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This article was published in 1978
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Development of an Anthelmintic
Laboratory to Field.
D.R. Johns, B.V.Sc.,M.R.C.V.S., Smith, Kline & French Laboratory, Cobbitty
In the highly competitive marketing of anthelmintics industry is always on the alert for the development of a new compound which may have advantage over existing products. Such a development is an exercise in precision covering many disciplines and the results must suit the short and long term policy of the company as well as meet the requirements of the regulatory authorities in each of the many countries which will eventually market the product.
Despite certain restrictions on its application to the development of a biological product the PERT and critical path method of development has been generally accepted as the most efficient. Millions of dollars may be involved in reaching a point where a reappraisal must be made of safety and efficacy data and a decision to halt must be accepted.
Briefly, development involves the role of the Chemist, Biochemist, Toxicologist, Parasitologist and Veterinarian as well as application to target animals, field application and close liaison with marketing administration.
Initially both chemist and biochemist consider parasite biochemistry in relation to the known and accepted activity of anthelmintics already in use and either reorganise the basic molecule or decide to follow anti-parasite leads shown by unrelated compounds. This lead to the bench synthesis of small gram quantities of material which is passed on to the toxicologist or parasitologist who perform most of the basic work in small laboratory animals. In the case of the benzimidazole compound albendazole the LD50> ran range from 0.9 g/kg in guinea pigs to 10.0 g/kg in hamsters.
Chronic toxicity studies must be carried out in two species generally the rat and the dog when four groups of rats, i.e. 20 males and 20 females or four groups of beagle dogs, i.e. 4 males and 4 females are given in the feed or by capsule a range of daily doses e.g. 2, 10 and 30 mpk for 90 days. At 30 days complete haematology clinical chemistry and urinalysis is carried out and at 90 days every animal is post-mortemed and every organ is submitted to detailed histopathology.
Concurrently the toxicologist will submit the compound to rats and mice teratology studies where a range of doses is given to pregnant rats and mice from day 6 through to day 15 after which they are examined for weight and number of foetuses, external anomalies and resorption sites. Depending on national requirements either 2 year rat feeding or a 3 generation rat study may be carried out.
Parallel toxicology and dose response studies are then carried out in the target species, e.g. sheep and cattle. In the case of albendazole the dose ranged from 26.5, 37.5., 53.0 and 75.0 mpk using 2 rams and 2 ewes per group. This resulted in a no effect level of 37.5 mpk Then followed a chronic toxicity study in which double and treble the intended field use dose was given to groups of 10 sheep every ten days for 90 days whilst observing for anorexia, diarrhoea, ataxia and wool skin junction reaction. At the end of 90 days 50% of each group including controls was slaughtered and examined for macroscopic lesions in all viscera and body cavities. Lambs were also given multiple doses to observe the effects on secondary wool follicle development.
Reproduction studies are then run on the target species. The ram is multiple dosed and semen ejaculates examined at 3, 21, 29 and 56 days post dosing for motility, consistency, volume, proportion of dead to alive sperm after which testicular tissue is submitted for histopathology.
The effect on the ewe is examined by running up to 600 ewes with fertile rams fitted with sire sine harness. Ewes are allotted to replicate groups and multiple dosed on days 17, 21, 24 and 28 of foetal development.
These observations are then carried to the field where larger groups of up to 500 ewes are given multiple doses at +30 days following introduction of the rams. Control ewes are dosed with placebo. At lambing and particularly during the period at which the lambs born would have been from 10 to 24 days of development when the ewe was dosed each lamb is examined and if necessary post-mortemed by a veterinarian.
Anthelmintic efficacy is studied concurrently at several places throughout the world, South Africa being the most stringent in its requirements using the non parametric method. However, some confirmatory work is required in Australia and this entails Research Station involvement in the growth and maintenance of infective larvae, their administration to worm free sheep, then slaughtered, parasite recovery and counting and growth and maintenance of snail colonies.
Safety in use is further studied by dosing target animals in the field. In general dosing is carried out in sheep and cattle of all ages, class and breeds under as many geographical, pasture and management conditions as possible. Between 10,000 and 25,000 sheep are dosed and from 1 to 2 thousand head of cattle. Animals are also concurrently dosed with other compounds which are found in the stock owners armamentarium.
National Regulatory Authorities must be provided with exact and detailed data showing the metabolites of the active ingredient and the nature of the residues remaining in the tissues and for what period these residues exist following administration.
Residue studies are therefore conducted using the hot method in which the parent molecule is labelled with 2-14 or the cold method, i.e. a chemical procedure.
C-14 residues in the general metabolic pool do not allow for complete accuracy in determining the metabolic rate hence the necessity for a cold method. Residue levels are determined by slaughtering dosed animals at 1, 2, 4, 6, 10, 20, 30 and 45 days following dosing and submitting fluids (plasma and urine) and tissue extracts to thin layer chromatography and combustion analysis for radioactivity.
All metabolites are then further characterised so that a reliable method can be devised to identify the marker residue which has reduced to 0.1 p.p.m., or lower at the end of the withdrawal period.
Finally the formulating chemist must present such data as Generic and chemical name of the compound, molecular and structural formulae and chemical and physical properties. He must provide a flow sheet describing the preparation of the compound, an assay method, details of particle size, the formulation and a method of testing the active ingredient in the final formulation. He must test for stability at varying temperatures and humidity to establish shelf life and be must liaise with those responsible for packaging and field testing to know the acceptance by the animal, the ability of the product to flow through dosing equipment and if it does show any settling he must know how much pack movement is required for reconstitution.
The accumulated data which may have taken from 3 to 5 years to collect is finally submitted to the National Health and Medical Research Council for examination by
1. The Pesticides and Agricultural Chemicals Sub Committee - Residues,
2. The Poisons Scheduling Sub Committee,
3. The Non Statutory Technical Committee on Veterinary Drugs which in turn guides the decisions to be made by the States in respect of registration.
When all this has been accomplished it is hoped that a safe and effective product can now be presented to the consumer by the company marketing group. Such development has been known to involve the expenditure of at least 6 million dollars.