AU701413B2 - Anthelmintic compositions - Google Patents

Description

9068 YOSP.996 -la- Anthelmintic Compositions TECHNICAL FIELD OF THE INVENTION This invention relates to injectable compositions containing the anthelmintic abamectin.

BACKGROUND

Abamectin is difficult to formulate, particularly for injectable compositions as it is insoluble in water. If a water miscible co-solvent is used it may release the abamectin too quickly leading to an ineffective application of the active ingredient and possible damage to the animal.

Indeed, calves under 10 weeks old cannot be treated with an aqueous injectable composition containing abamectin and a co-solvent as this may be toxic.

10t OBJECT It is an object of this invention to provide an improved injectable composition containing an anthelmintic, or one which will at least provide the public with a useful choice.

STATEMENT OF INVENTION In one aspect the invention provides an injectable solution containing abamectin, together with a vegetable oil and a co-solvent chosen from the group comprising alcohols having 4 or more Scarbon atoms.

Preferred alcohols are benzyl alcohol, ethyl benzyl alcohol, phenethyl alcohol, and other aromatic monohydric alcohols.

The most preferred co-solvent is benzyl alcohol. More preferably the benzyl alcohol is present in the range of 1-30% by weight.

Preferred vegetable oils are sesame oil, soya bean oil, corn oil, and rape oil.

The most preferred vegetable oil is sesame oil.

Preferably the abamectin is present in the range of from 0.5-5% by weight of the formulation.

9O68YOSP.996 -2- Optionally, a wetting agent such as ethyl oleate may be used to assist in dissolving the anthelmintic in the solution.

In another aspect the invention provides a method of treating animals for helminthiasis by injecting a composition as described above at a rate of 100-300 gg/kg of the animal's live weight.

DRAWINGS

Figure 1: is a chart showing Faecal Egg Counts prior to treatment and for two weeks after treatment (Trial 1).

Figure 2: is a chart showing Faecal Egg Counts prior to treatment and for three weeks after treatment (Trial 2).

DEFINITIONS

FEC stands for Faecal Egg Count FECR stands for Faecal Egg Count Reduction PREFERRED EMBODIMENTS S 15 These and other aspects of this invention, which should be considered and always novel aspects, will be apparent from the following examples.

We have found that the particular formulations containing abamectin, as set out in the following examples, can increase the length of activity of the anthelmintic on both external parasites and internal parasites.

20 Previous formulations of abamectin have been based on glycol solvents, glycerol formal, surfactants and/or water. These have had the disadvantage that the abamectin was rapidly released in the animal, giving a shorter activity and which was also sometimes toxic. This resulted from the rapid absorption into the blood stream of the abamectin and excretion of the drug. It is particularly noted in the case of young animals, who have a low tolerance to these high levels of abamectin in the blood stream. Indeed, calves under 10 weeks old cannot be treated with an injectable composition containing abamectin and a water miscible co-solvent as this may be toxic.

Abamectin is sparingly soluble in vegetable oils such as sesame oil, soya bean oil, corn oil or rape oil. Pure sesame oil has a relatively high viscosity, which makes it unsuitable for use as a 9(If0YOSP.996 -3solvent for injection using typical veterinary syringes. We have found that by using sesame oil together with a co-solvent chosen from the class of alcohols having 4 or more carbon atoms, and more preferably benzyl alcohol it is possible to make a stable injectable solution containing abamectin which allows the abamectin to remain in solution even when stored in cold conditions, whilst at the same time allowing the controlled release of the drug into the animal's body, for use against internal and external parasites.

Example 1: Trial Solution 1 abamectin 1% Benzyl alcohol Sesame oil to 100% In this example the abamectin was dissolved in a mixture of benzyl alcohol and sesame oil.

Example 2: abamectin 2% Benzyl alcohol 15 Ethyl oleate Sesame oil to 100% We have found that we can produce long acting injectable solutions containing between 0.5-5% of abamectin, by using from 1-30% benzyl alcohol and a vegetable oil such as sesame oil, soya bean oil, corn oil or rape oil, and by optionally using from 5-30% of a wetting agent such as 20 ethyl oleate.

a a

TRIALS

Trial 1 9068YOSP 996 -4- Evaluation of the efficacy of Trial Solution 1 Injection in the control and treatment of naturally occurring infection with common pathogenic nematodes in cattle.

