AMINO ACID COMPOSITION AND USE THEREOF IN TREATING TUMOR GROWTH AND METASTASIS

Abstract: The present invention provides a method for the prophylactic and/or therapeutic treatment of tumor growth and/or metastasis comprising administering to a human being or other mammal a medicament or nutritional formulation comprising at least one amino acid selected from the group consisting of glycine, alanine, serine, and physiologically acceptable salts of glycine, alanine and serine in an amount which is effective for preventing and/or inhibiting tumor growth and/or metastasis. Also provided is a method for inhibiting increased cell proliferation comprising administering to a human being or other mammal a medicament or nutritional formulation comprising at least one amino acid selected from the group consisting of glycine, alanine, serine, and physiologically acceptable salts of glycine, alanine and serine in an amount which is effective for inhibiting increased cell proliferation.

Description: Amino Acid Composition and Use thereof in Treating Tumor Growth and Metastasis The present invention relates to the use of certain amino acids in the preparation of a medicament or nutritional formulation with anti-tumor and/or anti-metastatic effect. Cancer is a disease in which individual cells begin by prospering selfishly at the expense of their neighbours but in the end destroy the whole cellular society. Cancer is widely accepted as a disease that is highly dependent on cell proliferation. In the multi-step model of carcinogenesis, cell replication is necessary for tumor development at every stage: fixing both spontaneous and chemically-induced mutations in DNA in the initiating step; the clonal expansion of initiated cells in the promotion phase; and providing further stimuli for cell growth in the progression phase. A carcinogen which sows the seed of cancer by causing latent genetic damage is called a tumor initiator. Tumor promoters are substances which are not themselves mutagenic but which cause cancer in tissues previously exposed to a tumor initiator. Tumor promoters are also referred to as non-genotoxic or epigenetic carcinogens, since they do not directly interact with DNA. An increase in DNA synthesis and cell proliferation had been speculated to increase error susceptibility rate for genetic base pairing which can lead to progression of the cancer process. WY- 14,643 is a member of the class of compounds known collectively as peroxisome proliferators which are characterized by increased number and size of peroxisomes as well as associated peroxisomal enzymes in hepatocytes. Additionally, they cause hepatocellular adenoma and carcinoma in rodents to varied degrees. WY- 14,643 is one of the more potent carcinogens in this class causing tumors in 100 % of animals in one year. Although the mechanism by which peroxisome proliferators cause cancer remains unknown, a considerable amount of evidence suggests that they act via non-genotoxic mechanisms involving tumor promotion. Peroxisome proliferators are a group of non-genotoxic carcinogens which include a number of hypolipidemic drugs, solvents, and industrial plasticizers. Several studies have implicated increased hepatocyte proliferation as a key factor in the genesis of liver cancer by these chemicals. For example, the potency of these compounds as carcinogens has been shown to correlate with their ability to cause sustained cell proliferation; WY-14,643 increased cell proliferation for as long as the compound was administered while diethylhexyl phthalate (DEHP), a much less potent carcinogen, did not. Further, a link between the induction of basophilic foci, which exhibit very high rates of cell proliferation compared to surrounding tissue and the incidence of tumour formation resulting from feeding WY- 14,643 in the diet has been established. Additionally, elevated rates of hepatocyte replication are important in the promotion of previously initiated cells. WY- 14,643 and nafenopin cause a greater number of preneoplastic lesions in livers of older rats than in livers of younger rats suggesting that peroxisome proliferators act as tumor promoters on spontaneously initiated cells which are more numerous in older rats. While many studies suggest that increased hepatocyte replication is crucial in the development of peroxisome proliferator-induced liver cancer, the mechanism by which these chemicals stimulate mitogenesis is unknown. It has now surprisingly been found that glycine prevents the increase in hepatocyte proliferation caused by WY- 14,643. However, the increase in peroxisomal β-oxidation due to WY- 14,643 was not affected by glycine indicating that the pathways responsible for cell proliferation and peroxisome proliferation are distinct. In view of the above-mentioned effects, there are provided pharmaceutical compositions, formulations and diets comprising glycine as well as methods of using glycine. For use in the compositions, formulations, diets and methods of the invention, glycine is conveniently employed in free amino acid form, in the form of glycine precursors, in particular alanine or serine, likewise in free amino acid form, in physiologically acceptable salt form of said amino acids, or in form of mixtures of said amino acids and/or physiologically acceptable salts thereof. Glycine is preferably used in free amino acid form, in physiologically acceptable salt form or in the form of a mixture of glycine in free amino acid form with glycine in physiologically acceptable salt form; most preferably glycine is in free amino acid form. Glycine may also be used in the form of dipeptides according to the invention. The term "amino acid of the invention" as used hereinafter is meant to refer to glycine, alanine