Active principle based on avermectins, related strain and process for the preparation thereof, as well as veterinary composition containing it

Abstract: By carrying out the fermentation of Streptomyces avermitilis ATCC 55278, a dried mycelium is obtained which, orally administered to animals, particularly to ovines, has an antiparasitic activity.

Description: The present invention refers to antiparasitic products for zootecnic-veterinary use and, more specifically, to an active principle based on avermectin, to the process for the production thereof, to the relevant strain, as well as to the composition for veterinary use containing it. The avermectins form a family of eight macrolide antibiotics with 16 carbon atoms, having the following structural formula: wherein: The avermectins are obtained by fermentation starting from Streptomyces avermitilis (ATCC 31271) and are further obtained in purified form by the process disclosed in USP No. 4,310,519. However, the individual avermectins, even if separated among them, exhibit a remarkable instability, and, therefore, only an avermectin B 1 derivative obtained, after purification of the other seven homologues, by means of catalytic reduction of the 22-23 position bond of the aforesaid avermectin, a derivative that is named ivermectin and the chemical name of which is 22, 23-dihydroavermectin B 1 , has found practical use. The ivermectin is at present the main form, used in the zootecnic-veterinary field of these macrolide antibiotics and is administered for veterinary prophylaxis, exclusively in a form of an injectable speciality. One of its uses, in a form of a gastroprotected tablet indicated for canine filariosis, is known. Of course, the purification complexity aimed at the isolation of only the avermectin B 1 , as well as the considerally high cost of the catalytic reduction carried out in the presence of rhodium chloride and triphenyl phosphine, and, finally, the problems involved in preparing and utilizing injectable sterile forms, make this speciality remarkably expensive. In addition, the need of injective administration is not by itself a negligeable drawback. On the other hand, the outstanding instability of avermectins in an acid environment excluded from the beginning their therapeutical use in forms which were not injectable or conveniently protected. Taking also into account that the original producing microorganism, Streptomyces avermitilis ATCC 31271, cultivated and fermented according to the teachings of USP No. 4,310,519, gives rise to a very low output of avermectin, the objective limits to a wider and more general utilization of the ivermectin itself are evident. It has now surprisingly been found, and it is the main object of the present invention, that by orally administering to animals, e.g. ovines, the mycelium, obtained by fermentation, merely dried, without any purification procedure and/or separation of avermectins and/or chemical modification of them, excellent results, so far the veterinary activity is concerned, are achieved since the total disappearance of gastrointestinal strongyles and a not negligeable reduction of other parasites is observed in the treated animals. In another embodiment of the present invention it has been found that it is possible to increase the productivity of total avermectins using a strain modified as compared with the ATCC 31271, used in the prior art, the strain, according to this invention, being the Streptomyces avermitilis GRL 1926 (ATCC 55278) mutant, obtained, in a way known by itself, from the above mentioned one through a series of genetic mutation operations brought about by chemical and physical agents. In a further embodiment, the present invention is realized with a composition having antiparasitic activity, useful in zootecnic-veterinary field and, in particular, in the treatment of ovines, suitable for oral administration, the composition being characterized in that it contains, as active principle, dried mycelium obtained as such by the fermentation of the aforesaid strain, and that the quantity of dried mycelium is such to provide a dose of at least 0.00001%, preferably between 0.00001% and 0.00008%, of total avermectins per kg of animal's live weight. The following example illustrates the preparation of avermectins with the strain in accordance with the present invention and the obtaining of the respective