TRIAL ACTIVITY AND DESIGN Day 1 Animals selected and allocated randomly: cattle untreated control 100 cattle treated with Dectomax* 100 cattle treated with Trial Solution 1 Animals weighed Samples taken for the FEC from 15 control animals and 15 animals in each treated group. Animals giving samples identified so that they can be followed throughout the trial.

Larval development test on group samples conducted.

Animals treated as per treatment protocol.

Day 7 S. S.

S

Samples for the FEC from each of 45 identified animals taken If eggs are present in the faeces, conduct larval development test on group samples for confirmation of the species involved.

Day 14 Samples for the FEC from each of 45 identified animals taken.

If eggs are present in the faeces, conduct larval development test on group samples for confirmation of the species involved.

Some of the animals in the control group treated due to high egg counts.

is a registered trade mark of Pfizer Pty Limited) (*Dectoinax PARASITOLOGY ANALYSIS Faecal samples will be analysed for gastrointestinal nematodes and lungworms. Larval differentiation test will also be performed whenever the eggs are present. Only initial larval differentiation results are available at the moment.

RESULTS

Mean FEC and of reduction per group prior to treatment and for 2 weeks after the treatment: Day 1 Day 7 Day 14 9068 YOSP.99( abamectin 490* 0(100%) 3.3 (99.8%) doramectin 610* 10.7(98.9%) 350(86.8%) control 586* 1056.6 1896.6 *:no statistical difference between the treatment Figure I shows the graphical representation of these results.

Trial 2 Evaluation of the efficacy of Trial Solution 1 Injection in the control and treatment of naturally occurring infection with common pathogenic nematodes in cattle.

TRIAL ACTIVITY AND DESIGN Day 1 Animals selected and allocated randomly: 10 4 4 15 44* 4 4 .4 100 cattle to each group: C controls A abamectin copper injection B doramectin copper injection G abamectin (Trial Solution 1) I ivrmnectin copper injection Animals weighed.

Samples taken for the FEC.

Larval development test on group samples conducted.

Animals treated as per treatment protocol.

Day 7 Samples for the FEC taken.

Larval development test on group samples conducted.

Day 14 Samples for the FEC taken.

Larval development test on group samples conducted.

Day 21 Samples for the FEC taken.

Larval development tests on group samples conducted.

9068YOSP.99 -6- PARASITOLOGY ANALYSIS Faecal samples were analysed for gastrointestinal nematodes and lungworms. Larval differentiation test were performed whenever the eggs are present.

RESULTS

Mean FEC and of reduction per group prior to treatment and for 3 weeks after the treatment: Day I Day 7 Day 14 Day 21 abamectin+ 340 0(100%) 0(100%) 0(100%) Cu abamectin 160 0(100%) 0(100%) 0(100%) doramcctin 345 15(88.88%) 5(97.91%) 25(92.53%) ivermectin 170 5(96.29%) 25(89.58%) 40(88.05%) control 290 135 240 335 Figure 2 shows the graphical representation of these results.

Trial 3 4 a A trial was undertaken to assess the efficacy of Trial Solution 1 injection and Stand-by pour-on formulations against natural infections of gastro-intestinal nematode parasites of cattle.

Twenty-four clinically healthy Friesian and Friesian cross male calves, 6-7 months of age, and of similar body condition, harbouring natural infections were restrictively randomised into 4 groups. Group 1 remain as the untreated control, Group 2 was injected subcutaneously in the left side of the neck with Abamectin L A (200gg/kg), Group 3 was treated with the Stand-by pour-on formulation along the mid line of the back and Group 4 was treated with Ivomec* Pour-on formulation (500ptg/kg) in accordance with the manufacturers recommendation (MSD AgVet). On days 7 and 8 post treatment the calves were necropsied for worm counts. At necropsy all the treated groups harboured significantly fewer adult, late 4 t h and early 4 t h stage worms of the main cattle parasites viz. Ostertagia spp. and Cooperia spp. as well as Haemonchus contortus Only in the case of Cooperia spp was there a significant difference between treated groups there being fewer adult worms present in the group treated with injectable abamectin There were also significantly fewer adult and late 4 th stage 9068YOSP.996 -7worms of Trichostrongylus axei in the treated groups but not early 4 th stage worms. All three treated groups also showed significant efficacy against adult Dictyocaulus viviparus, Chabertia ovina (both p<0.01), Capillaria spp. and Trichuris spp. (both p<0.05).

