IE51300B1 - Protozoal growth-inhibiting diamines - Google Patents

Abstract

Compounds of the formula wherein R1 is hydrogen or carboxy; Y is -CH2F, -CHF2, -CF3 or -C IDENTICAL CH; and Z is -(CH2)3-NH2, -CH(CH)3- (CH2)2-NH2 or -CH=CH-CH2- NH2; with the proviso that, when R1 is hydrogen, Z is -CH(CH3)-(CH2)2- NH2 and Y is not -CF3; or a lactam thereof when R1 is carboxy; and their salts, are useful in inhibiting the growth of protozoa. They may be administered to animals, e.g. in their drinking water, and, if desired, together with an antiprotozoal agent.

Description

Ihis invention relates to ccnpositions containing a-substituted amines and α-substituted-amino acids which are useful in inhibiting the growth of protozoa in animals and particularly in poultry.

Polyamines have been implicated in many aspects of cell division. Impairment of the biosynthesis of polyamines by means of enzyme inhibitors is believed to cause a decrease in cell proliferation in mammals. Although the physiological role of polyamines has not been clearly delineated, there is evidence to suggest their involvement with cell division and growth, H.G. WilliamsAshman et al.. The Italian J. Biochem. 25, 5-32 (1976), K1300 A. Raina and J. Janne, Med. Biol. 53, 121-147 (1975) and D.H. Russell, Life Sciences 13, 1635-1647 (1973).

Polyamines are also known to be essential growth factors for certain microorganisms, as for example E. coli, Enterobacter, Klebsiella, Staphylococcus aureus, C. cadaveris, Salmonella typhosa and Haemophilus parainfluenza. There is evidence to suggest that polyamines are associated with both normal and neoplastic mammalian cell growth, there being an increase in both the synthesis and accumulation of polyamines following a stimulus causing cellular proliferation. It is also known that there is a correlation between polyamine formation and the activity of the decarboxylase enzymes of ornithine, S-adenosylmethionine, arginine and lysine. The term polyamine is taken to include the diamine putrescine and the polyamines spermidine and spermine. Putrescine is the decarboxylation product of ornithine, catalyzed by ornithine decarboxylase. Putrescine formation may also occur by decarboxylation of arginine to form agmatine, which is hydrolyzed to give putrescine and urea. Arginine is also involved in ornithine formation by action of the enzyme arginase. Activation of methionine by the enzyme S-adenosylmethionine synthetase forms S-adenosylmethionine which is decarboxylated. The propylamine moiety of the activated methionine may then be transferred to putrescine to form spermidine. Alternatively, the propylamine moiety may be transferred to spermidine to form spermine. Hence, putrescine serves as a precursor to spermidine and spermine. Additionally, putrescine has been shown to have a marked regulatory effect upon the polyamine biosynthetic pathway. Also an increased synthesis of putrescine has been shown to be an early indication that a tissue will undergo renewed growth processes. Cadaverine, which is the decarboxylation product of lysine, has been shown to stimulate the activity of S-adenosylmethionine decarboxylase and is known to be essential to the growth processes of many micro-orcanisms, for example, H. parainfluenza.

A rationale of polvamine metabolism has been suggested by Cohen, Science 205, 964 (1979). Tbe apparently unique role of polyamine metabolism in trys5 anosomes, and the dependence of trysanosomes upon ornithine decarboxylase as a source of putrescine, supports our observations that certain specific irrever sible inhibitor of ornithine decarboxylase are useful in inhibiting or preventing the growth of protozoa and protozoal infections.

The compounds which have been found to have this anti-protozoal activity are Q-substituted-o-amino-acids of formula Ia and amines of formula Ib R Y f I H2N — CH—CH2— CH2— C-COOH Ia CH Y* I I H2N —CH—CH2—CH2—CH—NH2 Ib salts thereof, and lactams of the formula Ia compounds. In these formulae, R is H or CH^f Y is CHjF, CHFj, CF^ or C=CH; and Y' is CHjF, CHFj or C=CH.

