NZ236475A - Composition for the treatment of animals infected with endoparasites comprising a pyraclofos derivative and optionally a benzimidazole carbamate type compound - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £36475 Priority Dats(a): l.cn"2-: $?\.

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Publication Date: P.O. Jo urn.;!, i\:;: 12)"? 5? 2364 NEW ZEALAND 1 i No.: Date: PATENTS ACT, 1953 I4DECI990SJ ^ p j COMPLETE SPECIFICATION ANTIPARASITIC COMPOSITION FOR ANIMAL USE we, SMITHKLINE BEECHAM CORPORATION, a corporation organized under the laws of the Commonwealth of Pennsylvania, United States of America, of One Franklin Plaza, Philadelphia, Pennsylvania 19101, United States of America, and TAKEDA CHEMICAL INDUSTRIES, LTD., a corporation organized under the laws of Japan, of 3-6 Doshomachi 2-chome, Chuo-ku, Osaka, Japan hereby declare the invention for which >4X/ we pray that a patent may be granted to &}&/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 2364 Z ANTIPARASITIC COMPOSITION FOR ANIMAL USE The present invention relates generally to an antiparasitic composition and its use for treating animals infected with internal and external parasites, such as helminths, nematodes, ticks and others. More specifically the invention relates to a composition containing albendazole and the organophosphate, pyraclofos, and its use in treating infections due to benzimidazole-resistant parasitic infections.

Background of the Invention Parasitic infections of animals, particularly domesticated animals such as cattle, horses, sheep and swine, poultry or pet animals, have long been a problem for owners and breeders alike. The major groups of animal parasites are found among the Protista, the helminths (round worms and flat worms) and the arthropods. Helminthiasis is an infection of the host animal caused by parasites called helminths.

Helminthiasis is a serious epidemic in domesticated animals such as swine, sheep, horses, cattle, goats, dogs and cats and in poultry. Among helminths, the group of parasites known as nematodes or round worms cause 236475 3 universal and serious infections in various species of animals. The genera of parasites which are most commonly found to affect the above-mentioned animals are Haemonchus, Trichostronavlus. Ostertaaia. Nematodirus. Cooperia. Ascaris. Bunostomum. Oesophaaostomum.

Chabertia. Trichuris. Stronqylus. Trichonema. Dictvocaulus. Capillaria. Heterakis. Toxocara. Ascardia. Oxvuris. Ancvlostoma. Uncinaria. Toxascaris and Parascaris. Whereas some nematodes, such as Cooperia and Oesophaaostomum. primarily attack the intestinal canal, others such as Haemonchus and Ostertaaia are present most abundantly in the stomach. Dictvocaulus and others are found in the lungs. Still other parasites may inhabit other tissues and organs of the body, such as the heart, blood vessels, and subcutaneous and lymph tissues. The parasitic infection, known as helminthiasis, causes anemia, malnutrition, body weight loss and severe damage to .the intestinal wall and other tissues and organs and, if left untreated, may lead to death of the host animal.

Other parasitic infections with various species of arthropods, e.g., ticks and mites, fleas, lice, flies, mosquitoes, while the cause of less severe health problems for the animals, such as itching, uncleanliness and discomfort, are also carriers of internal parasites such as helminths, for example, trematodes, cestodes, and nematodes which cause fatal diseases in the animals.

I 236 4 75 4 A variety of anthelmintic compositions presently exist to treat internal parasites, and other compositions exist to treat the external parasites.

Among such compositions are various compounds called benzimidazoles and benzimidazole carbamates. See, for example, U. S. Patent Nos. 3,956,499; 3,574,845 and 3,915,986. Among such compounds are albendazole [methyl 5-(n-propylthio)-lH-benzimidazol-2-ylcarbamate], fenbendazole [methyl 5-(phenylthio)-lH-benzimidazol-2-ylcarbamate], oxibendazole [methyl 5-(n-propoxy)-1H-benzimidazol-2-ylcarbamate], oxfendazole [methyl 5-(phenylsulfinyl)-iH-benzimidazol-2-ylcarbamate], parbendazole, cambendazole, mebendazole, flubendazole, ricobendazole, luxabendazole and others. These benzimidazoles are commercially available. For example, albendazole is presently marketed for use as a cattle dewormer under the brand name "Valbazen" [SmithKline Beecham Animal Health Laboratories].

However, various species of helminths have developed resistance to several of these benzimidazoles. Particularly albendazole alone is becoming an ineffective control agent against ever increasing populations of benzimidazole-resistant nematodes. Therefore several benzimidazoles have been formulated in combination with 2364 75 various organophosphate compounds for similar use. For example, US Patent 4,436,737 provides anthelmintic compositions containing benzimidazoles and the organophosphate, profenofos. Profenofos has the formula C^H^ClBrOjPS. The combination product (albendazole and profenofos) was employed against benzimidazole-resistant helminths, such as Haemonchus contortus and Trichostronqvlus colubriformis.

South African patent 6602811 provides an anthelmintic composition containing an organophosphate and oxibendazole or thiabendazole.

Japanese patent 59033204 discloses a fungicide containing an organophosphate and thiabendazole.

European Patent 181525 discloses an anthelmintic combination of tetramisole with several organophosphates, including chlorpyrifos.

U. S. Patent 3,992,533 refers to thiophosphoric acid phenyl esters as a broad insecticidal agent against plant pathogenic pests.

UK Patent Application 2,094,625 refers to an anthelmintic composition containing alkyl S-substituted benzimidazolyl carbamate and O-ethyl-O-(4-bromo-2-chloropheny1)-3-propyl phosphorothioate.

U.S. Patent 3,244,586 refers to the organophosphate chlorpyrifos, a compound of formula C^CljNOjPS. 2364 6 U. S. Patent 4,531,005 refers to a number of organophosphate compounds, including pyraclofos, useful as insecticides for plant.

U. S. Patent 4,531,005 refers to 4-pyrazolyl phosphorous acid esters useful as intermediates for the-production of 4-pyrazolyl phosphorothioates. These latter compounds are noted as agricultural insecticides and acaricides.