mycelium. In this example, reference is made to the strain duly registered at the American Type culture Collection No. 55278 and therein stored in accordance with the Budapest Treaty provisions. EXAMPLE ATCC 55278 strain is grown in 5 Erlenmeyer 250 ml flasks, containing 50 ml of culture medium A, incubated at 28°C for two days on a table rotating at 200 rpm (2,5 cm eccentric). At the end of this period, a 6 l fermenter containing 4 l of culture medium B is inoculated with the 4 flasks contents under the following operation conditions: temperature: 22-30°C stirring: 200-1200 RPM air: 0.1-1.3 l/l/minute time: 80-220 hrs At the end of this period, the culture is cooled and centrifuged to collect the mycelium, which is again suspended in 3 l cool water and again centrifuged. The moist mycelium cake is then vacuum dried at 30-75°C for 16-48 hrs. At the end of the drying period, the mycelium is ground in a mill to 100-500 mesh granulometry, giving thereafter the following analytical composition data (as micrograms/gram of dry mycelium): avermectin A1: 1.000-2.000 avermectin A2: 8.000-4.550 avermectin B1: 8.500-5.000 avermectin B2: 3.000-6.000 The total avermectins average contents 1 gram of dried mycelium is therefore 19.000 micrograms. The compositions of the above two culture medium A and B are herebelow indicated: Culture medium (g/l) A B saccharose 20 90 soluble distillates 15 5 peanut flour 8 17 yeast extract 2 15 calcium carbonate 15 3 lard oil 1 22 Burkholder solution 2 4 spring water up to 1 litre With the dried mycelium of the present invention, experimental tests in vivo for verifying the antiparasitic activity have been carried out: TEST No. 1 12 sheeps weighing about 50 kg and of 7-12 months age were administered with 1 gram of dry mycelium (equal to 19 mg total avermectins) obtained as per the previous example. After 9 and 16 days from the administration, the faeces of the treated sheeps and of 10 untreated sheeps were analyzed, so as to assess the presence of gastrointestinal strongyles (SGi), taenias and trichnuri. The presence of the SGI is measured in terms of UPG (eggs per grams of faeces). Tables 1, 2 and 3 which follow report the tests results, wherein (-) stands for the absence and (+) for the presence of the parasite. PRESENCE OF PARASITES BEFORE THE ADMINISTRATION TREATED ANIMALS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) TRICH. TAENIAS 1 7 300 + - 2 8 420 + - 3 8 380 - + 4 9 530 - + 5 7 550 - + 6 11 450 - + 7 9 280 - - 8 12 310 - + 9 12 750 - + 10 10 430 - + 11 8 370 - + 12 7 290 + + UNTREATED CONTROLS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) TRICH. TAENIAS 13 9 180 - + 14 11 230 - + 15 11 450 - + 16 11 250 + + 17 9 310 - + 18 9 420 - - 19 9 340 - + 20 8 510 - + 21 10 430 - + 22 10 230 - + PRESENCE OF PARASITES AFTER 9 DAYS FROM THE ADMINISTRATION TREATED ANIMALS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) TRICH. TAENIAS 1 7 150 + - 2 8 380 + - 3 8 180 - + 4 9 210 - + 5 7 350 - + 6 11 400 - + 7 9 180 - - 8 12 350 - + 9 12 450 - + 10 10 230 - + 11 8 120 - + 12 7 290 + + UNTREATED CONTROLS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) TRICH. TAENIAS 13 9 380 - + 14 11 430 - + 15 11 320 - + 16 11 510 + + 17 9 470 - + 18 9 520 - - 19 9 240 - + 20 8 440 - + 21 10 510 - + 22 10 290 - + PRESENCE OF PARASITES AFTER 16 DAYS FROM THE ADMINISTRATION TREATED ANIMALS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) TRICH. TAENIAS 1 7 0 - - 2 8 0 - - 3 8 0 - + 4 9 0 - - 5 7 0 - + 6 11 0 - - 7 9 0 - - 8 12 0 - + 9 12 0 - + 10 10 0 - - 11 8 0 - + 12 7 0 + + UNTREATED CONTROLS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) TRICH. TAENIAS 13 9 350 - + 14 11 230 - + 15 11 520 - + 16 11 470 + + 17 9 550 - + 18 9 320 - - 19 9 210 - + 20 8 360 - + 21 10 460 - + 22 10 190 - + TEST No. 2 In this test the active principle dose has been halved, by orally administering 0.5 grams of dry mycelium, obtained by the procedure of the above Example, to 12 sheeps weighing about 60 kg and of 12 to 24 months age. In this case, the faeces were analyzed after 15 and 30 days from the administration, both for the treated sheeps and for 10 control sheeps, in order to assess the adult-killing action, that is, if the antiparasitic action is extended also to the adult forms of the parasite and not only to the eggs. The analytical results are reported in the following tables 4, 5 and 6. PRESENCE