No adverse reactions to the treatments were observed at the time of their administration. There were no visible lesions at the injection site of Group 2 calves at slaughter 7 days after treatment.

(*Ivomec is a registered trade'mark of Merck Co., Inc. and is used on compositions containing ivermectin) Experimental Procedures Animals: Twenty four clinically healthy Friesian and Friesian cross male calves, 6-7 months of age, and of similar body condition, harbouring natural infections of gastro-intestinal nematodes were obtained from commercial properties and held on pasture for 7 days prior to the commencement of the trial. The calves were individually identified by ear tags on arrival at Wallaceville Animal Research Centre, weighed and sampled for faecal egg counts to ensure they were carrying adequate worm burdens.

Animal Welfare: Animals handling procedures were in compliance with local regulations and conducted with the approval of the Wallaceville Animal Ethics Committee AEC No. 440.

Parasite burdens: On arrival on day -7 the animals were weighed and faecal sampled and a bulk faecal culture was undertaken to identify the composition of the calves worm burdens.

20 Treatments: On Day 0 the calves were re-weighed and randomly allocated to four treatment groups on the basis of faecal egg count, live weight and overall body condition (see Table 1).

Treatments were administered according to individual live weights using disposable 5.0, 10.0 or 20.0 ml graduated plastic syringes (see Appendix 2 Table Ib). Group 2 was injected in the left side of the neck near the base of the ear with Abamectin L.A. (200 gg/kg); Group 3 was 25 treated with the Stand-by Pour-on formulation (500 gg/kg MSD Ag Vet) in accordance with the manufacturer's recommendation and Group 1 remained as the untreated control.

Table 1: Treatment Allocation 906XYOSP.996 -8- 2 n=6 Abamectin injectable Day 0 200Lg/kg 3 n=6 Stand-by pour-on Day 0 4 n=6 Ivomec Pour-on Day 0 500Lg/kg Husbandry Feed and water: The animals were maintained on pasture for the duration of the trial with ready access to drinking water. The groups treated with pour-on anthelmintics were kept on separate pastures for approximately 12 hours after treatment to avoid the transfer of the pour-on formulations to those in other groups. At all other times the calves were maintained as one herd.

Reactions to treatments: All animals were observed for adverse reactions to the anthelmintics immediately after treatment and when they were returned to pasture. At time of slaughter Group 2 calves (Abamectin injectable) had the left side of the neck skinned in order to assess any reaction to the treatment.

Faecal Sampling: All animals were faecal sampled on Day Day 0 and Day 5. Faecal egg counts were carried out using a modified McMaster technique in which each egg counted represents 50 eggs per gram faeces.

Necropsies: The calves were necropsied on Days 7 and 8 following anthelmintic treatment.

15 The groups were arbitrarily divided into two with half of each group slaughtered on each day.

The animals were euthanased by stunning with a captive bolt pistol followed by exal sanguination and severing of the nerve chord. Worm counts of the gastro intestinal tracts and lungs were carried out using the method described by Brunsdon, (1972) and in accordance with the World Association for the Advancement of Veterinary Parasitology guidelines (Wood et al.

20 (1995)). Lungs and the abomasal mucosa were processed using the techniques described by Downey (1981) and Oakley (1989).

Briefly the lungs together with the heart and the gastrointestinal tract were removed and the abomasum, small intestine and large intestine ligatured and separated. The lungs were infused after the method of Oakley (1989) using mains pressure water directed through the pulmonary artery. Approximately 8 litres were passed through each lung. The bronchial tree was then opened with scissors and examined to ensure no worms remained. The total contents and washings of the abomasum and small intestine were also collected separately, made up to 8 litres and a 1/10 aliquot passed through a 451 sieve and preserved with formalin for counting.

