Paraherquamide and Dihydroparaherquamide As Antiparasitic Agents

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~" ' MM II II II MM Ml I II Ml II II I II J European Patent Office © Publication number: 0 301 742 B1 Office europeen- desj ^brevets .

@ Date of filing: 18.07.88

Paraherquamide and dihydroparaherquamide as antiparasitic agents.

© Priority: 28.07.87 US 78615 © Proprietor: MERCK & CO. INC. 126, East Lincoln Avenue P.O. Box 2000 @ Date of publication of application: Rahway New Jersey 07065-0900(US) 01.02.89 Bulletin 89/05 @ Inventor: Matas, Maria T. D. © Publication of the grant of the patent: C/Olld 4 03.03.93 Bulletin 93/09 E-28010 Madrld(ES) Inventor: Punsola, Luis © Designated Contracting States: C/Mayor 39 La Selva del Campo CH DE FR GB IT LI NL E-43470 Reus Tarragona(ES) Inventor: Del Val, Sagrarlo Mochales © References cited: Pez Volador, 14 EP-A- 0 146 414 Madrid 7(ES) Inventor: Ostllnd, Dan A TETRAHEDRON LETTERS, vol. 22, 1981, 613 Encampment Drive Bound Brook pages 135-136, Pergamon Press Ltd., GB; M. New Jersey 08805(US) YAMAZAKI et al.: "The structure of paraherquamide, a toxic metabolite from penlcllllum paraherquei" © Representative: Hesketh, Alan, Dr. European Patent Department Merck & Co., Inc. Terllngs Park Eastwlck Road 00 Harlow Essex, CM20 2QR (GB) CM

00 Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid (Art. 99(1) European patent convention). Rank Xerox (UK) Business Services (3. 10/3.5x/3.0. 1) EP 0 301 742 B1

Description

BACKGROUND OF THE INVENTION

5 Paraherquamide and dihydroparaherquamide are known compounds and are disclosed in Yamazaki et al in Tetrahedron Letters 22 135-136 (1981). Paraherquamide is indicated as being a fungal metabolite of Penicillium paraherquei. Dihydroparaherquamide is prepared from paraherquamide by catalytic hydrogenation. No uses for the compounds are suggested.

w SUMMARY OF THE INVENTION

This invention is concerned with the use of paraherquamide and dihydroparaherquamide as antiparasitic agents. Thus, it is an object of this invention to describe such uses. A further object is to describe compositions containing such compounds as the active ingredient thereof. Further objects will become 75 apparent from a reading of the following description.

DESCRIPTION OF THE INVENTION

Paraherquamide has the following structure: 20 Ha

I 25

30 Dihydroparaherquamide has the following structure:

35 II

Paraherquamide is isolated as a fungal metabolite of Penicillium paraherquei using standard fermentation and isolation techniques. The analytical and structural characteristics of paraherquamide are described in detail in Yamazaki et al Tetrahedron Letters 22 135-136 (1981). 45 Dihydroparaherquamide is prepared from paraherquamide by catalytic hydrogenation over palladium on a carbon support. The analytical characteristics of dihydroparaherquamide are also given in Yamazaki et al. It has been unexpectedly discovered that paraherquamide and dihydroparaherquamide are potent endo- and ecto-antiparasitic agents against parasites particularly helminths, ectoparasites, insects, and acarides, infecting man, animals and plants. 50 The disease or group of diseases described generally as helminthiasis is due to infection of an animal host with parasitic worms known as helminths. Helminthiasis is a prevalent and serious economic problem in domesticated animals such as swine, sheep, horses, cattle, goats, dogs, cats and poultry. Among the helminths, the group of worms described as nematodes causes widespread and often times serious infection in various species of animals. The most common genera of nematodes infecting the animals 55 referred to above are Haemonchus, Trichostrongylus, Ostertagia, Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia, Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostoma, Uncinaria, Toxascaris and Parascaris. Certain of these, such as Nematodirus, Cooperia, and Oesophagostomum attack primarily the intestinal tract while