There remains a need in the art for effective antiparasitic compositions for use in treating or preventing parasitic infections, particularly those parasites which are naturally resistant or which have developed resistance to prior art compositions.

Summary of the Invention As one aspect, the invention provides an antiparasitic composition for the prevention, treatment control and/or removal of internal and external parasites from animals which can be administered to animals in effective dosages which do not cause adverse toxic effects in animals. In one embodiment, a composition of this invention comprises pyraclofos as the active ingredient thereof, in a further embodiment, the composition comprises the combination of pyraclofos and a 236 4 7 7 benzimidazole as the active ingredients. In a still further embodiment, the benzimidazole of the composition is albendazole.

As a further aspect, the invention provides a 5 formulation enabling internal administration of the antiparasitic compositions of this invention for the treatment of endoparasitic infection.

Yet other aspects of this invention include appropriate dosage units of the antiparasitic 10 formulations of this invention.

Still a further aspect of this invention is a method for prevention, treatment or control of the endoparasitic infections of animals, caused by helminths, for example, flatworms and roundworms. This method 15 comprises administering to the animals an antiparasitic composition or formulation as described above.

Still a further aspect of this invention is a method for prevention, treatment or control of the ectoparasitic infections of animals, caused by insect and 2 0 arachnids. This method comprises orally administering to the animals an antiectoparasitic composition comprising pyraclofos or a related compound in a suitable carrier.

Other aspects and advantages of the present invention are described further in the following detailed 25 description of preferred embodiments thereof. f~\ ->• 8 Detailed Description of the Invention The present invention provides an antiparasitic, particularly anthelmintic, composition and method of using the composition in the prevention, treatment, control and/or removal of various parasitic infections in animals, caused by both internal parasites, such as helminths and external parasites, such as arthropods. Among the utilities of the present composition is the treatment of parasitic infestations caused by parasites which are resistant to other antiparasitic compositions, particularly the popular anthelmintic compound, albendazole.

The inventors have discovered that the organophosphate compound, pyraclofos, a known plant insecticide, can be effectively employed as an active ingredient in compositions for the removal or control of endoparasites in animals. It has also been surprisingly discovered that pyraclofos can be internally, preferably orally, administered to control animal ectoparasites, as well. Pyraclofos can be formulated for safe internal administration to an animal, either alone or in combination with known antiparasitic compounds, for example, benzimidazoles or benzimidazole carbamates, such as albendazole. 2 3 6 4 7 5 The chemical name for pyraclofos is 0-[l-(4-chlorophenyl)-lH-pyrazol-4-yl]-0-ethyl-S-propyl phosphorothioate. The structural formula of this compound is 0 , N— / —CI II P- c2h5o l_o y c3h7s' -N (I) This compound and methods for producing it are described in detail in U. S. Patent 4,531,005, particularly Example 10 18 therein. This patent is incorporated herein by reference for the purpose of providing information on this compound. Pyraclofos can be obtained under the trade name "Boltage" [Takeda Industries, Japan].

Pyraclofos can be present as the sole active 15 ingredient of an antiparasitic composition. More desirably, the inventors have discovered that in combination with a selected benzimidazole, an anthelmintic composition is formed which is characterized by superior efficacy and non-toxicity in animals. 20 This composition including pyraclofos and a benzimidazole is particularly efficacious against parasites which are substantially resistant to benzimidazoles in general, or albendazole specifically, namely the nematodes, Haemonchus contortus. Ostertaqia 25 circumcincta. and Trichostrongvlus colubriformis. 236475 While pyraclofos is specifically disclosed in the compositions and formulations exemplified herein, it may be possible to substitute for pyraclofos in the above compositions, certain compounds hereafter referred to as "pyraclofos-related compounds". The term "pyraclofos-related compounds" means pyrazolyl phosphoric esters having the general formula (II) wherein R1 is a lower alkyl group; R2 is a lower alkoxy group or a lower alkylthio group; R3 is a hydrogen atom or a lower alkoxy carbonyl group; X is an oxygen atom or a sulfur atom; Y is a lower alkyl group, a lower alkoxy group, a lower alkylthio group, a halogen atom, a nitro group or a trifluoromethyl group; and n is an integer of 0, l, 2 or 3.

Referring to general formula (II), R1 for a lower alkyl group means a straight-chain or branched alkyl group of 1 to 4 carbon atoms, such as methyl, ^ 23 6 4 n ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and so on. R2 for a lower alkoxy group means an alkoxy group of 1 to 4 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isotuboxy, sec-butoxy and so on. R2 for a lower alkylthio group means an alkylthio group containing 1 to 4 carbon atoms, such as methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio and so on. R3 for a lower alkoxycarbonyl group means an alkoxycarbonyl group of 2 to 5 carbon atoms, such as methoxycarbonyl, ethoxycarbonyl and so on. Of the groups denoted by Y, the lower alkyl group, lower alkoxy group and lower alkylthio group means the samfe groups as defined for R1 and R2, respectively, although t-butyl may also be used as the lower alkyl group. The halogen atom, also represented by Y, includes fluorine, chlorine, bromine and iodine. The symbol n denotes the number of substituents represented by Y and where two or more such substituents are present, they may be the same or different. Furthermore, where the group Y is a lower alkoxy group and n is equal to 2, the two alkoxy groups may, taken together, represent an alkylidenedioxy group, such as methylenedioxy, propylidenedioxy, etc., which also falls within the scope of the invention. 236475 12 Among the pyraclofos-related compounds that may be useful in the invention as substitutions for pyraclofos, the compounds having substituents of Formula II as listed in Table 1 below, may be of particular importance. Furthermore, the compounds of formula (II) wherein R1 is ethyl and R2 is n-propylthio are particularly desirable. '3 236 VO o in n- CM m in cr\ CM CM o CO n r> CO in rH -P vo in r* «3" vo flj C in in in in in in in in in O (0 • • • • • « . • • .