906XYOSP.996 -9- The contents and washings of the abomasum and small intestine were also collected separately, made up to 8 litres and a 1/10 aliquot passed through a 45p sieve and preserved with formalin for counting. The contents of the large intestine were made up to 8 litres and half passed through 635 1 sieve and preserved with formalin for counting. The abomasal mucosa of each animal was incubated in 1.5 litres of physiological saline at 37 0 C over night (Downey, 1981) and then treated as with the contents.

Nematode Counts: Nematodes present in the total contents of the washings from the lungs, the 1/2 aliquot sample of the large intestine, a 1/5 subsample of the abomasal contents, abomasal mucosa and a 1/10 subsample of the small intestine were identified to developmental stage and genus and counted. Counts were multiplied by the appropriate aliquot factor to give total numbers present in each organ. The first 20 adult male worms of the genera Ostertagia and Cooperia in each sample (depending on availability) were identified to species using the spicule morphology to determine the species composition of the respective worm population on a proportional basis.

Statistical Methods: Data for both faecal egg counts and worm counts were transformed to Loge(X+100) to normalise their distributions before analyses were carried out. FEC's and **worm counts were analysed by 1-way ANOVA's using the Minitab 10.5 statistical package.

FEC's and worm counts given in tables are the back transformed values. The percentage efficacy were calculated as: (Mean of Control Mean of Treatment/Mean of Control) x 100 Results: 0* Faecal culture: Results of the pre-treatment faecal culture indicated that the following genera of gastrointestinal nematodes were present in the experimental herd Haemonchus contortus, Ostertagia spp., Trichostrongylus spp., Cooperia spp., and Oesophagostomum spp. (Table 2).

Table 2: Generic composition of faecal egg output of trial animals pre-treatment.

96XY()'P 996 Osiertagia spp. 23 12 Juleinonchus cofltittus 6 3 Cuoperia spp 162 81 Ocsophagostoimi spp 4 2 Total 200 100 Faecal egg counts (FEC's) Pre- and post- treatment FEC's and percentage faecal egg count reductions (FECR) are presenrted in Table 3. FEC's of all three treated groups of day 5 were significantly lower than the controls (p<0.01) but not significantly different from one another. The FECR for the RInectable formulationi of abamectin was the higher than both of the pour-on treatments.

Table 3: Faecal Egg Coun[ts (Geometric means).

Pre-treatment Post-treatment 00*S 0 *0 40 0 0

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*000 00 S 0S 0S GrouIp I Control 2 Abami-ec LA 3 Suirid-by Pon 4 Ivomrec P-on FEC 1- 8/02 546.0 537.9 513.7 FEC 2 15/02 FEC 3 20/02 FECR 4. 733.3 673.7 a -4 t 393.9 6 9 b 98.97 4 4 528.9 93.05 1 4 4 568.1 641.6 91.94 FECR =Faecal egg count reduction for FEC3 on samples taken 5 days post treatment. Values for FEC 3 for 20/02 with different super scripts are significantly different (p<0.0l).

Worm counts The geometric mean worm burdens recovered from the abomasum, small intestine, large intestine and the lunigs and the percentage reduction are presented in Tables 4-7. A small 9068Y()OP,996 11 number of parasites of sheep origin were present in the calves which had been in a mixed grazing management system. Treated animals had significantly fewer adult, late 4th and early 4th stage worms of Ostertagia spp, Haemonchus contortus, and Cooperia spp. Only in the case of Cooperia spp was there a difference between treatment groups there being significantly fewer adult and late 4th stage worms of Trichostrongylus axei in all treated groups but not early 4th stage larvae. Due to the trial design it cannot be verified that this is due to the lower efficacy of the treatments against this developmental stage at the calves may have acquired some larvae while grazing on pasture between the treatment date and the time of their slaughter. All three treatments also showed significant efficacy against adult Dictyocaulus viviip)rus, Chabertia ovina (both p<0.01) as well as Capillaria spp. and Trichuris spp. (both p<0.05). The efficacy of the treatments against a Nematodirus spp., and Oesophagostomum spp. could not be determined with any degree of accuracy as there were only present in small numbers.

Table 4: Mean abomasal worm burdens (Geometric data).