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others, such as Haemonchus and Ostertagia, are more prevalent in the stomach while still others such as Dictyocaulus are found in the lungs. Still other parasites may be located in other tissues and organs of the body such as the heart and blood vessels, subcutaneous and lymphatic tissue. The parasitic infections known as helminthiases lead to anemia, malnutrition, weakness, weight loss, severe damage to the walls of 5 the intestinal tract and other tissues and organs and, if left untreated, may result in death of the infected host. The compounds of this invention have unexpectedly high activity against these parasites, and in addition are also active against Dirofilaria in dogs, Nematospiroides, Syphacia, Aspiculuris in rodents, arthropod ectoparasites of animals and birds such as ticks, mites, lice, fleas, blowfly, in sheep Lucilia sp., biting insects and such migrating diperous larvae as Hypoderma sp. cattle, Gastrophilus in horses, and io Cuterebra sp. in rodents. The instant compounds are also useful against parasites which infect humans. The most common genera of parasites of the gastro-intestinal tract of man are Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, and Enterobius. Other medically important genera of parasites which are found in the blood or other tissues and organs outside the gastrointestinal tract are the filiarial worms such is as Wuchereria, Brugia, Onchocerca and Loa, Dracunuculus and extra intestinal stages of the intestinal worms Strongyloides and Trichinella. The compounds are also of value against arthropods parasitizing man, biting insects and other dipterous pests causing annoyance to man. The compounds are also active against household pests such as the cockroach, Blatella sp., clothes moth, Tineola sp., carpet beetle, Attagenus sp., and the housefly Musca domestica. 20 The compounds are also useful against insect pests of stored grains such as Tribolium sp., Tenebrio sp. and of agricultural plants such as aphids, (Acyrthiosiphon sp.); against migratory orthopterans such as locusts and immature stages of insects living on plant tissue. The compounds are useful as a nematocide for the control of soil nematodes and plant parasites such as Meloidogyne sp. which may be of importance in agriculture. 25 These compounds may be administered orally in a unit dosage form such as a capsule, bolus or tablet, or as a liquid drench where used as an anthelmintic in mammals. The drench is normally a solution, suspension or dispersion of the active ingredient usually in water together with a suspending agent such as bentonite and a wetting agent or like excipient. Generally, the drenches also contain an antifoaming agent. Drench formulations generally contain from about 0.001 to 0.5% by weight of the active compound. 30 Preferred drench formulations may contain from 0.01 to 0.1% by weight. The capsules and boluses comprise the active ingredient admixed with a carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate. Where it is desired to administer the instant compounds in a dry, solid unit dosage form, capsules, boluses or tablets containing the desired amount of active compound usually are employed. These dosage 35 forms are prepared by intimately and uniformly mixing the active ingredient with suitable finely divided diluents, fillers, disintegrating agents, and/or binders such as starch, lactose, talc, magnesium stearate or vegetable gums. Such unit dosage formulations may be varied widely with respect to their total weight and content of the antiparasitic agent depending upon factors such as the type of host animal to be treated, the severity and type of infection and the weight of the host. 40 When the active compound is to be administered via an animal feedstuff, it is intimately dispersed in the feed or used as a top dressing or in the form of pellets which may then be added to the finished feed or optionally fed separately. Alternatively, the antiparasitic compounds of this invention may be administered to animals parenterally, for example, by intraruminal, intramuscular, intratracheal, or subcutaneous injection in which the active ingredient is dissolved or dispersed in a liquid carrier vehicle. For parenteral administration, 45 the active material is suitably admixed with an acceptable vehicle, preferably of the vegetable oil variety such as peanut oil or cotton seed oil. Other parenteral vehicles such as organic preparation using solketal, glycerol formal, and aqueous parenteral formulations are also used. The active compound or compounds are dissolved or suspended in the parenteral formulation for administration; such formulations generally contain from 0.005 to 5% by weight of the active compound. 50 Although the antiparasitic agents of this invention find their primary use in the treatment and/or prevention of helminthiasis, they are also useful in the prevention and treatment of diseases caused by other parasites, for example, arthropod parasites such as ticks, lice, fleas, mites and other biting insects in domesticated animals and poultry. They are also effective in treatment of parasitic diseases that occur in other animals including humans. The optimum amount to be employed for best results will, of course, 55 depend upon the particular compound employed, the species of animal to be treated and the type and severity of parasitic infection or infestation. Generally good results are obtained with the compounds of the invention by the oral administration of from about 0.001 to 10 mg per kg of animal body weight, such total dose being given at one time or in divided doses over a relatively short period of time such as 1-5 days.

