Antineoplastic Activity of Clam Liver Extract," Blaschko, Nature, 1 163, 1 164 (September 14, 1968); "Antineoplastic Substances from the Sea: A Review," Cancer Chemotherapy Reports, Part 2, vol. 4, No. 3, 97 et seq. (July 1974).

The low incidence of cancer in elasmobranchs (sharks, skates and rays) has been noted, but no specific protective mechanism isolated. E.g., "High Ionic Strength: Its Significance in Immunosurveillance Against Tumor Cells in Sharks and Rays (Elasmobranchs)," Rosen et al., Medical Hypothesis,

6:441 -446 (1980). Rosen et al. presents a theory that high ionic strength of body fluid acts as a protective antineoplastic mechanism. "Dietary inhibitors of Mutagenesis and Carcinogenesis" Hayatsu et al. Mutation Research 202:429-46 (1988) considers certain foods believed to retard neoplastic changes. "Chemoprevention of Cancer," Lee W.

Wattenberg, Cancer Research, 45: 1-8 (1985) considers various cancer

prophylactic substances.

Summary of the Invention

A physiological therapeutic use of trimethylamine oxide has now been discovered. Chronic maintenance of subjects (including humans) on

trimethylamine oxide-class compounds can reduce or eliminate the incidence of cancer, or reduce its presence.

This invention includes a method of cancer prophylaxis comprising chronic therapeutic level administration of a trimethylamine oxide-class compound. In a particular embodiment therapeutic level administration includes daily dosages of trimethylamine oxide-class compound. In a given embodiment administration of at least about 35 mg/kg at least about every 1 to 5 days trimethylamine oxide-class compound is directed. In certain

embodiments dosages of at least about 100 mg/kg, or at least about 150 mg/kg, or at least about 250 mg/kg trimethylamine oxide-class compound are

preferred. In other embodiments, dosage from about or just below the "no effect" dose down to about 10% or about 5% of the no effect dose is employed. In particular applications, from about 10% to about 50% of the no effect dosage is employed. It is further contemplated that "no effect" dosage levels based upon drug blood levels or plasma levels are included in this invention. Thus, drug blood or plasma levels at from about or just below the "no effect" blood or plasma level to about 10% or about 5% of the no effect level is employed. In particular applications, from about 10% to about

50% of the no effect level is employed.

As expressed in blood levels, this invention includes maintenance of

blood levels of at least about 50mM to about 150mM, and particularly at least about 100mM blood trimethylamine oxide-class compound.

Representative trimethylamine oxide-class compounds of the method

are trimethylamine oxide, trimethylamine hydrochloride or choline.

In preferred embodiments, chronic administration comprises at least

about six months, and further at least about one year. In employment of the

method of the invention various methods of administration are contemplated including topical, oral, or parenteral including intramuscular, intraperitoneal,

intravenous or subcutaneous administration. In one embodiment of topical administration, administration comprises at least about 25 to about 500mM topical preparation.

This invention further includes a method of therapeutically treating subjects (animals including mammals, further including humans), cells, or other biological systems for mutagen or carcinogen challenge {e.g., tobacco

usage) comprising therapeutic level administration of trimethylamine oxide other trimethylamine oxide-class compounds. In a particular embodiment,

practice of the method includes daily dosages of trimethylamine oxide-class compound. Included dosages are at least about 35 mg/kg at least about

every 1 to 5 days trimethylamine oxide-class compound. In certain embodiments dosages of at least about 100 mg/kg, or at least about 150 mg/kg, or at least about 250 mg/kg trimethylamine oxide-class compound are preferred.

. As expressed in blood levels, this invention includes maintenance of

blood levels of at least about 50mM to about 150mM, and particularly at

least about 100mM blood trimethylamine oxide-class compound.

Representative trimethylamine oxide-class compounds of the method are trimethylamine oxide, trimethylamine hydrochloride or choline.