•H +J rH H rH rH H H rH rH cH V1 (11 >1 c co a vo a co Q co a o a vo Q o a o a o a J3 0 cm CM CM CM r> CM CM cm <n CL. U c c c c c c c c c c >• m i x CO co co w J CQ < Eh OS CO C/3 u o o u in X (M o o 0 u 1 co K (\l o o 0 u 1 <M "c C c" C c c e •w >l^> "W * - »w fO S in N. s.

N» K pw N. s.

S3 <\J sc ro » ro SB Kl ro X m s ro X u a U O o CJ o o U o o O w CO o CO CO CO a M S u in sc rj u sc CM u in a IM V in EC <M o in a (M u m se rj u rc <NJ o u •0 c 3 0 • cu o & 25 o o CM n in vo CO m in o H in rH O CM C2H5 SC3H7(n) 11 c2h5 oc2h5 12 C2H5 SC3H7(n) 13 C2H5 . SC3H7(n) 10 14 C2H5 SC3H7(n) C2H5 OC2H5 16 C2H5 SC3H7(n) 17 C2H5 SC3H7(n) 18 C2H5 SC3H7(n) 19 C2H5 SC3H7(n) C2H5 SC3H7(n) 14 H 4-Br 27 n D 1.6025 H 2-C1 24 n D 1.5516 H H H H H H H H H 0 2—CI 3-c1 3—CI 4-C1 4-CI 4-C1 4-F 4-F 4-1 24 n D 1. n D 1. 24 n D 1. 5493 5655 5922 24 11 D 1.5635 24 n D l. n D 1. n D l. 28 n D 1. n D 1. 5604 5933 5368 5695 6000 ho CM cr> CJl in cn vo r* n a\ in vo m r- (—i CM o *3* in CO VO CO c\ r> in in CO rH in CO in in in in in in in in in in in • • • • « • • • • • • rH rH rH rH rH rH rH rH rH rH a in a in Q o a CO Q o a o a O Q r- Q o a r- a CM CM CM CM CO CM CM CM CM CM CM c c e c C G c C C c c CM cm (N a r\j fM fM ro o fO co ro ro ro iH rH rH ro ro ro u h h o o o a a a a a O u 1 1 1 u u u c o o 1 1 i i i i i n r> •» CM CM 1 *3" ** "S* CN CM r> CO CO co co in c* c" c c e c c e c c" * «w» »W w SM* w in N. o. fs.

N.

N. r«- N. fx* X a a a a a a X a a a rg ro ro ro a ro ro ro ro ro ro ro U u U u O U U U o O O to co CO CO CO CO CO CO CO W in a CM CJ in a (M u a r\i u a rvi u in a <M CJ a IM u m a «VI o X rsi u a ru U m a C\J u a ru a rH CM CM cm n CM CM w CM vo <N cm 00 (N CTl CM O n h M in o r-1 in o cm £ CM CO rH in rH •<* CO O) VO in *0 CO r~ c\ vo r> n CM in in in in in in in • • • • • • • rH H H H H H rH O Q <0 Q r* Q c- a oo Q 00 Q oo a CM CM CN CM <M fNJ CM c n c c C c c CM CM r\3 CM CM CM fM <\i <M (NJ rH r \ CI *-N ,r—■» V 1 <M rg m rO K> 1 *3* rH rH ffi X K a u O a U i 1 w <r in I 1 1 pt« X •* VO 1 o n r> ■» * CM 1 CM CN CM CM CO o CO O O o o X X X X X sc X C c c* c* c c w N.

N. m N.

K N.

X X X X X X X . "> rvi rO ro ro ro u o o u V u a CQ co o CO CO CO CO in w m m m in in X X X X X X X «M tM r\j (VI CM (M cvi u V u o u o o CM n m vo r* 00 n en n n n n ro in o H 23 6 4 17 The presently most preferred benzimidazole for the combination compositions with pyraclofos or its related compounds is albendazole [methyl 5-(n-propylthio)-lH-benzimidazol-2-ylcarbamate], as 5 specifically disclosed in the compositions and formulations exemplified herein. However, it may be possible to substitute other known and commercially available benzimidizoles or prodrugs which metabolize in the animal into a suitable benzimidazole for albendazole 10 in these compositions. Among such other benzimidizoles are included the following: Fenbendazole [methyl 5-(phenylthio)-1H-benzimidazol-2-ylcarbamate] • Oxibendazole [methyl 5-(n-propoxy)-1H-15 benzimidazol-2-ylcarbamate] Oxfendazole [methyl 5-(phenylsulfinyl)-1H-benzimidazol-2-ylcarbamate] Parbendazole [methyl 5-(butyl)-lH-benzimidazol-2-ylcarbamate] Cambendazole [isopropyl 2-(4-thiazolyl)-5- benzimidazolecarbamate] Mebendazole [methyl 5-benzoyl-2-benzimidizolecarbamate] and 236475 18 Flubendazole [methyl 5-(4-fluorobenzolyl)-1H-benzimidazol-2-ylcarbamate]. Ricobendazole, and Luxabendazole are also known benzimidazoles. These benzimidazoles may be prepared by one of skill in the art with resort to known techniques such as those described in the art cited above.

Prodrugs which metabolize in vivo into selected benzimidazoles may also be employed to replace albendazole in the compositions of this invention. Among the known prodrugs are thiophanates [1,2-phenylenebis (iminocarbonothioyl)biscarbamic acid diethyl esters]. See, for example, U. S. Patent Nos. 3,745,187 and 3,769,308. Another prodrug which may be useful is Febantel, i.e., [2-[(methoxyacetyl)amino]-4-(phenylthio)-phenyl] carbonimidoyl] biscarbamic acid dimethyl ester. Also useful may be the Netobimin esters. These known compounds may also be prepared by known and conventional methods by one of skill in the art.

The antiparasitic compositions of the present invention containing pyraclofos are effective in substantially reducing the number and/or totally eliminating the internal and external parasites. These compositions are also effective upon topical application of warding off both internal and external parasites from ZS 6 19 animals. Additionally the compositions of this invention have the characteristic of substantial non-toxicity to animals when orally administered and ingested by animals at effective dosages.