and/or serine, in free amino acid form and/or physiologically acceptable salt form. The present invention therefore provides the use of at least one amino acid selected from the group consisting of glycine, alanine and serine, or physiologically acceptable salts thereof, in the preparation of a medicament or nutritional formulation for the prophylactic and/or therapeutic treatment of tumor growth and/or metastasis. The present invention also provides a method for the prophylactic and/or therapeutic treatment of tumor growth and/or metastasis comprising administering to a human being or other mammal a medicament or nutritional formulation comprising at least one amino acid selected from the group consisting of glycine, alanine, serine, and physiologically acceptable salts of glycine, alanine and serine in an amount which is effective for preventing and/or inhibiting tumor growth and/or metastasis. The invention further provides the use of at least one amino acid selected from the group consisting of glycine, alanine or serine, or physiologically acceptable salts thereof, in the preparation of a medicament or nutritional formulation for inhibiting increased cell proliferation. The present invention still further provides a method for inhibiting increased cell proliferation comprising administering to a human being or other mammal a medicament or nutritional formulation comprising at least one amino acid selected from the group consisting of glycine, alanine, serine, and physiologically acceptable salts of glycine, alanine and serine in an amount which is effective for inhibiting increased cell proliferation. "Tumor growth" as used herein refers to abnormal growth of tissue, which may be benign or malignant, whereby malignant tumors and cancer are used interchangeably herein. The amino acids of the invention are useful in the prophylactic and/or therapeutic treatment of tumors induced by either genotoxic or non-genotoxic carcinogens or both. They are particularly effective in the prophylactic treatment of tumors, particularly for the prophylactic treatment of tumors induced by non-genotoxic carcinogens such as peroxisome proliferators (e.g. hypolipidemic drugs, solvents, and industrial plasticizers) and/or cell proliferators. The amino acids of the invention are further particularly useful in the prophylactic and therapeutic treatment of malignant tumors such as liver cancer. Depending on the type of tumor and the developmental stage of the disease, the amino acids of the invention are useful in preventing the risk of developing tumors, in promoting tumor regression, in stopping tumor growth and/or in preventing metastasis. The nutritional formulation or medicament may be administered to the patient enterally or parenterally. The enteral administration route is preferred, particularly for subsequent or prophylactic treatment; particularly contemplated enteral administration routes are oral administration, nasal administration and/or tube feeding. The medicament or formulation is conveniently administered in the form of an aqueous liquid. The medicament or formulation in a form suitable for enteral application is accordingly preferably aqueous or in powder form, whereby the powder is conveniently added to water prior to use. For use in tube feeding, the amount of water to be added will depend, inter alia, on the patient's fluid requirements and condition. The medicament or formulation may be so formulated as to deliver to the patient from 1 to 80 g, preferably 1 to 60 g, particularly preferred 20 to 40 g of the amino acid of the invention per 24 hours. The amount of medicament or formulation to be administered depends to a large extent on the patients' specific requirements. Such daily amounts of amino acid of the invention are suitable for treatment of the desired effects as well as for prophylactic/pretreatment. In the case that the medicament or formulation comprises a single amino acid of the invention (in the L-configuration), it may be administered to the patient in an amount such that the concentration of that amino acid in the patients' plasma is elevated to between 0.4 and 2.0 mM, preferably from 0.5 to 1.2 mM. Whilst concentrations higher than this are anticipated, it is expected that significant clinical effects will be obtained if the concentration of the acid is increased, as a consequence of administration of the formulation or medicament, so that it lies in the range of from 0.6 to 0.9 mM. In traumatic, hypercatabolic patients it may even be beneficial to raise the plasma glycine, serine or alanine levels to about 0.2 to 0.3 mM which corresponds to plasma glycine levels of healthy individuals. The most preferred amino acid of the invention for incorporation into the medicament or formulation for use according to the invention is glycine or a physiologically acceptable salt thereof. Generally, it is indicated to use an amino acid of the invention in combination with one or more of the following components: (i) an omega-3 polyunsaturated fatty acid (PUFAs) where desired in admixture with an omega-6 PUFA; (ii) L-arginine or other physiologically acceptable compound associated with the synthesis of polyamines, or mixtures thereof; and (iii) a nucleobase source. Whereby the use of a medicament or nutritional formulation comprising an amino acid of the invention in combination with arginine or other physiologically acceptable compound associated with the synthesis of polyamines such as ornithine is preferred. Use of a medicament or nutritional formulation comprising an amino acid of the invention, arginine or ornithine and omega-3 polyunsaturated fatty acids (PUFAs) is also preferred. Nucleobase sources suitable for use in combination with the amino acids of the