In preferred embodiments, chronic administration comprises at least about one year. In employment of the method of the invention various methods of administration are contemplated including topical, oral, or parenteral including intramuscular, intraperitoneal, intravenous or subcutaneous administration. In one embodiment of topical administration, administration comprises at least about 25 to about 500mM topical preparation.

Still further, this invention includes a pharmaceutical preparation comprising trimethylamine oxide-class compound in unit dosage form. In particular embodiments, unit dosage form comprises at least about 0.5 gm to about 10gm trimethylamine oxide-class compound, and particularly at least about 2gm to about 5gm trimethylamine oxide-class compound. Optionally the dosage form comprises trimethylamine oxide or trimethylamine

hydrochloride, and is adapted to parenteral, or enteral administration. Such dosage form includes tablets, dragees, trochees, liquids, drops, suppositories,

capsules or ampoules, which in particular embodiments (as to tablet, dragee, trochee, or capsules further includes an exterior taste-aroma barrier. Detailed Description of the Invention

This invention will be better understood by reference to certain terms

as defined herein.

"Precursor" as used in reference to trimethylamine oxide means

trimethylamine and pharmaceutically acceptable salts thereof that are

metabolized or converted to trimethylamine oxide in mammalian systems.

Particular reference is made to trimethylamine hydrochloride. For example

see, "Urinary Excretion of Choline and Trimethylamines after Intravenous

Administration of Choline in Liver Diseases," J. de la Huerga et al., J. Lab.

Clin. Med._f 39:904-910 (1 951 ). "Metabolized or converted" is to be

expansively understood to include biological pathways of an animal (such as

via liver enzymes) and pathways within an animal (such as via intestinal

flora). Precursor also includes tertiary amines.

"Trimethylamine oxide-class" compound shall be understood as a

collective term for trimethylamine oxide and precursors, analogs, and

derivatives thereof. Weight is stated as based upon the weight of

trimethylamine oxide moieties (stated as "Active Weight") unless otherwise

noted. When applied to the weight of trimethylamine oxide precursors,

analogs, and derivatives thereof, conversion may be made based upon the

weight ratio between the weight of trimethylamine oxide and such precursor,

analog, or derivative. It is to be particularly understood that tertiary amines

are considered herein as precursor of trimethylamine or its analogs or

derivatives. "Chronic" as used in reference to administration of trimethylamine oxide-class compounds shall mean administration on at least about 5 days in

any thirty day period. "Chronic" as used in reference to maintenance levels

shall mean maintenance above a specified level for at least 10 days in any

thirty day period

"Therapeutic" is used in reference to the administration or maintenance

dosages of trimethylamine oxide-class compounds which result in a

trimethylamine oxide level sufficient to effect at least about a 50% reduction

in neoplastic occurrence as compared to an untreated control. Reduction in

occurrence is measured as either tumor free animals or in reduction of the

number of tumors total in a population.

It is understood that therapeutic use may be systemic or localized, and

dosage levels are measured accordingly. By way of example, topical uses,

designed to protect the integument from mutagenic effects of sunlight, would

address the drug level in or on skin. Systemic administration levels directed to protect against a systemic mutagen would address blood levels.

It is understood that both chronic and therapeutic administration of

trimethylamine oxide-class compounds are facilitated by use of depot and

reservoir type methods of administration as well as topical administration

including transdermal patches.

"No-effect" shall mean that dosage or blood (or plasma) level at which

no toxic effects are observed. In certain applications a level "just below" the

no effect level is noted. This recognizes the reasonable or anticipated

fluctuaction in no effect level. By way of example, ethanol has a varying effect based upon circadian rythm and surrounding circumstances. Just below an ethanol no effect level would be that where, even with a fluctuating

no effect level, no toxic effects are observed.