According to the present invention, a composition 20 comprises an anti-protozoal agent known per se and a compound of formula Ia (or a lactam or salt thereof) or Ib (or salt thereof). Such compositions of the invention may be used for inhibiting or preventing the growth of protozoa and protozoal infections.

Preferably, Y or Y* ls CHFj· Examples o£ compounds of formulae Ia and Ib are the following {the first two of which are particularly preferred): 2.5- diamino-2-fluoromethylpentanoic acid, 2.5- diamino-2-difluorojnethylpentanoic acid, 2.5- diamino-2-trifluoromethylpentanoic acid, 2.5- diamino-2-ethynylpentanoic acid, 2.5- diamino-2-fluoromethylhexanoic acid, 2.5- diamino-2-difluoromethylhexanoic acid, 2.5- diamino-2-trifluoromethylhexanoic acid, 2.5- diamino-2-ethynylhexanoic acid, 1-fluoro-2,5-hexanediamine, 1,1-difluoro-2,5-hexanediamine, and 3.6- hept-l-ynediamine.

Illustrative examples of salts used in this invention include non-toxic acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acids,and organic acids such as methanesulfonic, salicylic, maleic, malonic, tartaric, citric, cyclamic and ascorbic acids.

In addition to the salts indicated above, the term salts is taken to include those internal salts or zwitterions of those compounds which are amphoteric in nature. Moreover, whereas the optical configuration for the compounds described herein is not specifically designated, it is recognised that the o-carbon atom is a centre of asymmetry and that individual optical isomers of these compounds exist. Both the dand 1-optical isomers, as well as racemic mixtures, are contemplated as being for use in this invention.

Compounds of formula Ib in which Y' is CHjF or CHFj, and their salts, are described in GB-A-2003876.

The compound of formula Ib in which Y'is C=CH, i.e. 3,6-hept-l-ynediamine, and its salts are described in GB-A-2001058.

Compounds of formula Ia in which R is H and Y is CH2F, CHF2 or CHF^, and their lactams and salts, are described in Patent Specification No. 47081. Conpounds of formula Ia in which· R is H and Y is C=CH, and their lactams and salts, are described in Patent Specification No. 47951.

Compounds of formula la in which R is CHg and Y is CKjF, CHF2 or CF3 “ay be prepared in a manner analogous to that described in GB-A-2001960 for the corresponding compounds in which R is H, having regard to the nature of the product. The temperature ranges at lines 29 and 36 on page 7 of Patent Specification No. 47081 should be 0 to 100°C in the present case.

Compounds of formula Ia*in which R is CHj may be prepared bv methods analogous to those described in Patent Specification No. 47951 for the corresponding compounds in which R is H. The preferred strong bases used for carbanion formation, which will abstract a proton from the carbon atom adjacent to the acetylene moiety, are alkyllithiums (such as butyllithium),phenyllithium, lithium diisopropy20 lamide, lithium amide, tertiary potassium butylate and sodium amide. The alkylating reactant Ph-Ch=N-CHR(CH2)2-X, R being H or CHj and X a halogen such as chlorine or bromine, can be prepared by methods known in the art. Thus, the reagent Ph-CH=N-(CH2)3~Br can be prepared by reacting 3-brcsnopropylamine hydrochloride with benzaldehyde and an organic trialkylamine, in a solvent such as diethyl ether, tetrahydrofuran, dioxane, chloroform or dichloromethane. The reactant Ph-CH=NCH(CHj)-CH2-CH2~Br can be prepared by reacting 1-bromo30 3-butaneamine hydrobromide with, benzaldehyde and an organic amine such as triethylamine. l-Bromo-3-butaneamine hvdrobromide is a known compound which can be prepared from 3-aminobutanol, by treatment with concentrated HBr at a temperature of from 25 to 110°C for from 1 to 12 hours. 3-Aminobutanol is obtained from the β-keto-alkanoic acid ester of the formula CH3-CO-CH2coo-ch2-ch3.