invention comprise or consist of natural nucleobases, nucleosides, nucleotides, RNA, DNA, equivalents thereof and/of mixtures comprising one or more of these compounds. Natural nucleobases include the purines adenine and guanine as well as the pyrimidines cytosine, thymine and uracil. Where the nucleobase source is in the form of free nucleobases, it is preferably uracil. Natural nucleosides include the ribose nucleosides adenosine, guanosine, uridine and cytidine and the deoxyribose nucleosides deoxyadenosine, deoxyguanosine, deoxythymidine and deoxycytidine. Natural nucleotides include phosphate esters of natural nucleosides, such as the monophosphates adenylate (AMP), guanylate (GMP), uridylate (UMP), cytidylate (CMP), deoxythymidiylate (dTMP), deoxycytidylate (dCMP), and diphosphates and triphosphates of natural nucleosides such as ADP and ATP. A purified nucleobase source, such as yeast is preferred. However, other sources such as meat and the like may be used. Preferably the nucleobase source is RNA. Accordingly, the invention provides medicaments or nutritional formulations comprising effective amounts of: (a) an amino acid of the invention (component (a)) in association with one or more components selected from (b) omega-3 PUFAs where desired in admixture with omega-6 PUFAs (component (b)); (c) L-arginine or other physiologically acceptable compound associated with the synthesis of polyamines, or mixtures thereof (component (c)); and (d) a nucleobase source (component (d)). Said medicaments and nutritional formulations are hereinafter designated "diets of the inventions". One unit dose of such medicament or nutritional formulation preferably comprises 1.5 to 80 parts by weight of component (a) in association with the following amounts of one or more components selected from (b) to (d): 0.1 to 20 parts by weight of component (b), 3 to 40 parts by One unit dose of such a medicament or nutritional formulation preferably comprises 1.5 to 80 weight of component (c) and 0.1 to 4.0 parts by weight of component (d). Particularly preferred one unit dose comprises 1.5 to 80 parts by weight of component (a) in association with the following amounts of one or more components selected from (b) to (d): 2 to 5 parts by weight of component (b), 7.5 to 20 parts by weight of component (c) and 1.7 to 2.0 parts by weight of component (d). The amount of components (a) to (d) administered daily will conveniently correspond to 1.5 to 80 g for component (a), 0.1 to 20 g, preferably 2 to 5 g, for component (b), 3 to 40 g, preferably 7.5 to 20 g, for component (c) and 0.1 to 4.0 g, preferably 1.7 to 2.0 g, for component (d). With respect to component (d) the above dosage is indicated for RNA, DNA, nucleosides or nucleotides. For nucleobases one weight unit of nucleobases is regarded to be equivalent to 2.5 to 3.0 weight units of RNA, DNA, nucleosides or nucleotides. Where medicaments or nutritional formulations comprising an amino acid of the invention in combination with one or more of the above-mentioned components (b), (c) and (d) are used, such medicaments or nutritional formulations will conveniently comprise in one unit dose (a) 1.5 to 80 parts by weight of one or more amino acids selected from the group consisting of glycine, alanine and serine, in free form or physiologically acceptable salt form, or mixtures thereof, in combination with one or more compounds selected from the group consisting of (b) 2 to 5 parts by weight omega-3 polyunsaturated fatty acids; (c) 7.5 to 20 parts by weight L-arginine or L-ornithine, or mixtures thereof; and (d) 1.7 to 2.0 parts by weight RNA. Preferred medicaments or nutritional formulations comprise in one unit dose: (a) from 1.5 to 80 parts by weight of an amino acid selected from the group consisting of glycine, alanine and serine, in free form or physiologically acceptable salt form, or mixtures thereof, in association with (c) 3 to 40 parts by weight, preferably 7.5 to 20 parts by weight, of arginine or an equivalent amount of one or more other physiologically acceptable compounds associated with the synthesis of polyamines, or an equivalent amount of a mixture of arginine with such compounds. More preferably the medicaments or nutritional formulations of the invention comprise component (a) in combination with component (c) at a weight ratio of 1 :2 to 4: 1 , particularly preferred at a weight ratio of 1 : 1 to 2: 1. Further preferred medicaments or nutritional formulations comprise in one unit dose: (a) from 1.5 to 80 parts by weight of an amino acid selected from the group consisting of glycine, alanine and serine, in free form or physiologically acceptable salt form, or mixtures thereof, in association with (b) 0.1 to 20 parts by weight, preferably 2 to 5 parts by weight, of omega-3 PUFAs; and (c) 3 to 40 parts by weight, preferably 7.5 to 20 parts by weight, of arginine or an equivalent amount of one or more other physiologically acceptable compounds associated with the synthesis of polyamines, or an equivalent amount of a mixture of arginine with such compounds. Omega-3 PUFAs are conveniently protected against peroxidation. Physiologically acceptable ways of protecting omega-3 PUFAs against peroxidation are known in the art. They include physiologically acceptable micro-encapsulation of omega-3 PUFAs and the use of physiologically acceptable antioxidants. A typical example suitable for use as physiologically acceptable micro-encapsulation agents is starch. The micro-encapsulation can be effected in a manner known per se. The micro- encapsules may be coated in a manner known per se, by physiologically acceptable coating agents such as Gum Arabic. Typical examples of antioxidants suitable for use in the method of the invention include antioxidant