"Unit dosage" form shall mean trimethylamine oxide-class compounds

comprising from about 0.5gm to about 10gm Active Weight per unit dosage in a pharmaceutically acceptable carrier. Preferred unit dosages are from about 3 to 6gms Active Weight. Due to the characteristic odor of trimethylamine oxide-class compounds, unit dosage forms which mask this

odor are preferred. In topical forms, perfumes or barriers to the external

exposed surface are preferred. In tablet, capsule, lozenge, dragee or trochee

unit dosage forms, exterior barriers, such as polymers, or hard gelatins, are preferred alone or in conjunction with additional coatings such as zein or waxes (e.g., yellow wax, carnauba).

Applied therapeutic levels will be determined based upon the length of

time a subject is exposed to a source of neoplastic potentiation, and the severity and focus of such exposure. For example, uranium miners face long term systemic threat of cancer, particularly lung cancer. High systemic doses between about 35mg and about 350mg/kg or even about 1600mg/kg are be employed. However, since the half-life of particular trimethylamine oxide- class compounds in vivo are on the order of about 3 days, dosages may be administered only intermittently, that is about every 1 to 5 days, and preferably every 2 to 3 days. It is to be understood that trimethylamine oxide-class compounds of particularly long half-life or efficient depot characteristics are permissibly administered even less frequently. Benefit is obtained with a systemic maintained dose of about 25 to about 250mM in blood, and more particularly about 50 to about 150 mM in blood, and most

particularly above about 100mM in blood. Benefit is also obtained at dosages

or blood (or plasma) levels from about or just below the "no effect" amount

to about 10% or about 5% of the no effect amount. In particular applications, from about 10% to about 50% of the no effect dosage is beneficial.

Topical applications for localized protection provide greater latitude for

localized high concentrations. It is understood that topical applications for systemic protection would follow the dosages noted above. In a cutaneous application for cutaneous protection, maintenance of skin levels in the areas to be protected by topical preparations of at least about 25 to about 500mM, and more particularly about 50 to about 150 mM, and most particularly above about 100mM are preferred.

The methods and compositions of this invention possess valuable pharmacological properties. In the broadest embodiment the prevention against the deleterious effects of mutagens or carcinogens is disclosed along with cancer prevention. In another embodiment, administration of

trimethylamine oxide-class compounds avoids the neoplastic effect, particularly the acute effect, of mutagen or carcinogen exposure on carcinogen/mutagen challenged subjects, cells, or other biological systems.

In particular prophylaxis against melanoma, carcinoma,

adenocarcinoma, sarcoma, neuroblastoma, myeloma, lymphoma, or leukemia is set forth. By anatomical reference, prophylaxis against cancers of the esophagus, stomach, colorectal region, lung, oral cavity, larynx, cervix, breast, skin, and prostate are included within this invention. Further included

is cancer of the endometrium, with particular reference to endometrial cancer incident to unapposed estrogen therapy. Additionally included is thyroid

cancer prophylaxis, with particular reference to such cancer incident to radiation exposure.

Mutagens and carcinogens will be understood expansively to include

procarcinogens or proximate carcinogens that are themselves unreactive, but

are converted to primary or ultimate carcinogens in biological systems; and

further to include free radicals, cocarcinogens and promoters. This class further includes electromagnetic spectrum forces such as x-rays, radiation (and sources or materials, such as uranium or radon, which emit radiation,

e.g., oc, b, g rays), microwaves, magnetic fields, and UV light. Particular radiation cancers included in the prophylaxis of this invention are thyroid cancer and leukemia. In some instances, the cytochrome P₄₅₀-dependent monooxygenases of the endoplasmic reticulum are known to convert proximate carcinogens into reactive electron-deficient intermediates or "electrophils." Electrophils are understood to be reactive intermediates. The electrophils interact with electron-rich or nucleophilic centers in DNA (or

perhaps RNA) to produce mutation, potentially initiating carcinogenisis. A cocarcinogen or promoter is not a carcinogen alone but acts to facilitate growth of latent or dormant tumor cells.