S1300 The β-keto-alkanoic acid ester is treated with hydroxylamine to form the corresponding oxime, which is reduced with lithium aluminium hydride in ether or tetrahydrofuran at a temperature of from 25 to 50°C for from 1 to 12 hours. Subsequent hydrolysis of the ester moiety gives 3-aminobutanol.

The alkylating reaction temperature varies from -100 to 25°C.

Removal of the protecting groups is achieved by treatment with aqueous acid such as hydrochloric acid followed by aqueous base such as sodium or potassium hydroxide, or treatment with phenylhydrazine, hydroxyamine or hydrazine and then with aqueous base.

The preparation of 2-difluoromethyl-2,5-diaminopentanoie acid is described in Example 15 of Patent Specification No. 47081. lhe preparation of 2-ethynyl-2,5-diaminopentanoic acid is described in Exanple 5 of Patent Specification No. 47951. 1-Fluorohexane-2,6-diamine dihydrochloride is prepared by the procedure described in Example 5 of GB-A-2003876, but substituting 4-chthalamidcpentanoyl chloride for the 4-phthalamidobutyxyl chloride used in that Example. 3,6-Hept-l-ynediamine is prepared by the procedure of Example 5 of GB-A-2001058, but substituting 1-bromo3-iminobenzylbutane for the 3-bromopropyl-l-iminobenzyl specified in that Example.

The compounds described herein are useful in inhibiting the growth of protozoa in animals. The term animals is intended to include inter alia mammals, such as mice, rats, guinea pigs, rabbits, ferrets, dogs, cats, cows, horses and primates including man. Also encompassed within the term animals are both fish and fowl.

The term fowl is intended to include male or female birds of any kind including parrots and canaries, but is primarily intended to encompass poultry which are commercially raised for eggs or meat. Accordingly, the term fowl is particularly intended to encompass hens, cocks and drakes of chickens, turkeys and ducks.

The term protozoa is intended to include those members of the subphyla Sarcomastigophora and Sprozoa of the phylum Protozoa. More particularly, the term protozoa as used herein is intended to include those genera of parasitic protozoa which are important to man because they either cause disease in man or his domestic animals. These genera are for the most part found classified in the superclass of Mastigophora of the subphylum Sarcomastigophora and the class of Telosporea of the subphylum Sporozoa in the classification according to Baker (1969). Illustrative genera of these parasitic protozoa include Histomonas, Trypanosoma, Giardia, Trichomonas, Eimeria, Isopora. Toxoplasma and Plasmodium.

Excluded from the superclass of Mastigophora is the genus Leishmania, certain species of which cause the tropical disease of Leishmaniasis in man. Also, specifically excluded from the genus Trypanosoma, as used in this invention, are the species Trypanosoma cruzi, which can cause Chagas' disease in man, and the species Trypanosoma lewisi. The compounds described herein have been found not to be particularly effective against these species.

On the other hand, the compounds of Formula I are particularly useful in inhibiting the growth of Trypanosoma brucei, the causative agent for nagana, or the tsetse-fly disease of horses and cattle in central Africa. The compounds described herein are also remarkably effective in inhibiting the growth of Eimeria tenella, a species of protozoa causing coccidiosis in fowl.

Indeed, a preferred embodiment of the present invention is the use of these compounds to inhibit the growth of intestinal coccidia in commercial poultry.

The economic importance of intestinal coccidia is highly significant. Thus in 1972, the estimated loss to the poultry industry in the United States due to coccidial infections was approximately 47 million dollars. Due to the rapid development of drug resistance by coccidia, and due to the relatively high toxicity of some of the drugs used in the treatment of coccidiosis, there is a need for effective coccidiostats that are non-toxic and to which intestinal coccidia do not develop rapid drug resistance.