vitamins such as Vitamin C, Vitamin E or mixtures thereof. The amount of antioxidant added should be sufficient to prevent peroxidation of the omega-3 PUFAs. Such amounts can be easily calculated. In general, for convenience, any antioxidants employed to prevent peroxidation, will be employed in excess. It will be appreciated that the presence of any other agent administered in association with the omega-3 PUFAs may require adjustment of the amount of antioxidant to be employed. The omega-3 PUFAs may be employed in a form suitable for the physiological supply of omega-3 PUFAs, e.g. in free acid form, in triglyceride form, or in the form of physiologically acceptable natural sources of omega-3 PUFAs. Such natural sources include linseed oil and fish oils such as menhaden oil, salmon oil, mackerel oil, tuna oil, cod-liver oil and anchovy oil. Said natural sources, in particular, the fish oils, comprise substantial amounts of omega- 3 fatty acids. Where the omega-3 PUFAs are employed in triglyceride form, said triglycerides may comprise esters with other physiologically acceptable fatty acids. Preferred omega-3 PUFAs include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in free acid form, in triglyceride form or in form of natural sources having a high EPA and/or DHA content. When the amino acids of the invention are administered in the form of a medicament such a medicament will comprise from 1 to 99 g of the amino acid of the invention per lOOg. In general, favorable effects are obtained when administering the diets of the invention in the form of a formula diet, which may, depending on the circumstances be a complete formula diet (i.e. a diet supplying essentially all required energy, amino acids, vitamins, minerals and trace elements) or a diet supplement. The diet will conveniently be taken in aqueous liquid form. A formula diet accordingly may comprise a source of carbohydrates, lipids fat (fat source) and protein (nitrogen source), and at least one amino acid selected from the group consisting of glycine, L-alanine and L-serine, or physiologically acceptable salts thereof, characterized in that the acid or salt is present in the formula diet in an amount of about 0.5 to lOg per lOOg. The formula diet will preferably further comprise other nutritionally advantageous components such as vitamins, minerals, trace elements, fibers (preferably soluble fibers). Examples of suitable nitrogen sources include nutritionally acceptable proteins such as soy bean or whey derived proteins, caseinates, and/or protein hydrolysates. Suitable carbohydrate sources include sugars such as maltodextrins. Examples of suitable fat sources include triglycerides, as well as di- and monoglycerides. Examples of vitamins suitable for incorporation into the medicament or formulation of the invention include Vitamin E, Vitamin A, Vitamin D, Vitamin K, folic acid, thiamin, riboflavin, Vitamin B-, B 2 , B 6 and Bι 2 , niacin, biotin and panthotenic acid in nutritionally acceptable form. Examples of mineral elements and trace elements suitable for incorporation into the medicament or formulation include sodium, potassium, calcium, phosphorous, magnesium, manganese, copper, zinc, iron, selenium, chromium, and molybdenum in nutritionally acceptable form. In particular, the medicament or formulation will preferably comprise beta-carotene (Vitamin A), Vitamin E, Vitamin C, thiamine, Vitamin Bι , choline, selenium and zinc in nutritionally acceptable form. The term "soluble fiber" as used herein refers to fibers which are able to undergo substantial fermentation in the colon ultimately to produce short chain fatty acids. Examples of suitable soluble fibers include pectin, guar gum, locust bean gum, xanthan gum which may optionally be hydrolysed. For adults the total amount of soluble fibre per day will conveniently lie in the range of from 3 to 30g. It will be appreciated that omega-3 PUFAs may be administered in higher amounts than those indicated hereinabove, and that such higher amounts will in general not impair the desired effect or provoke undesired side effects. Compounds particularly suitable for use as component (c) in the formulation of the invention include L-arginine and L-ornithine, most preferably L-arginine. Component (c) may be employed in free form, physiologically acceptable salt form, e.g. in the form of a salt with phosphoric acid, citric acid, tartaric acid, fumaric acid, adipic acid or lactic acid, or in small peptide form. Preferably L-arginine in free form is employed. The term small peptides as used herein refers to peptides having from 2 to 6, preferably from 2 to 4 amino acids. As already indicated, omega-3 PUFAs will conveniently be administered in the form of fish oils, protected or not against peroxidation. Such fish oils also comprises omega-6 PUFAs. Omega-6 PUFAs have also a favorable effect on the immune response and on the resistance to infection upon surgery. Accordingly, diets of the invention will conveniently further comprise omega-6 PUFAs. For the purpose of the invention the omega-6 PUFAs may be in free acid form or in a form suitable for the physiological supply of omega-6 PUFAs, e.g. in triglyceride form. Examples of omega-6 PUFAs particularly appropriate for use according to the invention, include linoleic acid and arachidonic acid, linoleic acid being most preferred. Examples of suitable omega-6 PUFA sources are known in the art. They include fish oils and vegetable oils. Examples of omega-6 PUFA sources having a high linoleic acid content such as safflower oil, sunflower oil, soya oil, cotton oil and corn oil. Administration of a daily amount of omega-6 PUFAs in the range of from 1.5 to 5.0 g will in general suffice to attain a favorable effect. One