TMAO provides clear therapeutic response to challenge by mutagens and carcinogens, and prophylaxis against development of melanoma, carcinoma, adenocarcinoma, sarcoma, neuroblastoma, myeloma, lymphoma, or leukemia, with particular reference to mammary adenocarcinoma. Particular mutagens or carcinogens comprise a long list of known compounds

which is expanded on a daily basis. Implicated compounds include alkylating

substances such as nitrogen derivatives, nucleic-acid antimetabolites, and pesticides. A few better know representatives of this class include dimethylbenz-(a)-anthracene (also termed "7-12-dimethylbenz-(a)-

anthracene," "dimethylbenz[a]anthracene," and "DMBA"), benzopyrene,

aflatoxin, sunlight, benzene, coal tar, tobacco use, pyridine, dioxin, polychlorinated biphenyls, formaldehyde, asbestos, and nitrogen mustards. Further included are ionizing radiation and magnetic fields.

While not being bound by any particular mode of action, it is believed

that trimethylamine oxide-class compounds act in a manner analogous to free radical traps. That is, trimethylamine oxide-class compounds -- by some mechanism ~ interferes with a cascade of events leading to cancer initiation. By known pathways this may involve the the cytochrome P₄₅₀-dependent monooxygenases of the endoplasmic reticulum which convert proximate carcinogens into reactive electron-deficient intermediates or electrophils; or possibly by preventing electrophil interaction with electron-rich or nucleophilic centers in DNA (or RNA) thus avoiding nucleic acid mutation, thus avoiding carcinogenisis. Further postulated activity includes interference with the activity of cocarcinogen or promoter agents preventing such agents from facilitating growth of latent or dormant tumor cells. Additional potential mechanisms for the mode of activity of trimethylamine oxide-class compounds are set forth in "Chemoprevention of Cancer," Lee W. Wattenberg, Cancer Research, 45: 1 -8 (1985) which considers various cancer

prophylactic substances, the teachings of which are incorporated herein by reference.

It is a particular advantage of the present invention, in a number of embodiments, that the mechanism for trimethylamine oxide-class compounds is peculiar to the prevention of cancers, but largely insensitive to the type of

cancer prevented. That is, the determinative factor is not the type of

neoplasm which avoided, but the common mechanisms leading to neoplasm

initiation or early survival/proliferation.

The salutary effect of chronic therapeutic levels of trimethylamine

oxide-class dosages has been demonstrated in animal systems. For example, rats were challenged with dimethylbenz-(a)-anthracene, and therapeutically treated by using TMAO. Of the treated rats, 66% remained tumor free. In contrast, 100% of control animals developed tumors (mammary

adenocarcinoma). Further, of the treated rats exhibiting tumors, all had fewer total tumors than controls. The treated population exhibited one eighth the tumors as compared to a control population.

Therapeutic dosages (including blood or plasma levels) may be empirically derived using any of a number of well known tests. Even given the range of trimethylamine oxide-class compounds, the varying clearance times in subject animals, the number of methods of administration and the behavior, and disposition of challenging mutagens or carcinogens, dosages are readily determinable.

In one model, a cell culture is made directly from the subject to be

protected. In a checkerboard type assay, this cell culture is be challenged

with a specific mutagen or group of mutagens, and dosed with varying levels of trimethylamine oxide-class compound. Typical assays produce chromosome mutation, or mutation of genome. By calculation, one skilled in the art may then determine protective levels of trimethylamine oxide-class

compound to be maintained in a subject. The particular trimethylamine oxide- class compound is then administered until the desired level is reached. In systemic application this is conveniently be measured in blood or plasma. In topical protection applications, this is measured with reference to skin surface area. Further, based upon test clearance rates for the trimethylamine oxide-class compound in the subject, unit dosages and dosage regimen or frequency is easily established.