While the compositions of the invention are particularly useful for the treatment, prevention or management of helminthiasis, caused particularly by benzimidazole-resistant helminths, such compositions may also be useful, also, for the treatment of diseases 10 caused by other parasites. For example, compositions of the present invention containing as the active ingredient only pyraclofos and/or one or more additional pyraclofos-related compounds may be useful in treating and inhibiting Dirofilaria in dogs, Nematospiroides. Svphasia 15 and Aspiculuris in rodents, external arthropodal parasites such as ticks, mites, lice and fleas in animals and fowl, Lucilia and stinging insects in sheep, migrant dipteral nymphs such as Hvpoderma in cattle, Gastophilus in horses, and Cuterepra in rodents. 2 0 Of the compounds of the invention, pyraclofos and the broader class of compounds (II), particularly wherein R1 is ethyl and R2 is n-propylthio, exhibit remarkable control and inhibitory effects on pests feeding on pet stock, such as mosquitoes, fleas and lice, 2 5 and on pests parasitizing domesticated livestock, such as ticks, and yet may be safely administered in oral form to warm-blooded animals. 236 4 75 Depending upon the particular use for which a composition of this invention is directed, the antiparasitic compositions of the invention can be administered to animals in accordance with the procedures 5 known to the profession in the field of veterinary medicine. For example, in the treatment of ectoparasites and endoparasites of animals, e.g., ticks which feed through the surface of the animal's skin and helminths which parasitize internally, the compositions of this 10 invention may be administered to such animals internally.

While internal administration is preferably by the oral route, other modes of internal administration, including without limitation, direct administration into the rumen, intraperitoneal, intramuscular, intratracheal, 15 intravenous or subcutaneous administration, may also be employed.

Compositions of this invention may be prepared by dispersing the pyraclofos or related compounds, preferably in combination with albendazole, uniformly in 20 an inert vehicle or diluent. The term "inert vehicle" means any pharmaceutical vehicle that does not react with the pyraclofos or albendazole and can be safely administered to animals. 23 6 4 21 These compositions may be formulated into various oral dosage forms such as capsules, pills, tablets, granules, solutions, oral pastes and other standard oral formulations. For oral administration, the compositions of this invention can also be evenly admixed into the animal ration, dissolved or suspended in drinking water or admixed in one of the dosage forms described above. In certain cases, oral dosage forms preparations may be given to animals independently of feeding. The vehicle for dietary administration is preferably a component of the ration or a substance which can be a component of the ration. The vehicle or diluent suited to such compositions includes, among others, malt clearings, corn meal, brewers1 grains, crushed oyster shell, wheat bran, soluble molasses, corncob meal, soybean meal and crushed limestone. The active compound is intimately dispersed throughout such vehicle, for example by grinding, stirring, milling or rotational agitation.

For internal administration by oral and other internal delivery routes, the compositions may be prepared by dissolving or dispersing pyraclofos and/or albendazole in a liquid vehicle. Among desirable liquid vehicles are vegetable oils, for example, peanut oil, cottonseed oil, corn oil, water, and physiological saline 236475 22 solution. Presently most desirable for the combination formulation containing pyraclofos and albendazole is corn oil.

Preparations of the above-mentioned dosage 5 forms can be manufactured by conventional pharmaceutical procedures, by adding to the active ingredient(s) diluents, disintegrating agents, antifoams, lubricants, dispersants, surfactants, binders, and emulsifiers.

These optional ingredients may be selected from starch, 10 lactose, talc, magnesium stearate, colloidal silicon dioxide, microcrystalline cellulose, dicalcium phosphate, bentonite, calcium sulfate demihydrate, hydroxypropylmethylcellulose, corn starch, sodium starch glycolate, sodium laurylsulfate, Polysorbate 85 (Sanyo 15 Chemical), phenyl sulfonate CALX (ICI), Synperonic NP13 (ICI), Synperonic PE L44 (ICI), benzyl alcohol, propylene glycol, vegetable resin (wood resin) and so on. Emulsifiers, such as solketal, glycerol formal, benzyl alcohol, carboxymethylcellulose, Sorpol 1200 (Toho 20 Chemical), Polysorbate 80 (Sanyo Chemical), cetyl alcohol and Brij 58 (ICI), may be employed. The latter two emulsifiers are desirable for the combination compositions. Further an optional stiffening agent, e.g., Cabosil (Cabot) may also be added. 236475 23 The presently most desirable formulation of the combination of pyraclofos and albendazole employs corn or other vegetable oil as the liquid vehicle with optimal concentrations of 1% Brij 58, 1% cetyl alcohol and 1% 5 colloidal silica. This formula is preferred for intraruminal administration, but may also be employed orally. This formula, when the albendazole and pyraclofos are homogenized therein, readily suspends, remains .flowable at low temperatures, of about 5°C. 10 Optional components of this formulations included a flavoring agent and a dye.

Dosages of the active ingredients, pyraclofos alone or pyraclofos and albendazole in combination can vary widely in the total weight of the formulation. The 15 percentage content of the active ingredients may also be varied according to the species of host animal, type of parasite to be treated, severity of infection or infestation, and the body weight of the host. The amount of the composition producing the optimal effect is, of 2 0 course, dependent on the particular active compound, species of host animal to be treated, and the type and degree of parasitism or prevalence. Moreover, the rate of release of the active ingredient can be freely adjusted by various techniques such as 25 microencapsulation. 236475 24 From field testing with the combination of pyraclofos and albendazole, the effective ranges of dosages for internal administration include pyraclofos from about 5 to about 50 mgs/kg animal body weight and 5 albendazole from about 1.0 to about 10.0 mgs/kg animal body weight. Preferably, for administration to sheep pyraclofos is present in the combination at about 30 mgs/kg with albendazole at 3.8 mgs/kg. For cattle, the albendazole dosage may be increased to 10 mgs/kg. These 10 dosages can be adjusted for the effect of other ingredients if necessary.

Oral preparations contain generally about 1 to 80 percent by weight of the active compound (pyraclofos alone or in combination with albendazole) and preferably 15 about 10 to 3 0 percent by weight. Generally, however, the useful oral dose of the active compound is about 0.1 mg to 2,000 mg and preferably about 1 mg to 1200 mg per kilogram body weight of the animal and this dose is preferably administered in a single oral dose. One 2 0 presently desirable dosage contains about 40 mg/kg of the active compound.

