Studies of pharmacokinetics, anthelmintic efficacy and acute toxicity of pyrethrins in sheep and dogs and pyrethroids analysis in air

PhD Thesis

"Pyrethrins" is the collective term used for the six insecticidal constituents of pyrethrum plant (Crysanthemum cinerariaefolium). The active principles are esters viz cinerin I, cinerin II, jasmolin I, jasmolin II, pyrethrin I and pyrethrin II. Pyrethroids are synthetic compounds resembling pyrethrins. These insecticides are usually analyzed using chromatographic methods. Natural pyrethrins are also known to possess anthelmintic properties. The current study was undertaken to develop a method for the analysis of pyrethroids in air samples and also generate data on the disposition, toxicity, and anthelmintic efficacy in sheep and dogs. The other objective was to determine by means of an epidemiological survey the role of pyrethrum marc in helminth control programme in Molo, Kenya. A method was developed and validated for the analysis of bioallethrin, permethrin, alpha-cypermethrin and deltamethrin in air. The analytical method consisted of sampling of r:::: 1 m3 of air by adsorption of the analytes in XAD sampling cartridges. Ethyl acetate was used for extraction and the extracts were analyzed by gas chromatography with electron capture detection (GC/ECD). The desorption/extraction efficiency (EE) was determined with fortification of 5 ng and 50 ng and gave recoveries ranging from 81.9% to 97.6% (n=4). The limit of quantification (LOO) was deduced from method validation at a lower fortification level of 4.5 ng/m3 . An upper fortification level validated the analytical method at concentrations of 45 ng/m3 . The analytical method and its validation is easily transferable to other pyrethroids or pesticides active substances amenable to chromatography with selective detection. An epidemiological survey was conducted on the prevalence of gastrointestinal nematodes, general farm managements and worm control programmes in Molo, xv Kenya. Data was gathered from 40 small-holder farms with a total of 391 sheep in Turi, Kamara, Sachangwan and Kerisoi areas of Molo. Nematode infections were found in 55% of the sheep. The nematodes encountered were of the genera Haemonchus (59%) Oesophagostomum (16%), Trichostrongylus (12%), Cooperia (7%), Nematodirus (3.3%), Strongyloides (1.3%), Bunostomum (0.9%) and Ostertagia (0.8%). The number of eggs per gram of faeces (EPG) ranged from 100 to 4,700 with 90% of flocks having an average EPG less than 500. Levamisole, albendazole or febantel had been used in 87.5% of the farms. In most farms, anthelmintics were given only in response to perceived helminthiasis. There were no significant differences in worm burdens between farms using anthelmintics, pyrethrum marc or those using other helminth control measures. Nematode infection rates were influenced by age and the presence of coccidia. Infection rates were also influenced by the location of the farm and the source of water. The survey indicates that worm control programmes in Molo are based on rare and haphazard anthelmintic treatment. The efficacy of pyrethrum marc and albendazole (valbazen, Ciba Geigy) against experimental sheep gastrointestinal nematode infections were compared. Sheep were infected by inoculation of 10,000 larvae per animal given by oral drenching. The nematodes isolated in the infections were Haemonchus spp. (60.1%), Oesophagostomum spp. (13.9%), Trichostrongylus spp. (13.2%), Cooperia spp. (8.3%), Nematodirus spp. (3.5%), StrongylOides spp. (0.8%) and Ostertagia spp. (0.2%). One group was treated with a single oral dose of 0.2 ml kg-1 body weight of 2.5% albendazole while the other was fed pyrethrum marc at the rate of 36 mg pyrethrins/kg body weight at days 0, 2, 4, 6, 8 and 10. By day 4 post-treatment, the egg counts for every animal treated with albendazole had dropped significantly (p<0.05) to below detectable levels. Pyrethrum marc on days 4, 6, 8 and 10 caused 37%, 31%, 39% and 52% faecal egg counts reductions respectively. These reductions were statistically significant on days 8 and 10 post-treatment (p<0.05). xvi Pharmacokinetic parameters which included maximum concentration (Cmax) and the corresponding time (Tmax), elimination rate constant, plasma half-life (t~), area under the curve (AUC), mean residence time (MRT), total clearance and apparent volume of distribution (Vd) for pyrethrins were studied in sheep after administration of ¥ single oral or subcutaneous (s.c) doses. Pyrethrins were absorbed and detected in plasma within half an hour using GC/ECD analysis. Peak plasma concentration of 0.26 ~lg/ml (Cmax) was attained within 8 hours (Tmax) after oral administration. Thereafter, the levels of pyrethrins in plasma declined and at 96 hours post-administration the plasma levels were below detection limits. A similar pattern was observed for the subcutaneous route but the Cmax was 0.30 ~lg/ml at a Tmax of 6 hours. Absorption was higher following absorption from s.c. route than from the gastrointestinal tract (GIT). After absorption, pyrethrins were widely distributed in the body and also rapidly eliminated. The apparent volume distribution (Vd) was 2,447 and 1,720 litres for oral and s.c routes respectively. Mean residence time (MRT) was 9.8 hours (oral route) and 9.5 hours (s.c. route) Total clearance was 4,337 ml/min and 3180, ml/min for oral and s.c routes respectively. Pyretllrins were excreted unchanged Into milk within 48 hours at levels ranging from 0.01-0.003 to 0.02-0.05 flg/ml following oral and s.c. administration respectively. The efficacy of pyrethrins administered orally at 150 mg kg-1 against mixed helminth infestations was studied in 8 to 10 week old male and female puppies. The mean and range of pre-treatment hookworms EPG were 3,760 (2,100 to 6,300) and 4,660 (2,900 to 6,300) for the treatment and control groups respectively. The values for the ascarid eggs were 3,560 (2,900 to 4,600) for the treatment group and 4,320 (2,700 to 6,000) for control group. Treatment with pyrethrins caused a significant decrease in both hookworm and ascarid EPG counts (p<0.05). Faecal egg count depression showed that the efficacy was 76% against hookworms and 57% against ascarids. The XVll adult helminths recovered in the studies were identified as Toxacara canis and Dipylidium caninum species. Data on the acute toxicity of pyrethrins in 8 to 10 weeks old male and female puppies was generated following oral, s.c. and intravenous (i.v.) administration. The acute median lethal doses (LD5o)and 95% confidence intervals in mg kq' body weight II were 1,220 (803 to 1,853) orally, 996 (735 to 1,350) subcutaneously and 24 (14 to 31) intraveneously. Clinical signs of toxicity were observed within 3 to 6 minutes after intravenous route and 20-30 minutes after oral and subcutaneous administration. The toxic signs were in the central nervous system (CNS) and in the GIT and included imbalances and incoordinated movements, rapid respiration, shaking, twitching and jumping, followed tremors of the ears, neck and hind limbs. The tremors progressed to severe seizures, convulsions and some puppies died within 13 to 45 minutes. Other signs included hypersensitivity, profuse salivation and elevated body temperatures but some puppies recovered within 48-72 hours. Postmortem examination revealed haemorrhages and presence of fluids in the lungs and delayed blood coagulation. The present study shows that GC/ECD method of analysis is suitable for determination of pyrethroid in air. This study also indicates that pyrethrins are fairly safe when administered orally in puppies but toxic intravenously. Pyrethrins also have anthelmintic potential and are rapidly absorbed and eliminated when administered orally and subcutaneously.

University of Nairobi