unit dose of the medicaments or nutritional formulation defined above may accordingly further contain 1.5 to 5 parts by weight of omega-6 PUFAs. In addition to components (b), (c) and (d), and omega-6 PUFAs further components may be added to the diets of the invention and may have a beneficial effect on the activity of the amino acid of the invention. An example of such beneficial components are omega-9 PUFAs. A preferred natural source for such fatty acid mixtures are fish oils. For taste and other reasons, the fish oils will, in oral application forms, preferably be used in encapsulated form. Where the formula diet of the invention is intended for use as a nutritional supplement (e.g. prophylactic treatment), the amount of energy supplied by it should not be too excessive, in order not to unnecessarily suppress the patients appetite. The supplement should conveniently comprise energy sources in an amount supplying from 150 to 1000 Kcal/day, preferably 250-500 Kcal/day. For use as a complete formula diet (e.g. for promoting tumor regression, preventing metastasis, stopping tumor growth) the diets of the invention will conveniently supply from 600 to 1500 Kcal/day. The contribution of the nitrogen source, carbohydrate source and lipid source to the total daily caloric may vary within wide ranges. In preferred formulations of the invention the carbohydrate source provides for 40 to 70 % of the total energy supply and, the nitrogen and fatty acid source each for 15 to 30 % of the total energy supply of the formulation. For use as complete diet, the diet of the invention will conveniently be administered in aqueous liquid form in volumes in the range of from 500 ml to 3000 ml. For use as a supplement, the administration may be in powder or liquid form. The supplement will conveniently be administered in the form of unit doses suitable for administration of the supplement 1 to 4 times per day. Where the diets of the invention comprise energy sources, it is appropriate not to supply more than 1500 Kcal/day. Where acute treatment of patients following excessive ethanol exposure is necessary, the amino acid of the invention will conveniently be administered parenterally. Typical administration forms suitable for such acute treatment are e.g. the aqueous solutions disclosed hereinbelow. Typical pharmacologically acceptable formulation forms for oral administration will further comprise pharmacologically acceptable diluents, carriers, vitamins, spices, pigments and/or other adjuvants well known to the skilled person to be suitable for incorporation into such formulation. The diets and formulations of the invention may be obtained in a manner known per se, e.g. by admixing the ingredients. Typical formulations suitable for use according to the invention include aqueous solutions consisting essentially of 0.1 % to 90 % by weight of at least one amino acid selected from the group consisting of glycine, alanine and serine, and pharmaceutically acceptable salts thereof, the balance being distilled water. The amino acid of the invention may be present in a concentrated form of the solution in an amount of from 15 to 90% (by weight of the solution). Concentrated solutions are suitable for dilution to application forms or for use in acute treatment. Application forms having a lower content (e.g. 0.1 to 5 %) of the amino acid of the invention will in general be indicated for prophylactic purposes; concentrated forms of the solution having a higher content (e.g. 5 % to 40 % by weight) of amino acid of the invention will in general be more suitable for acute treatment. Other formulations suitable for inclusion in the medicament or formulation of the invention, in particular for parenteral application, include infusion solutions such as Ringer's injection solution, lactated Ringer's injection solution, crystalloids, colloids or other plasma substitutes, in association or enriched with about 0.1 to 5.0g per liter infusion solution of glycine, serine and/or alanine. Ringer's injection solution is a sterile solution, containing from 3.23 to 3.54g of sodium (equivalent to from 8.2 to 9.0g of sodium chloride), from 0.149 to 0.165 of potassium (equivalent to from 0.285 to 0.315g of potassium chloride), from 0.082 to 0.098g of calcium (equivalent to from 0.3 to 0.36g of calcium chloride, in the form of CaCl 2 -2H 2 O), from 5.23 to 5.80g of chloride (as NaCl, KCl and CaCl 2 -2H 2 O) and water in sufficient quantity to give 1000 ml solution. Lactated Ringer's Injection solution is a sterile solution containing from 2.85g to 3.15g sodium, as chloride and lactate), from 0.141 to 0.173g of potassium (equivalent to from 0.27g to 0.33g of potassium chloride), from 0.049 to 0.060g calcium (equivalent to from 0.18g to 0.22g of CaCl 2 .2H 2 O), from 2.31g to 2.61g of lactate, from 3.68 to 4.08g of chloride (as NaCl, KCl and CaCl 2 -2H 2 O) and water in sufficient quantity to give 1000 ml solution. The terms crystalloids and colloids in connection with fluid therapy are known in the art. They include plasma substitutes such as Haemaccel (polygeline based) and Gelofusine (gelatin based). The invention will be further understood by reference to the following specific description. EXAMPLE 1 MATERIALS AND METHODS Animals and Treatment Male Sprague-Dawley rats were housed 2-3 per cage and were given powder diet (Table 1 ) and water ad libitum TABLE 1 Dietary Content by Percent CONTROL GLYCINE WY- 14,643 WY-14,643 + GLYCINE Casein 20 15 20 15 Sucrose 50 50 49.9 49.9 Corn Oil 5 5 5 5 Alpha 5 5 5 5 Cellulose Mineral 3.5 3.5 3.5 3.5 Mix Vitamin Mix 1 1 1 1 DL- 0.3 0.3 0.3 0.3 Methionine Choline 0.2 0.2 0.2 0.2 Bitartrate Corn 15 15 15 15 Starch Glycine 0 5 0 5 WY- 14,643 0 0 0.1 0.1 Two different experimental protocols