For administration in feed premixes, the feed may desirably contain about 1 to 80 percent, preferably 10 to 30 percent by weight of the active compound (s), 25 i.e., either pyraclofos alone or pyraclofos with 236475 albendazole. Feed supplements for direct feeding to animals contain about 1 to 50 percent by weight, preferably about 5 to 25 percent by weight, of the active compound(s). Such feed supplements are added to animal 5 rations in amounts sufficient to insure concentrations of the active compound which are effective for the treatment or management of parasitism. The preferred concentration of the active compound is dependent on the above-mentioned factors and the species of compound but in 10 order to insure an adequate antiparasitic effect, it is generally recommended that the compound be used in a concentration of about 1.0 to 50.0 percent, preferably about 10 to 30 percent.

Preparations for administration by routes other 15 than oral contain generally about 1 to 80 percent by weight and preferably about 10 to 50 percent by weight of the active compound(s). For parenteral administration, the useful dosage is generally about 0.1 mg to 300 mg/dose and preferably about 1 mg to 30 mg/dose per 2 0 kilogram body weight of the animal.

It is preferable that animals be treated with repeated dosages of the compositions of this invention to inhibit reinfection or recurrence of original infection. Specifically, the compositions of the present invention 25 may be administered repeatedly over a comparatively short period, for example 1 to 5 days for pet animals. 236475 26 The following examples illustrate various aspects of the invention, but are not intended to limit the scope of the invention.

Example 1 - Preparation of Compositions of this Invention 5 The term "active ingredient" as used below means pyraclofos unless otherwise indicated.

A. Albendazole The benzimidazole Albendazole used in the following examples is the commercially available Valbazen 10 [SmithKline Beecham Animal Health, Lincoln, NE] containing 19 grams per liter of the compound in an aqueous suspension.

B. Pyraclofos Several solutions of pyraclofos may be prepared 15 as follows: Pyraclofos (20.0% W/W), phenyl phosphonate CALX (4.0 % W/W), Synperonic NP13 (6.0% W/W) and Solven 100 (70.0% W/W) are mixed and stirred to provide a solution, which is then diluted with water to give an emulsion of 250 ppm concentration. 2 0 An alternative solution is made by dissolving the active ingredient (30.0% W/W) in corn oil (70.0% W/W) to give a homogeneous solution for oral administration.

A granule formulations is prepared by dissolving the active ingredient (10.0% W/W) and either 25 wood resin (4.0% W/W) or Synperonic NP 13 (4.0% W/W) in 23 6 4 75 27 methylene chloride and the solution is added to granular gypsum (20~60 mesh; 86.0% W/W) in a mixer. After sufficient mixing, the solvent is removed, followed by drying to give granules.

An alternative granule formulation is prepared by the wet granulation method. The active ingredient (25.0% W/W) is mixed with calcium sulfate hemihydrate (75.0% W/W). The mixture is then dried with a tray or fluidized-bed dryer and filled into a suitable container. 10 A tablet formulation is prepared by adding to the active ingredient (50.0% W/W) an adequate amount of 10% starch paste [magnesium stearate (1.0% W/W), corn starch (5.0% W/W), sodium starch glycolate (2.0% W/W), sodium laurylsulfate (1.0% W/W) and microcrystalline 15 cellulose (41.0% W/W) to prepare a wet mass for granulation. The mass is granulated and dried with a tray or fluidized-bed dryer. The dry granules are sieved and the remaining components are added. The mixture is compression-molded to give tablets. 2 0 As necessary, the core tablets are further coated with a film-forming material, such as hydroxypropylmethylcellulose, in an aqueous or nonaqueous solvent system. A plasticizer as well as a suitable color may be incorporated in the film-forming 25 material solution. 236475 28 An oral liquid formulation is prepared by dissolving the active ingredient (5.0% W/W) in a mixture of Polysorbate 85 (5.0% W/W), benzyl alcohol (3.0% W/W) and propylene glycol (30.0% W/W). The solution is 5 adjusted to pH 6.0 ~ 6.5 with phosphate buffer and diluted with water (57.0% W/W) to a specified final volume. The liquid is filled into a drinking water vessel for use.

An oral paste formulation is prepared by 10 dispersing aluminum distearate (5.0% W/W) at 50 - 60°C in a mixture of coconut oil (85.5% W/W) and Polysorbate 85 (3.0% W/W). The dispersion is cooled to ambient temperature with continued stirring, followed by dispersion of saccharin sodium (2.5% W/W) and active 15 ingredient (4.0% W/W) to give a paste.

Several injectable formulations are prepared as follows: One injectable formulation is prepared by dissolving 20.0 % W/W of the active ingredient in a mixture of 36.0 % W/W ethanol and 10% W/W surfactant 20 (Synperonic PE L44) and the solution is diluted with 34.0 % W/W propylene glycol to a specified final volume. This solution is sterilized by the established pharmaceutical procedure, for example by filtration through a bacterial filter or autoclaving and aseptically filled into unit-25 dose containers. 23 6 4 7 29 Another injectable formulation is prepared by dissolving 10% W/W of the active ingredient in a mixture of 10% W/W Polysorbate 80 and 50% W/W glycerol formal, followed by addition of 1.0 % W/W benzyl alcohol. The 5 solution is diluted with 29% W/W water for inject ion to a specified final volume. This solution is sufficiently sterilized by the established pharmaceutical procedure and aseptically filled into unit-dose containers.

Another injectable formulation is prepared by mixing 10.0% W/W active ingredient, 0.5% W/W carboxymethylcellulose (CMC) and 89.5 % W/W physiological saline to give a uniform -suspension. The resulting injection is suitable for intraperitoneal or intravenous 15 administration.

Still a further injectable formulation is prepared by mixing 10.0% W/W active ingredient, 2 0.0% W/W Sorpol 1200 and 70.0 % W/W physiological saline to give a uniform suspension. The resulting injection is suitable 2 0 for intraperitoneal or intravenous administration.