An alternative means of determining an initial therapeutic dosage is to begin at about one-tenth the "no-effect" dose, that is the dosage at which no toxic effects are observed. The dosage is then increased, thereafter, to the desired level which is either a target dosage range, as stated above, or at or below the no-effect dosage. The foregoing methodology is set forth in Basic

& Clinical Pharmacology, Fifth Ed., pp60-68, Ed. Bertram G. Katzung, M.D., Ph.D. (Appleton & Lang, Norwalk, Connecticut), the teachings of which are

incorporated herein by reference. As to high risk subjects, "Rationale and Strategies for Chemoprevention of Cancer," Bertram et al., Cancer Research, 47:301 2-31 (1 987) (the teachings of which are incorporated herein by

reference) discloses administration at or just below the "no-effect" dose.

There are other well known protocols useful in the broader

determination of dosages for particular trimethylamine oxide-class

compounds. Further, those skilled in the art are able to reliably predict when

animal protocols in common test animals such as rats, mice, pigs and

monkeys are reasonably predictive of general (including human) application.

Particular reference is made to the Ames Salmomella/microsome assay.

Specific reference is made to the methodology employed in

"Carotenoid Dose Level and Protection Against UV-B Induced Skin Tumors",

Mathews-Roth et al., Photochemistry and Photobiology, 42:35-38 ( 1 985)

which sets for the specifics of determining prophylactic dosages of retinoids

in animals challenged with UV-B light, the teachings of which are

incorporated by reference. This methodology is useful in trimethylamine

oxide-class compound dosage determination. Similarly, reference is made to

the determination of dosages effective against DMBA challenge, "Carotenoids

and Cancer Prevention - Experimental and Epidemiological Studies,"

Mathews-Roth, M.M., Pure & Applied Chemistry, 57:71 7-722 (1 985) the

teachings of which are incorporated by reference. Mutagenic testing

methodology, which is employed to determine prophylaxis in test compounds

against known mutagens, is broadly set forth on Carcinogenesis and

Mutagenesis Testing, Ed. J.F. Douglas (Humana Press, Clifton, New Jersey,

1 984) the teachings of which are incorporated by reference. Topical use of an anticarcinogen is methodologically disclosed in "Inhibition of experimental oral carcinogenesis by topical beta carotene,"

Suda et al., Carcinogenesis, 7:71 1 -5 ( 1 986) the teachings of which are

incorporated by reference. All of the foregoing methods can be employed

with trimethylamine class compounds in dose determination.

The pharmacologically active compositions of this invention can be

processed in accordance with conventional methods of Galenic pharmacy to

produce medicinal agents for administration to patients, e.g., mammals

including humans.

The compositions of this invention can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic

carrier substances suitable for parenteral, enteral (e.g., oral or inhalation) or

topical application which do not deleteriously react with the active

compositions. Suitable pharmaceutically acceptable carriers include but are

not limited to water, salt solutions, alcohols, gum arabic, vegetable oils,

benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,

amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin,

perfume oil, fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone,

etc. The pharmaceutical preparations can be sterilized and if desired mixed

with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting

agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring,

flavoring and/or aromatic substances and the like which do not deleteriously react with the active compositions. They can also be combined where desired with other active agents, e.g., vitamins.

For parenteral application, particularly suitable are injectable, sterile

solutions, preferably oily or aqueous solutions, as well as suspensions,

emulsions, or implants, including suppositories. Ampoules are convenient unit

dosages.

Also for parenteral application, particularly suitable are tablets,

dragees, liquids, drops, suppositories, lozenges, or capsules. As noted above, exterior barriers to the aroma and/or taste of trimethylamine oxide-class

compounds, such as polymers (shellac) or hard gelatins, are preferred. A lozenge, syrup, elixir, or the like can be used, preferably wherein a sweetened or strongly aromatic vehicle is employed.

Sustained or directed release compositions can be formulated, e.g.,

liposomes or those wherein the active component is protected with differentially degradeable coatings, e.g., by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the new compositions and use the lyophilates obtained, for example, for the preparation of products for

injection.