were used in this study: (1) a twenty-four hour single dose protocol and (2) a three week feeding study. For the single dose study, animals were fed either control or glycine diet for three days. On the third day, rats were treated with either 100 mg/kg WY- 14,643 or olive oil control vehicle (i.g.). Twenty-four hours later, all rats were given 100 mg/kg 5-bromo-2'-deoxyuridine (BrdU, from Sigma) i.p. to label proliferating hepatocytes and were sacrificed one hour later. For the three week feeding study, rats were fed either control or glycine diet for three days; then half of each group was switched to diet containing 0.1 % WY-14,643 for three weeks. During the last three days, all rats were given drinking water containing BrdU (80 mg/100 mL) to label proliferating hepatocytes. Cell Proliferation Livers were perfused with Krebs-Henseleit buffer (pH 7.6) to remove blood and fixed with 4 % paraformaldehyde. A section of duodenum, which proliferates rapidly, was taken as a positive control for BrdU incorporation. Tissue sections were deparafinized, rehydrated and hydrolyzed in 4 N HC1 for 20 minutes at 37° C. Endogenous peroxidase was quenched with 0.03 % hydrogen peroxide containing sodium azide (DAKO Envision System, Peroxidase). A primary monoclonal antibody to BrdU was diluted 1 :200 (DAKO, clone Bu20a) and allowed to incubate at room temperature for 10 minutes. A peroxidase labeled polymer conjugated to goat anti-rabbit and goat anti-mouse was applied and allowed to incubate for 10 minutes at room temperature followed by 3,3 -diaminobenzidine in a buffered solution containing hydrogen peroxide for 8 minutes (DAKO Envision System Peroxidase). Sections were rinsed twice with phosphate buffered saline containing 1 % Tween 20 after each incubation. Slides were counterstained with hematoxylin and proliferating cells were identified from brown staining. Cell proliferation was quantitated by counting the percentage of BrdU-positive cells in 10 random high power fields. Acyl CoA Oxidase Activity Acyl CoA oxidase is an accepted measure of induction of peroxisomes. It was measured as formaldehyde formed from hydrogen peroxide generated by peroxisomal β-oxidation by coupling hydrogen peroxide with the peroxidation of methanol via catalase. Liver samples were homogenized in 10 volumes of 0.25 M sucrose buffer. A reaction mixture (pH 8.3) cont-aining 22.2 mg palmitate, 400 μL methanol, 7.7 mg CoA, 137.8 mg ATP, 40.7 mg MgCl 2 , 10 μL Triton X-100, 13,3 mg NAD + , 90 mg fatty acid free BSA, and 402,9 mg niacinamide (per 100 mL buffer) was warmed to 37° C. The reaction was started by adding 200 μL of homogenate to 1.4 mL of reaction buffer and was terminated after 10 minutes with 40 % TCA. TCA was added before homogenate to the blanks. The solution was centrifuged to pellet the protein and 0.5 ml of the supernatant was added to 0.2 ml of the Nash Reagent to measure formaldehyde. After 60 minutes of incubation at 37° C, absorbance of samples was read at 405 nm. Protein concentration in the homogenate was determined by the method of Lowry. Apoptotic Index Apoptotic bodies were identified based on morphological features of hematoxylin and eosin stained liver sections. The features used to determine apoptotic cells were condensation of cytoplasm, accumulation of condensed chromatin at the nuclear membrane, and fragmentation of the nucleus and cell, giving rise to apoptotic bodies which were phagocytosed by neighboring cells. The apoptotic rate is expressed as the percentage of apoptotic cells counted in 10 random high power fields (1000 hepatocytes counted per slide). TNFa Immunohistochemistry and Image Analysis Samples of liver from each rat were quick-frozen and 6 μm sections prepared for immunohistochemistry. The sections were fixed in acetone for 10 minutes, allowed to air dry and then blocked with normal goat serum (1:67 dilution in undiluted Automation buffer; Biomedia Corp., Foster City, CA; and 1% Nonfat Dry Milk) for 30 minutes. All further incubations were made in a solution of Automation buffer containing 1% bovine serum albumin. Endogenous peroxidase activity was quenched by incubation for 10 minutes in 3% hydrogen peroxide. Slides were incubated for 1 hour at 4°C with a 1 :75 dilution of polyclonal goat anti-mouse TNFα antibody (R&D Systems; Minneapolis, MN) or normal goat serum as control, washed in Automation buffer and then incubated for 1 hour with a 1 :200 dilution of rabbit anti-goat IgG biotinylated antibody (Vector Lab Inc.; Bulingame, CA). After washing, the antibodies were labeled using the Vectastain Elite Kit (Vector Laboratories) and localized with the peroxidase substrate 6,6'-diaminobenzidine (DAB) enhanced with NiCl 2 . Finally, the slides were counterstained with modified Harris' hematoxylin and dehydrated for mounting. A Universal Imaging Corp. Image- 1/AT image acquisition and analysis system (Chester, PA) incorporating an Axioskop 50 microscope (Carl Zeiss, Inc., Thornwood, NY) was used to capture and analyze the im unostained tissue sections at lOOx magnification. Color detection ranges were set for the red-brown color of the DAB chromogen based on an intensely labeled Kupffer cell. The extent of TNFα expression in the liver was defined as the percent of the field area within the default color range determined by the software. Data from each tissue section (5 random fields per section) were pooled to determine means. Statistics Results .are reported as means +_ s.e.m. for n=4 or 5 in each group. Treatment groups were compared using one-way ANOVA and Student-Newman-Keuls post-hoc tests, where appropriate. RESULTS Single dose study WY-14,643 (100 mg/kg) increased cell proliferation almost 8-fold in normally quiescent hepatocytes 24 hours after administration