C. Pyraclofos plus Albendazole Three experiment combination products are prepared by homogenizing in vegetable oil, containing 1% Brij 58, 1% cetyl alcohol and 1% colloidal silica, 10 23 6 4 75 mg/kg pyraclofos and 3.8 mg/kg albendazole. The other formulations were similar except that they contained 2 0 mg/kg pyraclofos and 3.8 mg/kg albendazole and 30 mg/kg pyraclofos and 3.8 mg/kg albendazole, respectively.

Example 2 - Combination Field Testing The following test was performed to evaluate the anthelmintic efficacy of graded doses of pyraclofos with and without the addition of albendazole when used to treat sheep infected with benzimidazole resistant strains 10 of adult Haemonchus contortus. Ostertaaia circumcincta and Trichostronavlus colubriformis.

Albendazole alone and the combination product are as described in Example 1 above. Pyraclofos was used in solution of vegetable oil at lOOg/L. 15 Benzimidazole resistant (BZ-R) strains of H. contortus (H/C), O^. circumcincta (0/C) and T. colubriformis (T/C) were obtained from animals in the field. Infective larvae were harvested from incubation within 10 days of infection.

Fifty six nine-month old merino weaners of both sexes were made worm-free by treatment orally with ivermectin (0.2 mg/kg) at least 2 weeks prior to the initiation of the trial and maintained in above ground 2364 75 31 mesh floored pens. All sheep are uniquely identified by a numbered plastic ear tag, and fed ad libitum on a diet of pelleted lucerne with vitamin-mineral supplement.

Eight treatment groups of seven sheep were used as set 5 out in Table 2. Trial schedule was as follows: all sheep were infected on days 1, 2 and 3. At each of these three consecutive days all sheep received the following doses of infective larvae: H/C 3000 0/C 2000 T/C 3000 Infective larvae were administered by intra oesophageal tube, each species being present in approximately 100 mL of water. The fecal egg count on all sheep was performed 15 on day 23. On the basis of ranked body weight within each gender, sheep were randomly assigned to eight groups of seven sheep on day 24.

Treatments were administered on day 25. Individual dose volumes were determined and each dose 2 0 prepared and placed in calibrated syringe labelled with the ear tag number of the intended recipient. All treatments were administered by direct percutaneous intraruminal puncture. 23 6 4 75 32 Seven days after treatment individual fecal egg counts and group bulk larval culture were undertaken. Sheep were slaughtered for total worm counts (TWC) on day 36. The abomasum and small intestine were dissected out 5 and TWC determined by standard techniques.

The results of treatment are illustrated in Table 2 below. In the Table, W stands for Wether, E represent Ewe, 1 represents Merino short wool lambs, 2 represents crossbred long wood lambs, H/C represents H^_ 10 contortus. 0/C represents O. circumcincta. and T/C represents T. colubriformis. 33 TABLE 2 Group Treatment Sheep Gender/ Body FEC Number Breed Weight Day 1 Day 2 (kg) (EPG) 1 Untreated 385 W1 24.50 ,400 477 W2 27.50 ,800 482 W1 22.50 23,000 483 W1 . 50 54,800 484 El 23.50 31,400 956 E2 16.00 32,200 959 E2 21.00 54,800 Mean 22.21 33,914 Std deviation 3.34 14,190 152 Albendazole 897 El 19. 00 22,800 3.8 mg/kg 899 W1 22. 00 27,600 486 W1 21.50 ,400 913 E2 19.50 43,200 925 E2 .00 7,200 934 W2 28.00 14,800 950 E2 24 .00 16,800 Mean Std deviation 22.71 2.95 22,114 10,454 • • FEC Day 8 (EPG) 24,200 56,400 39,000 50,800 37,000 50,600 43,400 44,200 11,228 520 440 1,080 280 1,440 120 640 646 429 Total Worm Counts Day 14 H/C O/C T/C 3, 960. 00 40. 00 8, 440. 00 9, 640. 00 0. 00 6, 440. 00 4, 880. 00 40. 00 7, 480. 00 6, 720. 00 320. 00 12, 840. 00 6, 040. 00 80. 00 8, 960. 00 1, 640. 00 40. 00 11, 240. 00 2, 720. 00 240. 00 7, 720. 00 , 085. 71 108. 57 9, 017. 14 2, 481. 42 112. 56 2, 090. 07 200. 00 80. 00 if 480. 00 80. 00 0. 00 280. 00 320. 00 40. 00 1» 960. 00 120. 00 40. 00 1, 200. 00 320. 00 40. 00 1, 120. 00 40. 00 120. 00 2, 280. 00 80. 00 100. 00 920. 00 165. 71 68 . 57 if 320. 00 107 . 82 51. 11 616. 61 tno 04 CT) CJl 3 Pyraclofos 896 El 17.50 mg/kg 481 E2 24.00 899 W2 26. 50 936 W2 22.00 949 E2 24.50 951 E2 21. 00 986 W2 24.60 Mean 22.86 Std deviation 2.75 104 Pyraclofos 394 El 26.00 mg/kg 487 El .00 708 E2 18.00 774 W2 26.00 904 W2 22.00 957 E2 23.00 984 W2 .50 Mean 22.21 Std deviation 2.80 Pyraclofos 388 W1 21.50 mg/kg 392 El 17.50 398 El 22.00 400 El 24.50 900 W2 26.00 905 W2 24.50 941 E2 23.00 Mean 22.71 Std deviation 2.58 34 54 000 28,000 1,840.00 120.00 ,200.00 400 40 480.00 80.00 7,400.00 11 400 1,360 0.00 40.00 6,640.00 14 000 7,800 1,360.00 0.00 7,040.00 21 000 9,400 600.00 40.00 6,560.00 27 200 3,600 200.00 0.00 6,680.00 43 600 42,200 2,960.00 40.00 8,800.00 29 514 13,200 1,062.86 45.71 7,617.14 14 537 14,683 979.76 39.59 1,276.22 16 600 240 40.00 0.00 680.00 37 800 3,200 40.00 80.00 2,600.00 26 800 240 0.00 0.00 960.00 33 200 400 40.00 40.00 3,280.00 39 800 2,000 480.00 160.00 2,840.00 26 000 680 80.00 80.00 1,860.00 22 400 440 0.00 40.00 680.00 000 1,029 97.14 57.14 1,800.00 8 004 1, 053 158.36 51.75 1,013.06 34 200 80 0.00 0.00 0.00 14 000 40 0.00 0.00 80. 00 14 800 120 0.00 0.00 280.00 800 360 40.00 40.00 1,760.00 16 800 120 0. 00 0.00 320.00 26 200 200 0.00 0.00 1,520.00 34 400 40 0.00 80.00 80.00 23 029 137 0.71 17.14 577.14 081 104 14.00 29.14 683.47 VA} ro OJ CD -fc- CJl 6 Pyraclofos 386 mg/kg 3 67 + 390 Albendazole 391 5 3.8 mg/kg 921 944 947 Mean Std deviation 107 Pyraclofos 389 mg/kg 396 .+ 485 Albendazole 709 3.8 mg/kg 917 940 956 Mean Std deviation 8 Pyraclofos 393 30 mg/kg 487 + 926 Albendazole 931 3.8 mg/kg 932 981 983 W1 W1 El El E2 W2 W2 El El W2 W2 E2 W2 W2 El W2 E2 E2 W2 W2 W2 Mean Std deviation 22.00 20.50 21.50 19.50 23.00 26.00 30.50 23.29 3.51 24.00 22.00 19.00 25. 50 16.50 24.50 30.00 23.07 4.10 22,600 9,400 12,000 20,600 25,200 25,800 16,800 18,914 6,930 ,200 28,400 45,200 22,400 27,400 23,800 15,800 26,171 8,715 240 80 200 80 120 0 0 103 85 0 0 40 0 80 0 160 40 57 0.00 0.00 0.00 0.00 0.00 40.00 0.00 .71 14.00 0. 00 0.00 0.00 0. 00 0.00 0.00 0.00 0.00 0.00 0.00 80.00 120.00 0.00 80.00 0.00 0.00 40. 00 47.81 0.00 0.00 0.00 0.00 80.00 0.00 0.00 11.43 27.99 280.00 800.00 320.00 280.00 240.00 680.00 280.00 411.43 211.35 400.00 200.00 0.00 0.00 320.00 200.00 440.00 222.86 164.03 22.50 20.00 16.50 23.50 26.00 24.50 25. 50 22.64 3 .12 13,400 37,800 21,400 22,800 43,800 14,400 16,200 24,257 11,056 0 0 0 0 0 40 0 6 14 0.00 0.00 0.00 0.00 0.00 0. 00 0.00 0. 00 0. 00 0.00 0.00 0.00 0.00 0.00 0. 00 0.00 0.00 0. 00 80.00 40.00 40. 00 0.00 0.00 80. 00 ro 160.00 57.14 0> 51.75 _ CJl 23 64 75 36 Example 3 - Biological Field Testing with Pyraclofos Only Against Endoparasites A composition in capsule form containing pyraclofos 0-[1-(4-Chlorophenyl)pyrazol-4-yl] 0-ethyl S-n-propylthiophosphate, was administered orally to dogs parasitized with canine filariae to evaluate its anti-endoparasitic effect.