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With the preferred compounds of the invention, excellent control of such parasites is obtained in animals by administering from about 0.025 to 0.5 mg per kg of body weight in a single dose. Repeat treatments are given as required to combat re-infections and are dependent upon the species of parasite and the husbandry techniques being employed. The techniques for administering these materials to animals are 5 known to those skilled in the veterinary field. When the compounds described herein are administered as a component of the feed of the animals, or dissolved or suspended in the drinking water, compositions are provided in which the active compound or compounds are intimately dispersed in an inert carrier or diluent. By inert carrier is meant one that will not react with the antiparasitic agent and one that may be administered safely to animals. Preferably, a carrier io for feed administration is one that is, or may be, an ingredient of the animal ration. Suitable compositions include feed premixes or supplements in which the active ingredient is present in relatively large amounts and which are suitable for direct feeding to the animal or for addition to the feed either directly or after an intermediate dilution or blending step. Typical carriers or diluents suitable for such compositions include, for example, distillers' dried grains, corn meal, citrus meal, fermentation residues, is ground oyster shells, wheat shorts, molasses solubles, corn cob meal, edible bean mill feed, soya grits and crushed limestone. The active compounds are intimately dispersed throughout the carrier by methods such as grinding, stirring, milling or tumbling. Compositions containing from about 0.005 to 2.0% weight of the active compound are particularly suitable as feed premixes. Feed supplements, which are fed directly to the animal, contain from about 0.0002 to 0.5% by weight of the active compounds. 20 Such supplements are added to the animal feed in an amount to give the finished feed the concentration of active compound desired for the treatment and control of parasitic diseases. Although the desired concentration of active compound will vary depending upon the factors previously mentioned as well as upon the particular compound employed, the compounds of this invention are usually fed at concentrations of between 0.00001 to 0.002% by weight in the feed in order to achieve the desired 25 antiparasitic result. In using the compounds of this invention, the individual compounds may be prepared and used in that form. Alternatively, mixtures of the individual compounds may be used, or other active compounds not related to the compounds of this invention. The compounds of this invention are also useful in combatting agricultural pests that inflict damage 30 upon crops while they are growing or while in storage. The compounds are applied using known techniques such as sprays, dusts or emulsions, to the growing or stored crops to effect protection from such agricultural pests. The following examples are provided in order that this invention might be more fully understood; they are not to be construed as limitative of the invention. 35 EXAMPLE 1

Four "parasite-free" sheep were exposed to infection with infective L3 of H. contortus, O. circumcincta, T. axei, T. colubriformis, C. curticei, C. oncophora, and Oe. columbianum. Twenty-four days after the last 40 oral dose of infective larvae, when the infections were patent, three of the sheep were randomly assigned to treatment with either 6, 3 or 1 mg/kg body weight of paraherquamide as a single oral dose. The fourth sheep was maintained as an untreated control. All treatments were administered as solutions at a volume of 0.2 ml/kg P.O.: 6 mg/kg as a solution containing 30 mg/ml; 3 mg/kg as a solution containing 15 mg/ml; 1 mg/kg as a solution containing 5 mg/ml. The vehicle used was a mixture of dimethylsulfoxide: propylene 45 glycohmiglyol (1 .5:5:3.5). Four days following treatment fecal worm egg counts of the 3 treated sheep fell from a level of 4,000- 20,000 EPG to a barely detectable or undetectable level while the untreated control sheep egg count remained at = 9,000 EPG. Eight days following treatment the treated and control sheep were sacrificed and their residual worm 50 burdens were recovered and identified using standard procedures. The worm burdens of the treated sheep were compared with that of the untreated control and the evaluations are tabulated as Table 1 .

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EXAMPLE 2

A suspension of a crude extract of paraherquamide was prepared in 5% acetone 0.2% Triton® aqueous solution. Using a standard test procedure, this material was tested in two replicates against first instar Lucilia sericata at a concentration of 1 ml whole broth equivalent (the prepared material is diluted equally with serum). All larvae in both replicates were completely immobile and presumed dead within 2 hours. EP 0 301 742 B1

EXAMPLE 3

The same suspension used in Example 2 was used at a concentration of 2 ml whole broth equivalent to screen for activity against tick larvae. For this test Dermacentor albipictus larvae were immersed in 2 ml 5 whole broth equivalent paraherquamide for 10 minutes. The liquid was then removed with vacuum retaining the ticks on drug-impregnated filter paper. The compound was rapidly and completely active.

Claims

io 1. The use of paraherquamide or dihydro-paraherquamide or a mixture thereof, for the manufacture of a composition useful for the prevention and treatment of endo- and ecto-parasite infections.

2. The use as claimed in Claim 1 wherein the composition manufactured is orally administrable and contains the active ingredient at a dose of 0.001 to 10 mg per kg of body weight of the animal to be is treated.

3. A composition useful for the prevention and treatment of endo-and ecto-parasitic infections which comprises an inert ingredient and paraherquamide or dihydroparaherquamide or a mixture thereof.