from 0.7 + 0.3 % to 5.1 +_0.3 %. While the glycine diet did not affect basal rates of hepatocyte proliferation, feeding the diet containing 5 % glycine for three days prior to treatment with WY-14,643 resulted in an increase in cell replication to values of only 1.9 + 0.4 %. This value was not significantly different from either the control or glycine basal rates of cell proliferation but was significantly less than the 8-fold increase in hepatocyte replication characteristically caused by WY- 14,643. A defining characteristic of all peroxisome proliferators is an induction of peroxisome specific enzymes that follows their administration. Acyl CoA oxidase, the hydrogen peroxide producing enzyme catalyzing the rate limiting step of peroxisomal β-oxidation, reaches peak values which are 8-fold greater than controls after 18 days of treatment with WY- 14,643, but increases can be detected within one day. Twenty-four hours after treatment with WY- 14,643, acyl CoA oxidase was increased 2.5-fold. The glycine diet had no effect on basal acyl CoA oxidase activity and surprisingly did not prevent the increase in acyl CoA oxidase due to WY- 14,643. Peroxisome proliferation after 3 weeks of treatment with WY-14,643 was even more extensive. WY- 14,643 caused a 6-fold increase in acyl CoA oxidase activity, an increase which was not prevented by the addition of glycine to the diet. This interesting finding further supports the idea that peroxisome proliferation and cell proliferation are not tightly linked events. Three Week Feeding Study Diet consumption was monitored throughout the three weeks of feeding. There were no significant differences in diet consumption between the treatment groups studied; however, weight gain by the different groups varied considerably. Control and glycine fed rats did not differ in respect to body weight at any time during the feeding. In contrast, the WY- 14,643 diet resulted in an average weight gain of only about 60 % of control which is in agreement with results published previously in a chronic feeding study. The liver to body weight ratio for rats fed the control powder diet was about 5.1 + 0.6 % which is slightly higher than the 4-4.5 % for rats fed chow diet. Glycine-fed rats did not differ from controls while the WY- 14,643 diet caused a significant increase in liver to body weight ratio of about 70 %, a phenomenon not prevented by glycine. However, the addition of 5 % glycine to the WY- 14,643 diet prevented the increase in gross liver size caused by WY- 14,643. The WY- 14,643 diet results in an average liver size that is about 33 % larger than control and glycine while the addition of glycine to the WY-14,643 diet reduced livers to the size of controls. Replicating hepatocytes were determined as cells containing brown nuclei. Four BrdU positive cells were seen in the liver from a WY-14,643-treated rat in the three week study. However, BrdU-positive cells were rarely detected in livers from control, glycine or WY- 14,643 + glycine rats. Basal rates of cell proliferation (control diet) averaged 1.3 % while WY- 14,643 increased hepatocyte replication about 5-fold. The glycine diet resulted in rates of cell proliferation that did not differ from control, while the addition of glycine to the WY- 14,643 diet completely prevented the increase in hepatocyte replication caused by WY- 14,643 alone after three weeks. Thus, glycine clearly prevents both early and sustained increases in cell replication due to WY- 14,643. Since peroxisome proliferators such as nafenopin have been shown to inhibit apoptosis, which could contribute to an increase in liver size due to exposure to these chemicals, rates of apoptosis were also determined in livers from the three week study. The percentage of apoptotic cells ranges from 0.20% to 0.35% and were not significantly different for any treatment group. Therefore, changes in apoptosis are not a major contributor to changes in liver size due to WY- 14,643. TNFα has been shown to be involved in WY-14,643-induced cell proliferation; therefore, in order to determine if the glycine containing diet was preventing TNFα expression in response to WY- 14,643, immunohistochemical staining for TNFα was performed. Control levels of TNFα expression in liver sections were 0.4% of the total area of liver tissue and 0.7% in livers from rats fed the glycine containing diet. Feeding WY- 14,643 in the diet for three weeks increased staining for TNFα 2-fold, and this increase was largely prevented by the glycine- containing diet. DISCUSSION Glycine prevents increases in hepatocyte proliferation due to WY-14,643 WY- 14,643 unlike less carcinogenic peroxisome proliferators such as DEHP increases hepatocyte replication for as long as the compound is administered. The characteristic 8-fold increase in cell proliferation that occurred 24 hours after peroxisome proliferator treatment and the sustained 6-fold increase that was observed after three weeks of WY- 14,643 was prevented in this study by glycine. Interestingly, three weeks of dietary glycine caused a 50% reduction in basal levels of cell proliferation compared to control. Glycine diet did not prevent peroxisome proliferation due to WY- 14,643 at either 24 hours or 3 weeks which supports the hypothesis that peroxisome proliferation and cell proliferation occur via different mechanisms. Additionally, rates of apoptosis did not differ among the groups, consistent with results obtained in other chronic feeding studies with WY- 14,643. WY- 14,643 increased TNFα expression in liver about 2-fold over control while glycine-containing diet largely prevented this increase. EXAMPLE 2: ENTERAL COMPOSITIONS In the following compositions MM stands for "mineral mixture", SM for "trace element