For topical application, there are employed as nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water. Transdermal applications, such as patches, are also particularly noted. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, linaments, salves, etc., which are, if

desired, sterilized or mixed with auxiliary agents, e.g., preservatives,

stabilizers, wetting agents, buffers or salts for influencing osmotic pressure,

etc. For topical application, also suitable are sprayable aerosol preparations

wherein the active ingredient, preferably in combination with a solid or liquid

inert carrier material, is packaged in a squeeze bottle or in admixture with a

pressurized volatile, normally gaseous propellant, e.g., a freon.

Generally, the compositions of this invention are dispensed in unit

dosage form comprising from about 0.5gm to about 10gm Active Weight per

unit dosage in a pharmaceutically acceptable carrier. Preferred unit dosages

are from about 3 to 6gms Active Weight. They are incorporated in topical

formulations in concentrations of about 0.1 to 10gm weight percent.

As noted above, dosage of the compositions according to this

invention generally are about 35 to about 350 mg/kg every 1 to 5 days.

Particular indications for treatment are for persons with personal or family

histories of cancer, or employment in high cancer risk occupations (i.e.

uranium miners, farmers) or with high risk behaviors such as smoking

tobacco or lengthy exposure to sunlight.

It will be appreciated that the actual preferred amounts of active

compositions in a specific case will vary according to the specific

compositions being utilized, the particular compositions formulated, the mode

of application, and the particular situs and organism being treated. Dosages for a given host can be determined using conventional considerations, e.g.,

by customary

comparison of the differential activities of the subject compositions and of a

known agent, e.g., by means of an appropriate, conventional pharmacological

protocol. In particular, those subjects with compromised liver or renal

function will require close monitoring to be sure that circulatory and

interstitial levels are properly maintained, and that excretion levels are not

misleading. In the particular case in which trimethylamine oxide precursors

are employed, it must be ascertained whether the necessary metabolic

pathways are functioning in the animal being treated. For example, if choline

is administered, relying for transformation to trimethylamine oxide on gut

flora, and the subject is also taking antibiotics, there may be insufficient gut

flora for the required conversion.

Claims

EXAMPLE Cancer ProphylaxisTwenty-four 150gm female Sprague-Dawley rats each received adosage of 20mg dimethylbenz-(a)-anthracene in 1 ml sesame oil administeredby gavage according to the protocol of Higgins et al., Nature, 189:201 (1961 ). Typically this treatment results in 100% of test animals developing mammary adenocarcinoma within 30 to 50 days. In this experiment, all ratswere fed normal rat chow from which antioxidants had been omitted.Twelve rats received injections (I. P.) of TMAO (0.6ml injections of 390mg/ml) every 3.5 days beginning 3 days prior to dimethylbenz-(a)- anthracene, challenge. The control group received 0.6ml injections of isotonic saline at the same time points.On day 67, following 20 TMAO (or saline) injections all rats were sacrificed and examined for mammary tumors. The experiment was successfully completed in 9 TMAO treated and 9 control rats.In the TMAO treated group, 6 of the 9 rats were completely tumor free. In the three TMAO treated rats with tumors, there were a total of 5 tumors. In the control rats, all 9 had mammary tumors, for a total of 42 tumors.Thus therapeutic efficacy or prophylaxis is clearly established. Claims:

1 . A method of cancer prophylaxis comprising chronic therapeutic level

administration of trimethylamine oxide, analogs, derivatives and precursors thereof (collectively "trimethylamine oxide-class compound").

2. The method of Claim 1 wherein said therapeutic level administration

comprises daily dosages of trimethylamine oxide-class compound.

3. The method of Claim 1 wherein said therapeutic level comprises administration of at least about 35 mg/kg at least about every 1 to 5 days trimethylamine oxide-class compound.

4. The method of Claim 3 wherein said therapeutic level comprises administration of at least about 100 mg/kg trimethylamine oxide-class compound.

5. The method of Claim 4 wherein said therapeutic level comprises administration of at least about 150 mg/kg trimethylamine oxide-class compound.

6. The method of Claim 5 wherein said therapeutic level comprises administration of at least about 250 mg/kg trimethylamine oxide-class

compound.