OF PARASITES BEFORE THE ADMINISTRATION TREATED ANIMALS UNTREATED CONTROLS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) 1 13 250 13 12 200 2 12 600 14 12 350 3 15 1000 15 12 300 4 14 800 16 13 50 5 14 250 17 12 550 6 12 450 18 12 200 7 15 250 19 23 40 8 22 800 20 24 200 9 23 150 21 23 10 10 23 100 22 24 130 11 12 800 12 13 200 13 22 400 PRESENCE OF PARASITES AFTER 15 DAYS FROM THE ADMINISTRATION ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) UNTREATED CONTROLS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) 1 13 0 13 12 500 2 12 0 14 12 250 3 15 0 15 12 100 4 14 0 16 13 50 5 14 0 17 12 350 6 12 0 18 12 400 7 15 0 19 23 150 8 22 0 20 24 100 9 23 0 21 23 110 10 23 0 22 24 150 11 12 0 12 13 0 13 22 0 PRESENCE OF PARASITES AFTER 30 DAYS FROM THE ADMINISTRATION ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) UNTREATED CONTROLS ANIMAL NR. AGE (months) SGI (EGGS PER GRAMS OF FAECES) 1 13 0 13 12 200 2 12 0 14 12 150 3 15 0 15 12 300 4 14 0 16 13 250 5 14 0 17 12 400 6 12 0 18 12 450 7 15 0 19 23 50 8 22 0 20 24 50 9 23 0 21 23 310 10 23 0 22 24 400 11 12 0 12 13 0 13 22 0 The test on coltivated larvae has assessed the following percentage of nematodes: HAEMONCHUS 39% TRICHOSTRONGYLUS 36% STRONGYLOIDES 9% OSTERTAGIA 10% OESOPHAGOSTOMUM 3% UNIDENTIFIED 3% From the experimental data previously reported it clearly results the effectiveness of the dry mycelium as parasiticide, whereby the advantages of the present invention can be, in order of importance, summarized as follows: a) the avermectins which, as already noted, are unstable in acid environment and cannot therefore withstand the action of the gastric acid environment, in the form of the present invention, i.e. bound to the mycelium as directly obtained by fermentation, reach unchanged the animal's intestine, where they are liberated, thus performing an outstanding antiparasitic activity; b) long and expensive isolating procedures of only one avermectin from the mycelium and its subsequent catalytic conversion into ivermectin are avoided; c) thanks to the new strain Streptomyces avermitilis ATCC 55278, the total yield of avermectins in the mycelium resulting from the fermentation is higher; d) an active principle orally administerable to animals is provided for. It is to be understood that the present invention should not be considered as limited to the ovines, but it finds similar use for other animals, in particular to breeding animals subject to the same kinds of parasites sensitive to the action of avermectins. As regards the preparation of the composition for zootecnic-veterinary use, the usual veterinary technics are employed utilizing the customary administration vehicles.

Claims: Oral active principle characterized in that it comprises as such the mycelium obtainable from the preparation of avermectins by fermentation of Streptomyces avermitilis ATCC 55278. Active principle according to claim 1 characterized by the fact that said mycelium is in dried form. Streptomyces avermitilis ATCC 55278. Process for the preparation of the active principle according to claims 1 and/or 2 characterized in that the fermentation of Streptomyces avermitilis ATCC 55278 is performed and the resulting mycelium is collected and dried. Process according to claim 4 characterized in that the fermentation of said strain is carried out in a culture medium having the following composition: saccharose 90 g/l soluble distillates 5 g/l peanut flour 17 g/l yeast extract 15 g/l calcium carbonate 3 g/l lard oil 22 g/l Burkholder solution 4 g/l spring water up to 1 litre and the fermentation takes place under the following conditions: temperature 22-30° C stirring 200-1200 RPM air 0.1-1.3 l/l/minute time 80-220 hrs Process according to claims 4 and/or 5 characterized in that the mycelium resulting from fermentation is vacuum dried at a temperature between 30 and 75° C for 16-48 hrs. Composition for oral administration and for zootechnic-veterinary use containing an active principle according to claims 1 and/or 2. Composition according to claim 7 for use as an antiparasitic agent.

Inventors: CAPPELLETTI M LEONARDO, LUCARELLI GIACOMO, MEARELLI GIUSEPPE, CAPPELLETTI, M. LEONARDO, LUCARELLI, GIACOMO, MEARELLI, GIUSEPPE
Applicants: EURORESEARCH SRL, GNOSIS SRL, EURORESEARCH S.R.L.
Application year: 1993
Main classification: A61K35
IPC codes: C12P19, C12P17, C12N1, A61K31, A61K35