It is not exactly understood how the compounds of formula (I) are able to inhibit the growth of protozoa. Inter alia, the compounds described herein are irreversible inhibitors of ornithine decarboxylase and Sadenoxylmethionine decarboxylase. As irreversible inhibitors of these enzymes, these compounds inhibit polyamine formation which may be required for protozoal cell division. In any event, the practice of this invention is not limited to any particular mode or theory of action whereby the compounds of formula (I) are able to effectively inhibit the growth of protozoa.

The effect ot the compounds of general formula (I) upon the growth of protozoa, and more particularly upon the growth of coccidia, can be demonstrated using Eimeria tenella and two week old male white leghorn chicks as the test animals. The birds are kept in batteries and both the infected and non-infected birds are housed in separate rooms to assure the maintenance of coccidia-free birds.

Each experimentally infected bird receives 100,000 sporylated oocysts via gavage. The test compound is administered in the particular dosage desired through the drinking water and drug-free mash is provided ad libitum.

To evaluate the effect of the active ingredient on E. tenella infections, the chicks are sacrificed with carbon dioxide, necropsied, generally at day five of the study, and cecal lesions evaluated.

The inhibition of protozoal growth can also be determined using Trypanosoma brucei brucei, which is the causative agent of bovine trypanosomiasis (nagana) in Africa. The related species Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense are the causative agents for African sleeping sickness in humans.

In general, drug activity is tested against established infections of a pleomorphic EATRO 110 isolate of T. b. brucei in mice. Test animals are infected with 5 x 10^ parasites twenty four hours prior to testing. Control animals so infected generally die 5 days subsequent to innoculation. The compound to be tested is administered to the test animals via their drinking water in varying dosages. Animals cured of the infection remain parasite30 free more than 30 days after the deaths of the control animals as indicated by an examination of blood smears.

The compounds described herein are employed in amounts that are effective in inhibiting protozoal growth. These amounts will depend, of course, upon various factors, such as the type and nature of the protozoal infection, the activity of the specific compound, the age, sex and species of animal treated and whether the treatment is prophylactic or therapeutic. In general, the compounds described herein can be orally or parenterally administered at a daily dose ranging from 5 mg/kg to 7 g/kg. Preferably, in the case of Trypanosoma infections the dosage range is from about 600 mg/kg to 2 g/kg. In the case of Eimeria infections the dose can be lowered, ranging from about 15 mg/kg to 1 g/kg.

Due to the low toxicity of the compounds described herein, the compounds can be safely administered ad libitum via the drinking water of the test animals in the treatment of coccidiosis in fowl. Generally speaking, concentrations of the active ingredient ranging from about 0.01% to 2% are suitable, depending primarily upon the nature of the protozoal infection to be treated whether prophylactic or therapeutic, the severity of the infection and the period of treatment.

Thus, for example the compound 2-difluoromethyl-2,5diaminopentanoic acid can be effectively administered to chickens for the treatment of coccidiosis one day prior to infection as a 2% solution. Alternatively, a prophylactic course of treatment can be utilized 8 days prior to infection utilizing concentrations of 2-difluoromethyl-2,5diaminopentanoic acid as low as 0.015% in the drinking water of chickens. Preferably, a prophylactic concentration of from 0.06% to 1.0% is preferred.

This prophylactic treatment for the inhibition of protozoal growth provides one of the principle advantages to the use of the decarboxylase inhibitors described herein. Thus, in the case of coccidia infections in chickens, for example, Eimeria tenella grows intracellularly in the epithelial cells of the caecum as a trophozoite stage. Subsequently, these cells undergo a form of multiple mitosis to form a large number of merozoites.

These merozoites are released as the host cell lyses and serve to extensively infect fresh cells. The result is that the wall of the caecum is badly damaged, leading to severe blood and fluid loss and finally death. Moreover, during the life cycle of E. tenella, resistant oocysts are produced which are voided in the faeces of chickens. Chickens being coprophagous in nature, the disease is rapidly spread by contamination of their food supply. Accordingly, coccidial infections in commercial flocks, when they occur, are epidemically treated with massive doses of currently available chemotherapeutic agents, that are primarily cidal in nature. Consequently, medicated feeds are now routinely employed in commercial flocks, so that all commercial fowl now receive almost constant medication to prevent outbreaks of coccidiosis from occurring.