For two days prior to oral administration of the composition, the microfilariae in peripheral blood were counted. One ml of blood was taken from the lateral saphenous vein and the parasites were collected by the methylene blue sedimentation method (1500 rpm, 10 minutes) and examined under the microscope.

The dogs were then fasted overnight and for the following three days, the dogs were orally administered by gastric gavage with 10, 100 or 1000 mg composition/kg body weight/day. Blood sampling and examination as described above was performed on days 1, 3, 5, and 8 counting from the last day of oral treatment. The feces were also examined macroscopically for internal parasites.

In any of the 10, 100, and 1000 mg/kg groups, blood samples from the lateral saphenous vein of animals surviving the treatment were negative for microfilariae. The. discharge of roundworms (Toxocara canis) in feces was found in one case in the 10 mg/kg group. 236475 37 Thus the results shown in Table 3 below indicate that pyraclofos alone is effective in reducing the number of microfilariae in the blood of dogs infected with canine filariae and, also in controlling dog roundworms. In the following table, condition* represents color of hair and temper of dog, and the microfilariae** were measured on the day before administration (day 0), and days 1, 3, 5, and 8 after the last day of administration. A + sign indicates that microfilariae were detected; (+) indicates sparse microfilariae. A -sign indicates that microfilariae were not detected. Results provide a report on the general condition of the dog and other observations.

• • Group Dosage Animal $ fmg/kg) #/sex Condition* 1 10 Al/F tan ferocious A2/M tan/white 2 100 Bl/M black tractable B2/F black, legs tan vicious B3/F beagle tractable 3 1000 Cl/M light tan vicious C2/F beagle ' tractable 38 TABLE 3 Wt. (kg) Dosing Volful) 56 Microfilariae* 0 13 5 8 Results Good condition 13 104 Roundworms on feces (day 2); good condition. 14.5 1200 + + + (+) — Good condition 480 + + - - - Good condition 18 1440 + + - (+) - Good condition 12 9600 Salivation good cdtn. 11 8800 + + + - - Salivation good cdtn. 236475 39 Example 4 - Biological Field Testing with Internally Administered Pyraclofos Only Against Ectoparasites A. Using dogs (10.0 - 14.0 kg, mongrel) and cats (3.0 ~ 4.0 kg, hybrid) infected with fleas, an injection prepared from pyraclofos, CMC and physiological saline according to Example 1 was intraperitoneally or intravenously administered in predetermined amounts.

For evaluation of efficacy, the death of 80% or more of fleas in the fur coat and on a white sheet spread beneath the animal during 6 hours after administration was regarded as a positive response (+, effective) and otherwise as no response (-, ineffective). The results are shown in Table 4.