Mean worm count and efficacy for treatment groups 12- Genus stage Group 1 Group 2 Group 3 Group 4 of development Ostertagia spp Adult (5th) 6921.2 a 33.4 b (99.5) 6.9 b (99.9) 6.9 b (99.9) Late 4th 466.9 a 6.9 b (98.5) 24.6 b (94.7) 6.9 b (98.5) Early 4th 1837.6 a 34.8 b (98.1) 44.2 b (97.6) 35.7 b (98.1) Trichostrongy lus caxei Adult 3259.7 a 20.1 b (99.4) 23.2 b (99.3) 81.7 b (97.5) Late 4th 1087.7 a 20.1 b (98.2) 28.5 b (97.4) 61.9 b (94.3) Early 4th 3252.6 822.5(74.7) 1023.2 (68.5) 1614.2 (50.4) Haemonchus conlortus Adult (5th) 2 7 7 .1a 0 .0b (100) 0.0b(100) 0.0b (100) Late 4th 312.3 a 0.0 b (100) 28.5 b (90.9) 0.0 b (100) Early 4th 1360.3 49.7 b (96.3) 54.3 b (96.0) 39.9 b (97.1 For rows values with different super scripts and significantly different (p<0.01).

r Table 5: Mean small intestinal worm burdens (Geometric data).

Mean worm count and efficacy for treatment groups 906XYO P 996 13 Genus stage Group 1 Group 2 Group 3 Group 4 of developm-ent Cooperia sp Adult (5th) 5442a 286C(95 37.6b(93.2) 3055.8 b(94.1) Lae th738. a0.0 b(100) 84.9 b(98.8) 49.1 b(99.3) Eary th23164 23. b(98.9) 45lb(98.0) 462.3 b(98.0) Tri hos!rangy lus spp 15.1 0.0 0.0 0.0 Adult Late 4th 12.2 0.0 0.0 0.0 Early 4th 30.8 0.0 0.0 0.0 NeInatoirits spp 453.4 0.0 195.9 159.6 Adultt Capiliaria spp ActLIt (5 th) .70.lIx O.OY' O.OY 1.2 For rows values with dlifferent super scripts are significantly different Rows with non-annotated values are not significantly different.

a, b (p<0.0O1); x, y Table 6: Mean large intestinal worm burdens (Geometric data).

14- Mean worm count and efficacy for treatment groups Genus stage Group 1 Group 2 Group 3 Group 4 of dev'elopmcnt Qesophagosto milti/ 1.3 0.0 0.0 0.0 Aduilt Tric~iuris spp Adult (5th) 24.8 X 23 1.y Chohertia OVI'f1(1 1.0 a0.0 b00b 0.0 b Adult For rows values with different super (p<0.05).

scripts are significantly different a, b (p<0.0 x, y Table 7: Mean lungworin burdens (Geometric data).

Mean worm count and efficacy for treatment groups Genus stage Group 1 Group 2 Group 3 Group 4 of dev'elopment Dictyvocaulus vvprs10.9 a0.0 b(100) 0.0 b(100) 0.0 b(100) Early adl Its ValueS with different super scripts are significantly different (p<0.0l).

Species composition (Ostertagia and Cooperia).

9(06Y( p.99Y6 Four species of Ostertagia and three species of Cooperia were identified from the worm burdens of the calves in the trial i.e. 0. ostertagi, 0. lyrata, 0. leptospicularis, 0. kolchida, C.

.oncophora, C. mcmasleri, and C. punctata. 0. ostertagi and C. oncophora were the predominant species present representing approximately 60% and 75% of the respective genera (Table All three anthelmintic treatments appeared equally effective against all species of Ostertagia and similar proportions of all Cooperia species remained after treatment.

Table 8: Species composition of the Ostertagia and Cooperia worm populations (based on the identification of adult nmales).