The fact that the decarboxylase inhibitors herein described can be prophylactically administered, enables the host to overcome either a subsequent natural or artificial induced infection enzymatically via an inhibitory mechanism rather than via a cidal action. Thus, in the case of an E. tenella infection, the infection is curtailed in a manner that enables the host to avail itself of its own body defense mechanisms. The resulting antibodies which are produced via such a controlled infection, serve to further permanently immunize the host from future E. tenella infections.

The pharmaceutical compositions that are particularly suited for the prophylaxis or treatment of protozoal infections in fowl comprise the heretofore described a-substituted amines or the α-substituted-a-amino acids in combination with a pharmaceutically acceptable carrier. Advantageously, the antiprotozoal compositions are prepared by admixing the active compound with an inert carrier material. Typical carriers include talc, clay, pumice, silica, chalk, diatomaceous earth, walnut shell flour and equivalents thereof. Alternatively, the active ingredient can be admixed with a commercial feedstuff or vitamin and mineral pre-mix particularly adapted for fowl.

In most cases a concentrated aqueous solution of the active ingredient is employed in the management and treatment of coccidiosis in fowl. The compounds described, for the most part, are highly soluble, particularly in the form of their salts. Such solutions may advantageously contain preservatives, such as parabens, benzyl alcohol, phenol or thimerosal. In addition, isotonic agents, sugars, stabilizing or buffering agents can be usefully employed.

The compounds of formula (I) can be used in conjunction with other known drugs currently in use for the chemotherapy and chemoprophylaxis of disease caused by parasitic protozoa. Generally, this has the effect of decreasing the amount of enzyme inhibitors administered. Such drugs include, among others: quinapyramine; Berenil, Diminazene aceturate; Pentamidine isethionate; Primaquine; Tryparsamide; Amicarbalide; Amprolium; Amphotericin B; quinine; Monensin; Minocycline, 7-dimethylamino-6-demethyl-6-deoxytetracycline; Clindamycin, 7-deoxy-7(S)-chlorolincomycin; Buquinolate; Robenidine; and Nicarbazin. In some instances the compounds of formula (I) actually enhance or potentiate the effects of these drugs.

Of particular interest in this regard is the compound 2,5-diamino-2-difluoromethylpentanoic acid which has been shown to act synergistically with the antiprotozoal agents Antrycide, quinapyramine, Pentamidine isethionate and Amicarbalide. Thus, the 2,5-diamino-2-difluoromethylpentanoic acid concentration can be reduced by about four-fold when used in combination with subcurative doses (less than 1.0 mg/kg) of these drugs.

Additionally, the compounds of formula (I) can be used in combination with other known cytotoxic agents for the chemotherapy and chemoprophylaxis of parasitic diseases, particularly trypanosomiasis. Such cytotoxic agents include the antineoplastic antibiotic Bleomycin as well as other well-known cytotoxic agents, as for example, cyclophosphamide, methotrexate, prednisone, 6-mercaptopurine, procarbozine, daunorubicin, vincristine, vindesine, vinblastine, chlorambucil, cytosine arabinoside, 6-thioguanine, thio ΤΕΡΑ, 5-fluorouracil, 5-fluoro-215 deoxyuridine, 5-azacytidine, nitrogen mustard, l,3-bis(2chloroethyl)-l-nitrosourea (BCNU), 1-(2-chloroethyl)-3cyclohexyl-l-nitrosourea (CCNU), busulfan or adriamycin.