20 4. The composition of Claim 3 which is a drench formulation and the active ingredient is present at from 0.001 to 0.5% by weight.

5. The composition of Claim 3 which is a finished feed formulation and the active ingredient is present at from 0.00001 to 0.002% by weight. 25 6. The composition of Claim 3 which is a feed premix containing from 0.005 to 2% by weight of the active ingredient.

7. The composition of Claim 3 which is a feed supplement containing from 0.0002 to 0.5% by weight of 30 the active ingredient.

8. The composition of Claim 3 which is a parenteral formulation containing from 0.005 to 5% by weight of the active compound.

35 9. A method for the prevention and treatment of endo- and ecto-parasite infections in non-animal situations which comprises administration of paraherquamide or dihydro-paraherquamide or a mixture thereof.

Patentanspruche 40 1. Verwendung von Paraherquamid oder Dihydroparaherquamid oder einem Gemisch davon zur Herstel- lung eines Mittels, das geeignet ist zur Verhinderung und Behandlung von Endo- und Ektoparasiten- Infektionen.

45 2. Verwendung nach Anspruch 1 , wobei das hergestellte Mittel oral verabreichbar ist und den Wirkstoff in einer Dosis von 0,001 bis 10 mg/kg Korpergewicht des zu behandelnden Tieres enthalt.

3. Mittel, das zur Verhinderung und Behandlung von Endo- und Ektoparasiten-lnfektionen geeignet ist und das einen inerten Bestandteil und Paraherquamid oder Dihydroparaherquamid oder ein Gemisch davon 50 enthalt.

4. Mittel nach Anspruch 3, das eine Arzneimittelzubereitung ist und bei dem der Wirkstoff in einer Menge von 0,001 bis 0,5 Gew.-% vorhanden ist.

55 5. Mittel nach Anspruch 3, das eine fertige Futtermittelzubereitung ist und bei dem der Wirkstoff in einer Menge von 0,00001 bis 0,002 Gew.-% vorhanden ist.

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6. Mittel nach Anspruch 3, das ein Futtermittel-Vorgemisch ist und den Wirkstoff in einer Menge von 0,005 bis 2 Gew.-% enthalt.

7. Mittel nach Anspruch 3, das ein Futtermittel-Zusatz ist und den Wirkstoff in einer Menge von 0,0002 bis 5 0,5 Gew.-% enthalt.

8. Mittel nach Anspruch 3, das eine parenterale Zubereitung ist und den Wirkstoff in einer Menge von 0,005 bis 5 Gew.-% enthalt. io 9. Verfahren zur Verhinderung und Behandlung von Endo- und Ektoparasiten-lnfektionen in anderen Situationen als bei Tieren, das die Verabreichung von Paraherquamid oder Dihydroparaherquamid oder einem Gemisch davon umfa/St.

Revendicatlons 15 1. Utilisation de paraherquamide ou de dihydroparaherquamide ou d'un melange de ceux-ci pour fabriquer une composition utile pour la prevention et le traitement d'infections par des endo- et des ecto-parasites.

20 2. Utilisation selon la revendication 1 , dans laquelle la composition fabriquee est administrable oralement et contient I'ingredient actif a une dose de 0,001 a 10 mg par kg de poids corporel de I'animal a traiter.

3. Composition utile pour la prevention et le traitement des infections par des endo- et des ecto-parasites, qui comprend un ingredient inerte et du paraherquamide ou du dihydroparaherquamide, ou un melange 25 de ceux-ci.

4. Composition selon la revendication 3, qui est une formulation en breuvage et dans laquelle I'ingredient actif est present a raison de 0,001 a 0,5 % en poids.

30 5. Composition selon la revendication 3, qui est une formulation d'aliment fini et dans laquelle I'ingredient actif est present a raison de 0,00001 a 0,002 % en poids.

6. Composition selon la revendication 3, qui est un aliment de base contenant de 0,005 a 2 % en poids de I'ingredient actif. 35 7. Composition selon la revendication 3, qui est un supplement alimentaire contenant de 0,0002 a 0,5 % en poids de I'ingredient actif.

8. Composition selon la revendication 3, qui est une formulation parenterale contenant de 0,005 a 5 % en 40 poids du compose actif.

9. Procede de prevention et de traitement d'infections par des endo- et des ecto-parasites non applicable aux animaux, qui comprend I'administration de paraherquamide ou de dihydroparaherquamide ou d'un melange de ceux-ci. 45