Mean species composition and range Species Group 1 Group 2 Group 3 Group 4 0. oslertagi 60.2 (45-80) 0. lyrata 27.6 (10-45) O. leptospicularis 6.5 (0.10) 0. kolchida 5.7 (0-10) 0. oncophora 74.4 77.0 75.0 78.3 (61-80) (75-78) (67-80) (70-88) O. mcmasteri 10.1 15.4 13.8 13.3 (4-28) (0-22) (0-29) (9-18) O. punctata 15.5 7.6 11.2 8.4 (5-30) (0-25) (0-33) (0-15) No adult males found Only adult male found r r r Injection sites 16- No adverse reaction to either the injectable or pour-on formulations was observed at the time of treatment. At the time of slaughter the injection sites of group 2 calves were examined by removing the skin from the neck of each animal. No reactions to treatment were found.

ADVANTAGES

The relatively high viscosity of vegetable oils such as sesame oil makes them unsuitable for use as a solvent for injection using typical veterinary syringes. However, a co-solvent chosen from the class of alcohols having 4 or more carbon atoms, and preferably benzyl alcohol, decreases the viscosity of the vegetable oil such that the vegetable oil becomes a suitable solvent for injection.

The active ingredient (abamectin) remains in solution in formulations of this invention even when stored for long periods or in cold conditions.

The formulations of this invention also allow the controlled release of the abamectin in the blood stream of the animal.

INIUSTRIAL APPLICATION The formulations shown above are effective in the treatment of helminthiasis in cattle and other domestically important animals, including, but not limited to, pigs and goats.

VARIATIONS

Although we prefer to use sesame oil in these formulations other vegetable oils such as soya bean oil, corn oil and rape oil are possible as are other viscous vegetable oils.

20 Finally, it will be appreciated that various other alterations and modifications may be made to the foregoing without departing from the scope of this invention.

REFERENCES

oo* 17- Brunsdon, 1972: Inhibited development of Ostertagia spp. and Cooperia spp. in naturally acquired infection in calves. N.Z. Veterinary Journal 20: 183-189) Dow\ne. 1981: Recovery of Ostertagia from bovine abomasal mucosa by immersion in warm normal saline. In P. Nansen, R.J. Jorgenson, E.J.L. Soulsby, Eds Epidemiology and Control of Nematodiasis in Cattle. ECSC, EAEC, Brussels Luxembourg.

Oakley. 1989: The recovery of Dictycaulus viviparous from bovine lungs by lung perlusion: a modification of Inderbitzern's method. Research in Veterinary Science. 29, 395-6.

Wood Amaral Bairden K. et al, 1995: World Association for the Advancement of Veterinary Parasitology Second Edition of Guidelines for Evaluating the Efficacy of Anthelmintics in Ruminants (bovine, ovine, caprine). Veterinary Parasitology 58: 181-213 SY)'SP 996 18- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An injectable composition containing abamectin, together with a vegetable oil and a cosolvent chosen from the group comprising alcohols having 4 or more carbon atoms.

2. A composition as claimed in claim 1, wherein the co-solvent is chosen from the group comprising benzyl alcohol, ethyl benzyl alcohol, phenethyl alcohol, and other aromatic monohydric alcohols.

3. A composition as claimed in claim 2, wherein the co-solvent is benzyl alcohol.

4. A composition as claimed in claim 3, wherein the benzyl alcohol is present in the range of 1-30% by weight.

5. A composition as claimed in any one of claims 1-4, wherein the vegetable oil is sesame oil.

6. A composition as claimed in any one of claims 1-5, wherein the abamectin is present in the range of from 0.5-5% by weight of the formulation.

7. A composition as claimed in any one of claims 1-6, further including a wetting agent.

8. A composition as claimed in claim 7 wherein the wetting agent is ethyl oleate.

9. An injectable formulation comprising a composition as claimed in any one of claims 1-8.

10. A method of treating animals for helminthiasis by injecting a composition as claimed in claim 9 at the rate of 100-300 gg/kg of the animal's live weight.

11. A process for the preparation of a composition as claimed in claim 1 substantially as herein described with reference to the examples.

DATED this 20th day of September 1996 ASHMONT HOLDINGS LIMITED By their Patent Attorneys CULLEN CO.

Claims (1)

19- ABSTRACT An injectable composition containing abamectin, together with a vegetable oil and a co-solvent chosen from the group comprising alcohols having four or more carbon atoms. A composition of the invention is suitable for treating helminthiasis in animals. a* So