Of particular interest in the treatment of trypanosomiasis in general, and more particularly in the treatment of nagana in cattle, is the use of the enzyme inhibitor 2,5-diamino-2-difluoromethylpentanoic acid in combination with the antitumor antibiotic Bleomycin. This particular enzyme inhibitor appears to act synergistically with Bleomycin. Thus, mice infected with Trypanosoma brucei are cured after three days upon daily i.p. administration of Bleomycin at a dosage of 7 mg/kg. Similarly, trypanosome infections in mice are cured by the administration of a 1% solution of 2,5-diamino-2-difluoromethylpentanoic acid in the drinking water for 3 days.

The results of several combination experiments indicate that cures are consistently effected with 0.5 mg/kg of Bleomycin in combination with 0.5% of 2,5-diamino-2difluoromethylpentanoic acid administered via drinking water. Alternatively, cures are effected with concentrations of 0.25 mg/kg of Bleomycin in combination with only 0.25% of 2,5-diamino-2-difluoromethylpentanoic acid in the drinking water. A combination of 0.1 mg/kg of Bleomycin and 0.1% of 2,5-diamino-2-difluoromethylpentanoic acid has no effect.

Thus, the curative dosage combinations reflect a reduction in Bleomycin drug dosage of from 1/2 to 1/28 of the curative dose of the drug used singly, when used in combination with a subcurative dose (of from 1/2 to 1/4 of the curative dose) of 2,5-diamino-2-difluoromethylpentanoic acid.

Compositions for use in the invention, and the utility of the invention, will now be exemplified. All parts and percentages are by weight, unless otherwise specified.

For example, granules suitable for addition to the drinking water of poultry are prepared as follows: Grams 2-Difluoromethy1-2,5-diaminopentanoic acid 33.0 Corn Starch 18.5 Lactose 48.2 Zinc Stearate 0.3 100.0 513 0 0 The 2-difluoromethyl-2,5-diaminopentanoic acid is mixed with approximately 6 to 9 grams of lactose and passed through a fluid energy mill or micronizer to give a particle size of 1-25 microns. Water, 35 ml, is added to approximately 2.0 grams of the corn starch and blended to prepare a 5% starch paste. The micronized 2-difluoromethyl-2,5-diaminopentanoic acid - lactose powder, the remaining lactose and the remaining corn starch are well blended. The starch paste is added and blended, and the resulting mixture is passed through a No. 12 mesh screen. The resulting granules are dried at 38°C to a moisture content of approximately 3%, ground through a U.S. Standard No. 12 screen and lubricated by mixing with 0.3 grams of zinc stearate.

A 10% stock solution for use in the treatment of coccidiosis is prepared by dissolving 37.5 grams of 2difluoromethyl-2,5-diaminopentanoic acid in one gallon of water at room temperature. One part of this stock solution diluted with nine parts of water results in the preparation of a 1% medicated drinking water solution for poultry which is useful for the prevention of coccidiosis in poultry.

A medicated animal feed suitable for poultry is prepared utilizing the following ingredients. The birds are fed the medicated feed ad libitum.

Ground yellow corn....................... 60.3 Soy bean oil meal......................... 33.0 Alfalfa leaf meal......................... 1.0 Dicalcium phosphate....................... 3.0 Calcium carbonate......................... 1.0 Iodized salt.............................. 0.2 2-difluoromethyl-2,5-diaminopentanoic acid. .33 Vitamin-mineral-amino acid antibiotic mix to furnish the following per 100 pounds of feed: Oxytetracycline...................... 0.5 gm Penicillin (as procaine salt)........ 0.25 gm Manganese sulfate............. 8 gm DL-methionine........................ 22.7 gm Riboflavin........................... 130 mg DL-calcium pantothenate.............. 930 mg Niacin....... 1400 mg Pyridoxine........................... 130 mg Vitamin B12.......................... 1 m9 Choline chloride..................... 22.7 gm Vitamin A............................ 300,000 units Vitamin D3........................... 25,000 units The following illustrates the effect of 2-difluoromethyl-2,5-diaminopentanoic acid on Trypanosoma brucei brucei infections in mice.