mixture" and VM for "vitamin mixture". The composition of these three mixtures is as follows: MM VM Ingredients ε/lOOε Ingredients g/lOOg Maltodextrins 34.40 Maltodextrins 43.44 K citrate/phosphate 34.60 Sodium ascorbate 35.00 Magnesium dicitrate 8.20 Vitamin E- Ac. 50% 16.00 Calcium chloride 8.00 Niacinamide 1.55 Sodium citrate/chloride 9.00 Vitamin A- Acetate 1.20 Citric acid 3.50 Ca-D-Panthothenat 0.98 Choline tartrate 2.30 Vitamin Ki 1% 0.71 Vitamin B 12 0.1% 0.30 Vitamin D3 0.28 Vitamin Bg 0.20 SM Vitamin B 0.17 Vitamin B2 0.15 Ingredients g/lOOg Folic acid 0.02 Maltodextrins 47.79 Biotin 0.01 Molybdenum-yeast 18.00 Chromium-yeast 9.20 Zinc sulfate 7.00 Selenium-yeast 7.00 Ferrum(II) sulfate 6.92 Copper(II) gluconate 2.24 Manganese(II) sulfate 1.12 Sodium fluoride 0.70 Potassium iodide 0.03 Composition I Comprising Glycine Ingredients g/lOOg Water 77.40 Maltodextrins 12.28 Na/Ca caseinates 4.60 Glycine 3.00 Lipids: Palm oil 2.33 Sunflower oil 0.26 Emulsifier Nathin E 0.13 100.00 Composition II Comprising Glycine Ingredients g/lOOg Water 77.40 Maltodextrins 10.10 Na/Ca caseinates 4.60 Glycine 3.00 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palm oil 2.33 Sunflower oil 0.26 Emulsifier Nathin E 0.13 100.00 Composition Comprising Glycine and Arginine Ingredients g/lOOg Water 77.40 Maltodextrins 8.93 Na/Ca caseinates 4.60 Glycine 3.00 Arginine 1.17 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palm oil 2.36 Sunflower oil 0.23 Emulsifier Nathin E 0.13 100.00 Composition Comprising Glycine and Fish Oil (ω-3 fatty acids) Ingredients g/lOOg Water 77.40 Maltodextrins 10.10 Na/Ca caseinates 4.60 Glycine 3.00 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palm oil 1.32 Sunflower oil 0.23 Emulsifier Nathin E 0.13 Fish Oil EPAX 3000 TG 1.04 100.00 Composition Comprising Glycine and RNA Ingredients g/lOOg Water 77.40 Maltodextrins 9.96 Na/Ca caseinates 4.60 Glycine 3.00 Yeast extract RNA 0.14 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Palm oil 2.33 Sunflower oil 0.26 Emulsifier Nathin E 0.13 100.00 Composition Comprising Glycine, Arginine and Fish Oil (ω-3 fatty acids) Ingredients g/100g Water 77.40 Maltodextrins 8.93 Na/Ca caseinates 4.60 Glycine 3.00 Arginine 1.17 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palm oil 1.32 Sunflower oil 0.23 Emulsifier Nathin E 0.13 Fish Oil EPAX 3000 TG 1.04 100.00 Composition Comprising Glycine, Ai Ingredients K/100g Water 77.40 Maltodextrins 8.79 Na/Ca caseinates 4.60 Glycine 3.00 Arginine 1.17 Yeast extract RNA 0.14 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palm oil 2.33 Sunflower oil 0.26 Emulsifier Nathin E 0.13 100.00 Composition Comprising Glycine, RNA and Fish Oil (ω-3 fatty acids) Ingredients g/lOOg Water 77.40 Maltodextrins 9.96 Na/Ca caseinates 4.60 Glycine 3.00 Yeast extract RNA 0.14 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palm oil 1.32 Sunflower oil 0.23 Emulsifier Nathin E 0.13 Fish Oil EPAX 3000 TG 1.04 100.00 Composition Comprising Glycine, Arginine, RNA and Fish Oil (ω-3 fatty acids) Ingredients g/lOOg Water 77.40 Maltodextrins 8.79 Na/Ca caseinates 4.60 Glycine 3.00 Arginine 1.17 Yeast extract RNA 0.14 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palm oil 1.32 Sunflower oil 0.23 Emulsifier Nathin E 0.13 Fish Oil EPAX 3000 TG 1.04 100.00 As already set out above fish oil is a natural source for omega-3 PUFAs whereas sunflower oil is a natural source for omega-6 PUFAs.

Claims: CLAIMS: - 1. The use of at least one amino acid selected from the group consisting of glycine, alanine and serine, or physiologically acceptable salts thereof, in the preparation of a medicament or nutritional formulation for the prophylactic and/or therapeutic treatment of tumor growth and/or metastasis. 2. The use of Claim 1 , wherein the tumour is liver cancer. 3. The use of anyone of Claims 1 or 2, wherein the tumour is induced by a non- genotoxic carcinogen. 4. The use of any one of Claims 1 to 3, wherein the tumor is induced by a peroxisome proliferator and/or cell proliferator. 5. The use of any one of the preceding claims wherein the amino acid is glycine. 6. The use of any one of the preceding claims, wherein the medicament or nutritional formulation further comprises arginine. 7. The use of any one of the preceding claims wherein the medicament or nutritional formulation is adapted to be administered by enteral means. 8. The use of at least one amino acid selected from the group consisting of glycine, alanine or serine, or physiologically acceptable salts thereof, in the preparation of a medicament or nutritional formulation for inhibiting increased cell proliferation. 9. A method for the prophylactic and/or therapeutic treatment of tumor growth and/or metastasis comprising administering to a human being or other mammal a medicament or nutritional formulation comprising at least one amino acid selected from the group consisting of glycine, alanine, serine, and physiologically acceptable salts of glycine, alanine and serine in an amount which is effective for preventing and/or inhibiting tumor growth and/or metastasis. 10. A method for inhibiting increased cell proliferation comprising administering to a human being or other mammal a medicament or nutritional formulation comprising at least one amino acid selected from the group consisting of glycine, alanine, serine, and physiologically acceptable salts of glycine, alanine and serine in an amount which is effective for inhibiting increased cell proliferation.

Inventors: SCHNEIDER HEINZ, THURMAN RONALD G, SCHNEIDER, HEINZ, THURMAN, RONALD, G.
Applicants: NOVARTIS NUTRITION AG, SCHNEIDER HEINZ, THURMAN RONALD G, SCHNEIDER, HEINZ, THURMAN, RONALD, G.
Application year: 1997
Main classification: A61K31
IPC codes: A23L1, A61K31, A61P35