TABLE 4 Active Route of Animal Sex No. Response . ingred., adminis- Species mg/animal tration I. P. Dog Male 1 + 300 I. P. Cat Male 2 + Control I.P. Dog Female 1 Control I.P. Cat Female 1 B. An injection prepared according to Example 1 except that Sorpol 1200 was used in lieu of CMC was tested in the same manner as described in part A above. The results are shown in Table 5. The number of parasites before treatment ranged from 30 to 40. 236475 40 TABLE 5 Active Route of Animal Sex No. Response ingred., adminis- Species ma/animal tration 300 I.P.

Dog Male 2 + 1000 I.P.

Dog Male 1 + 3 I.V.

Dog Male 1 + I.V.

Dog Female 2 + 300 I.V.

Dog Female 1 + I.V.

Cat Male 1 + Control I.V.

Cat Male 1 - C. Using dogs (10.0 - 14.0 kg, mongrel) and 15 cats (3.0 - 4.0 kg, hybrid) infected with fleas, a solution prepared from pyraclofos and corn oil according to Example 1 was orally administered in predetermined amounts through a catheter.

The evaluation was made by the same method as 20 described in Example 5. The population of parasites before treatment ranged from 30 to 40 per animal. The results are shown in Table 6.

TABLE 6 Active Animal Sex No. Response ingred., Species ma/animal Dog Male 2 + 50 Dog Female 2 + 500 Dog Male 1 + 2000 Dog Female 1 + 50 Cat Male 2 + 500 Cat Female 2 + Control Dog Male 1 - Control Cat Female 1 - 236475 41 Example 5: Toxicity Study of Pyraclofos The acute toxicity values (LD50) in five-week old mice of ddY-SLC strain (male) are set forth below in Tatfle 7.

TABLE 7 Compound No.

LD50(mq/kg) Compound No.

LD;o(ma/kcn 1 >300 23 50-300 2 >300 24 >300 3 >300 >300 4 >300 26 >300 >300 27 50-300 6 >300 28 >300 7 >300 29 *=300 8 >300 >300 9 >300 31 >300 >300 32 >300 11 >300 33 >300 12 *300 34 >300 13 >300 *=300 14 >300 36 >300 >300 37 >300 16 >300 38 >300 17 >300 39 18 >300 40 19 >300 41 50-300 >300 42 >300 21 >300 43 >300 22 *=300 Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. For example, use of other appropriate carriers, diluents and optional agents for use in the 42 formulations may be part of this invention, as are obvious modes and intervals of administration. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

It is to be understood that the terms "animal" and "animals" used hereinabove and in the appended claims do not include humans. 236475

Claims (4)

WHAT WE CLAIM IS: 43

1. An orally endoparasitic composition and non-toxic amount of a administrable, anti-comprising an effective pyraclofos-related compound.

2. The composition according to claim 1 wherein said compound is pyraclofos.

3. An anti-endoparasitic composition comprising a combination of an effective non-toxic amount of a pyraclofos-related compound and an effective, nontoxic amount of a benzimidazole or prodrug thereof.

4. The composition according to claim 3 wherein said compound is pyraclofos.

5. The composition according to claim 3 or claim 4 wherein said benzimidazole is selected from the group consisting of albendazole, fenbendazole, oxibendazole, oxfendazole, parbendazole, cambendazole, mebendazole, flubendazole, ricobendazole, and luxabendazole. 23647 44

6. The composition according to claim 5 wherein said benzimidazole is albendazole.

7. The composition according to claim 3 or claim 4 wherein said prodrug is selected from the group consisting of thiophanate, febantel, and netobimin.

8. The composition according to claim 3 wherein said compound is pyraclofos and said benzimidazole is albendazole.

9. The composition according to claim 4 wherein said amount of pyraclofos is between 5 and 50 mg/kg animal body weight.

10. The composition according to claim 6 wherein said amount of albendazole is between 1 and lOmg/kg animal body weight.

11. The composition according to claim 8 wherein said amount of pyraclofos is between 5 and 50 mg/kg animal body weight and said amount of albendazole is between 1 and 10 mg/kg animal body weight. ?3S475 45

12. The composition according to claim 11 wherein said amount of pyraclofos is between 10-30 mg/kg animal body weight and said amount of albendazole is approximately 3.8 mg/kg animal body weight.

13. An anthelmintic composition comprising pyraclofos in an amount of between 5 to 50 mg/kg animal body weight and albendazole in an amount of between 1 to 10 mg/kg animal body weight.

14. The composition according to claim 13 comprising 10 to 3 0 mg/kg pyraclofos, 3.8 mg/kg albendazole, vegetable oil, 1% cetyl alcohol, 1% colloidal silica and an optional stiffener.

15. A therapeutic formulation for internal administration to an animal comprising the anti-endoparasitic composition of any one of claims 1 to 14.

16. A method for prevention, treatment, removal or control of the infections of animals by endoparasites comprising administering to an animal an effective amount of a composition comprising an effective anti-endoparasitic amount of a pyraclofos-related Q compound as the active ingredient thereof. < 5MAY199S \» 3 n 4 7 5 46

17. A method according to claim 16 wherein said compound is pyraclofos. control of the infections of animals by endoparasites comprising administering to an animal an effective amount of an anti-endoparasitic composition comprising a combination of an effective non-toxic amount of a pyraclofos-related compound and an effective, non-toxic amount of a benzimidazole or prodrug thereof.

19. A method according to claim 18 wherein said composition is a composition according to any one of claims 4 to 14.

20. A method for prevention, treatment or control of the infections of animals by ectoparasites comprising orally administering to an animal an effective amount of a composition comprising an effective and nontoxic amount of a pyraclofos-related compound as the active ingredient thereof.

21. A method according to claim 20 wherein said compound is pyraclofos.

18. a method for prevention, treatment or o 236475 47

22. A composition according to claim 1 or claim 3 substantially as hereinbefore described with reference to the foregoing Example 1.

23. A method according to claim 16 substantially as hereinbefore described with reference to either of Examples 2 or 3.

24. A method according to claim 18 substantially as hereinbefore described with reference to Example 2.

25. A method according to claim 20 substantially as hereinbefore described with reference to Example 4. DAT CD