Groups of five mice weighing 20-25 g are innoculated with T, b. brucei (EATRO 110 isolate; 5 x 103 organisms/mouse). The compound is administered via drinking water, ad libitum, 24 hours following infection. Results are expressed as average survival (in days) beyond the death of the control animals, based upon an average survival of control animals of five days. Berenil (diminazene aceturate) is included as a control trypanocide. The results are indicated in Table I below.

TABLE I Drug Treatment regimen Total Average Dose Survival (mg) (Days) None 0 0 2-difluoromethyl-2,5-diaminopentanoic acid 2% in drinking water, 6 days 600®. >30& 2% in drinking water, 3 days 300® >30 1% in drinking water, 6 days 300® > 30 1% in drinking water, 3 days 150® > 30 0.5% in drinking water, 3 days 75® 28.6 0.1% in drinking water, 3 days 15® 2 300 mg/kg p.o., daily 3 days 22.5 26.3 150 mg/kg p.o., daily 3 days 11.3 22.8 75 mg/kg p.o., daily 3 days 5.6 19.2 50 mg/kg p.o., daily 3 days 3.8 0 2-methyl-2,5-diaminopentanoic acid 2% in drinking water 3 days 300® 0 diminazene aceturate 2.5 mg/kg i.p. daily, 3 days 0.2 >30 £ Based upon a daily intake of 5 ml water/25 g mouse/day Considered curative. Animals survived >1 month beyond controls; blood smears were negative for parasites after 1 month. Attempts at subinoculation of brain suspensions into uninfected animals remained negative after >30 days.

The following Example illustrates the effect of a 2% solution of 2-difluoromethyl-2,5-diaminopentanoic acid in the drinking water of chickens infected with oocysts of Eimeria tenella.

Twenty chickens are infected per os at day 1 with 100,000 oocysts of E. tenella. Ten of the animals are given drinking water containing a 2% solution of 2-difluoromethyl-2,5-diaminopentanoic acid. The remaining animals serve as controls. By day 3 all of the control animals demonstrate clinical signs of the disease. On day 7 all of the animals are sacrificed, cecal lesions are macroscopically examined and quantified as follows.

= No detectable macroscopic lesions. +1 = Few scattered petechiae in the cecal wall; no thickening of the wall and normal cecal contents present. +2 = Lesions are numerous with noticeable loss in the cecal contents; cecal wall slightly thickened. +3 = Large amounts of blood and tissue debris present, i.e., cecal cores; cecal wall greatly thickened, little if any normal cecal contents present. +4 = Cecal wall greatly distended with much blood or cecal cores present. Cecal debris lacking or included in cores. (Dead bird also scores as +4.) TABLE II Lesion Scores In Individual Animals Controls +4 +3 +4 +4 +4 +4 +2 +4 +4 +4 (N « 10) Average Lesion Score For Group 3.6 2-difluoromethyl-2,5-diaminopentanoic acid treated (N = 10) +1 +1 +0 +1 +1 +3 +4 +4 +1 +1 1.7 300 Following essentially the same procedure as in the preceding Example, six chickens are administered a 2% solution of 2-difluoromethyl-2,5-diaminopentanoic acid (DFMO) in their drinking water for a period of 3 days. At day 1 this group of six chickens, in addition to two groups of ten chickens each, are all infected per os with 100,000 oocysts of E. tenella per chicken. One group of ten chickens serves as the control group, the other group of ten chickens receives a standard dose of Amprolium in their drinking water for the next 5 days. At present Amprolium is the coccidiostat of choice. On day 5 all of the animals are sacrificed and examined for evidence of disease using the lesion scoring index described in the preceding Example. The following results are obtained.

Treatment TABLE III Mean Lesion No. Chickens Days of Treatment Score_ Control 10 2% solution of DFMO 6 0.120% solution of Amprolium 10 3.70 0 0.3 The following Example illustrates the effect of varying doses of 2-difluoromethyl-2,5-diaminopentanoic acid on Eimeria tenella infections in chickens.