7. The method of Claim 1 wherein said therapeutic level comprises

maintenance of blood levels of at least about 50mM to about 1 50mM blood

trimethylamine oxide-class compound.

8. The method of Claim 7 wherein said therapeutic level comprises

maintenance of blood levels of at least about 100mM blood trimethylamine

oxide-class compound.

9. The method of Claim 1 wherein said therapeutic level comprises

administration of at least about 5% of the no effect dose.

10. The method of Claim 9 wherein said therapeutic level comprises

administration of at least about 10% of the no effect dose.

1 1 . The method of Claim 1 wherein said therapeutic level comprises

administration of from about or just below the no effect dose to at least

about 5% of the no effect dose.

1 2. The method of Claim 1 comprising administration of trimethylamine

oxide, trimethylamine hydrochloride or choline.

13. The method of Claim 12 comprising administration of trimethylamine

oxide.

14. The method of Claim 1 wherein said chronic administration comprises at least about one year.

15. The method of claim 1 wherein administration is topical, oral, or

parenteral including intramuscular, intraperitoneal, intravenous or subcutaneous administration.

16. The method of Claim 15 wherein topical administration comprises at

least about 25 to about 500mM topical preparation.

17. A method of therapeutically treating subjects, cells, or other biological systems for mutagen or carcinogen challenge comprising therapeutic level administration of trimethylamine oxide-class compound.

18. The method of Claim 17 wherein said therapeutic level comprises daily dosages of trimethylamine oxide-class compound.

19. The method of Claim 17 wherein said therapeutic level comprises

administration of at least about 35 mg/kg/ at least about every 1 to 5 days trimethylamine oxide-class compound.

20. The method of Claim 1 9 wherein said therapeutic level comprises administration of at least about 1 00 mg/kg trimethylamine oxide-class

compound.

21 . The method of Claim 20 wherein said therapeutic level comprises

administration of at least about 1 50 mg/kg trimethylamine oxide-class

compound.

22. The method of Claim 21 wherein said therapeutic level comprises

administration of at least about 250 mg/kg trimethylamine oxide-class

compound.

23. The method of Claim 1 7 wherein said therapeutic level comprises

maintenance of blood levels of at least about 50 to about 1 50mM blood

trimethylamine oxide-class compound.

24. The method of Claim 23 wherein said therapeutic level comprises

maintenance of blood levels of at least about 100mM blood trimethylamine

oxide-class compound.

25. The method of Claim 17 comprising administration of trimethylamine

oxide, trimethylamine hydrochloride or choline.

26. The method of Claim 25 comprising administration of trimethylamine

oxide.

V

27. The method of Claim 17 wherein said chronic administration comprises at least about one year.

28. The method of claim 17 wherein administration is topical, oral, or

29. The method of Claim 28 wherein topical administration comprises at least about 25 to about 500mM topical preparation.

30. The method of Claim 17 wherein said mutagen or carcinogen challenge comprises tobacco use.

31 . A pharmaceutical preparation comprising trimethylamine oxide-class

compound in unit dosage form.

32. The unit dosage form of Claim 31 wherein said dosage form comprises at least about 0.5 gm to about 10gm trimethylamine oxide-class compound.

33. The unit dosage form of Claim 32 wherein said dosage form comprises at least about 2.0 gm to about 5gm trimethylamine oxide-class compound.

34. The unit dosage form of Claim 31 wherein said dosage form comprises

trimethylamine oxide or trimethylamine hydrochloride.

35. The unit dosage form of Claim 34 wherein said dosage form comprises trimethylamine oxide.

36. The unit dosage form of Claim 31 adapted for parenteral, or enteral administration.

37. The unit dosage form of claim 36, wherein said form comprises tablets, dragees, trochees, liquids, drops, suppositories, capsules or ampoules.

38. The unit dosage form of claim 37 in tablet, dragee, trochee, or capsules form further comprising an exterior taste-aroma barrier.