Following essentially the same procedure as in the Exanple, page 19, the dosage of 2-difluorcnethyl-2,5-diaminopropionic acid (DFMO) is varied as shown in Table IV below. Treatment with DFMO is started at day -1. The chickens are infected per os at day 0, and treatment is continued for an additional 5 days or a total of 6 days. The. birds are sacrificed.at day 5 and examined for evidence of disease using the lesion scoring index described in the same Example.

TABLE IV Mean Lesion Treatment No. Chickens Days of Treatment Score Control 7 — 3.29 2% DFMO 6 6 0 1.0% DFMO 6 6 0.5 0.5% DFMO 6 6 1.00 The following Example illustrates the effectiveness of a low prophylactic dose upon Eimeria tenella lesions in chickens.

Following essentially the same procedure set forth in the Exanple, page 19, but varying the dose of 2-difluorcmathyl-2,5diaminopentanoic acid (DFMO) administered and the period of administration, the following results are obtained.

TABLE V Treatment No. Chickens Days of Treatment Mean Lesion Score Control 9 — 3.33 2% DFMO 9 -1 thru +5 0 1% DFMO 9 -1 thru +5 0 0.5% DFMO 9 -1 thru +5 0 0.25% DFMO 9 -8 thru +5 0.44 0.125% DFMO 9 -8 thru +5 0 0.0625% DFMO 9 -8 thru +5 0.66 The following Example illustrates the acquisition of a permanent immunity towards Eimeria tenella infections in chickens.

Birds that have previously been treated wtih 2-difluoro 5 methyl-2,5-diaminopentanoic acid at concentrations as low as 0.5% on days -8 through +5 relative to infection are challenged one week following completion of therapy as indicated in Table VI below. These results indicate that prophylactic therapy at low doses permits an adequate development of parasites in the absence of a disease state, thereby enabling the development of an immunity to subsequent E, tenella infections.

TABLE VI Treatment NO. of Chickens Days of Treatment Mean Lesion Score-Initial Mean Lesion Score-Final Control 9 — 0 2.50 1.0% DEMO 9 -8 thru +5 0.33 0 0.5% DFMO 9 -8 thru +5 0 0

Claims (12)

1. A composition which comprises an anti-protczoal agent known per se and a compound of the formula R Y I I H 2 N—CH—CH 2 —CH 2 —c—COOH Ia 5 or a lactam or salt thereof, wherein R is H or CE 3 and Y is CH 2 F, CHF 2 , CF 3 or C=CH.

2. A composition which comprises an anti-protozoal agent known per se and a compound of the formula CH, Y' I I H 2 N—CH— CH 2 — CH 2 — CH—NH 2 lb 10 or a salt thereof, wherein Y' is CEjF, CHFj or C=CH.

3. A composition as claimed in claim 1 or claim 2, wherein Y or Y* is CHF^.

4. A composition which comprises 2,5-diamino-2fluorcmethy Ipentanoic acid and en anti-protozoal agent known per se. 15

5. A composition which comprises 2,5-diamino-2difluoromethyIpentanoic acid and an anti-protozoal agent known per se·.

6. A composition as claimed in any preceding claim, wherein the anti-protozoal agent is quinapyramine, 20 pentamidine, amicarbalide or a bleomycin.

7. A composition as claimed in any preceding claim, for veterinary use, which additionally comprises a suitable carrier.

8. A composition as claimed in claim 7, wherein the 25 carrier is aqueous. 513 0 0

9. A composition as claimed in claim 8, wherein the carrier comprises non-sterile animals' drinking water.

10. A composition as claimed in any preceding claim, which comprises from 0.01 to 2.0% by weight of the 5. Compound.

11. A composition as claimed in claim 10, which comprises from 0.06 to 1.0% by weight of the compound.

12. A composition as claimed in claim 1 or claim 2, substantially as exemplified herein.