INTRODUCTION The nature of therapeutic agent and model of safety and efficiency evaluation, plays a vital role in preclinical animal toxicological data which are taking into account on a case by case basis in order to improve and help in phases of new drug development. Assessment of toxicity of a new drug can also be done early during evaluation with pharmacological assessment parallely and this information reduces financial expenditure through attrition reduction. Although there are limitations to preclinical studies at the beginning of clinical development . Our main focus is an attempt to organize preclinical trials to know the effectiveness of different tests employed to test for carcinogenicity, organ toxicity, biochemical dysregulation and which species of animals, animals group and size, duration of each test and at the same obtaining information for regulatory purposes.
TESTS FOR CARCINOGENICITY Carcinogenicity tests are performed in animals to identify tumorigenic potential of the drug and to assess the risks it might pose to humans despite their limitations. The evidence to show that a chemical molecule has a carcinogenic reaction in animals administered to or not can only be confirmed after a long-period study on the animals if the animals survive the whole study period. The complexity of carcinogenesis process makes it compulsory to have one long-term in vivo (lifespan) and another long or short-term in vivo study for better carcinogenic knowledge of the chemical molecule. The long-term in vivo assay lasts for 2 years while the short-term assay lasts from 6 months to 9 months depending on the assay used. Animal specie used is rodent which can be rats or mice. The number of animals used is from 50 to 100, for each sex of animal ( male and female) and there 4 group size for each sex with one control group and three dose group. After wards the animals are killed and necropsy carried out to observe the tumourigenic ability of the chemical molecule and the different types of tumour seen are recorded for each groups of animals in both sexes but if animals shows any sign of chronic illness that can cause death during the test, then a temporary examination may be carried out. Activated oncogenes (transgenics), inactivated tumour suppressor gene (p53+/-), inactivated DNA repair gene (XPA-/-) are introduced into the mice or rats in order to reduce the period of time for cancer cell growth and quicker carcinogen detection by standard bioassays. This may show results that are not desirable and would cause limitations of histopathological analysis (EPA guidelines) which is one of the drawbacks of these bioassays using Knockouts and transgenic mice model as described by Tennant RW, French JE and spaldling JW in 1995. The transgenic mice models are thorough for detecting carcinogens that are mutagenic and non-mutagenic plus tumour promoters (Tennant RW et al 2001), the knockout p53+/- mice model are sensitive to genotoxic carcinogens that are already known within 24weeks test period and insensitive to non-genotoxic carcinogens (spaldling JW et al 2000, Tennant RW et al 1999 and Gulezian D et al 2000)and the knockout XPA-/- mice model are also sensitive to genotoxic carcinogens that shows reactivity with the nucleotide excision repair pathway (De Vries A et al 1995 and 1998). Hayes AW 2007, also found out that at higher doses, carcinogencity is induced through mechanism linked with toxicity. For genotoxicity tests a minimum of three good laboratory practice is required which include two in-vitro test and one in-vivo test. The in-vitro tests are Salmonella mutagenicity assay (AMES TEST), a mammalian in-vitro cell test which can be the mouse lymphoma assay or chromosomal aberration test and the in- vivo tests can be rodent bone marrow micronucleus test or comet assay in rodent cells (module 3 guide, 2010)
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TESTS FOR ORGAN TOXICITY EVALUATION Organ toxicity testing is a method of evaluating the mechanism of action of chemical molecules and also demonstrate the alteration of functions of the organs and tissues caused by the chemical molecules which could be of important in a new drug development. Ames B N and Gold L S, 1990; Ames B N,1996 from their studies, reported that target ââ‚¬"organ toxicity and mitogenesis following succession are reasons for testing of over 50% of the chemicals in chronic bioassays at the maximum tolerated dose (MTD). They also discovered that at MTD, there is increased level of mitogenesis in both non-genotoxic and genotoxic agents and the rate of transmutation is made better. There is two doses levels introduced into animals used in preclinical studies to test for chemical molecules; the high dose which causes not more than ten percentage reduced in the weight of animal in relation to control groups and does not results in the death of the animal, signs of toxicity and other pathologic lesions is termed maximum tolerated dose (Hayes A W,2007) and the low dose is usually half of the high dose. General toxicology of acute toxicity is performed using a rodent species e.g rat and a non-rodent species e.g dog and the animals are dosed for a period of two to four weeks, twelve rodents and four non-rodents per sex per group with one control and at least three drug- treated group. After which a reproductive toxicology can be carried out using a rodent species e.g rats and non ââ‚¬"rodent species e.g rabbits, four groups of twenty male and twenty females are dosed for a about month prior to pairing for mating depending on the general toxicity data effects on organ weight or histopathology; if effects is seen on organ weight from general toxicity data, ten weeks dosing is recommend for the males because of spermatogenesis duration. For safety pharmacology, cardiovascular toxicity testing is carried out in non-rodent species only e.g dog and they are dosed for at least one month, to assess the safety of the chemical molecule on the heart. Renal toxicity testing is also carried out in rodent species e.g rat and non- rodent species e.g dog and they are dosed for at least one month, to assess the metabolism and rate of excretion of chemical molecule (pharmacokinetic) and safety from toxicity if accumulation occurs in the kidney. Respiratory toxicity testing and neurotoxicity (Functional Observational Battery) testing are also done for rodent species e.g rat and they are dosed for at least a month, to assess the effect of chemicals on organ and tissues of respiratory system e.g lungs, bronchi etc and nervous system by measuring locomotive activity, learning and memory activity, reflexes, hind-limb foot splay and grip strength, physiological parameter like temperature etc (lectures notes module 4 on non-clinical safety studies, reproduction toxicology, 2010). At necropsy, during post mortem examination precautions must be taking in handling unfixed tissue due to excessive manipulation that might complicate microscopic examination of tissues. Organ toxicity can also be study by perfusion and intravascular perfusion using karnovsky fixative to study neurotoxicity. Change in organ weight of kidney,liver can be used to determine organ toxicity, although weight change can occur without pathological change for example increase of about 25% liver weight may be seen without microscopic changes indicating hepatocellular hypertrophy.
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TESTS FOR BIOCHEMICAL DYSFUNCTION RECORD The development of bioinformatics from a genome sequence evaluation and explanation is now being employed as a diagnostic tool in understanding biology of disease condition. It can also be used in identifying the mechanism and testing of newly discovered medicine for treatment of disease. Direct and indirect damage are caused by radiation through ionization of the matter; indirect damage can induce free radical chain reaction that can cause injury and molecular damage with biochemical can then release biological mediators and hormones through rapid stimulation. This action can both enhance and suppress biological injury (Grosch D S and Hopwood L E 1979; Salter C A, 2001). Injury vulnerability to living organisms and biological as a result of irradiation in the presence of oxygen was first reported by Schwartz G N in 1988 as oxygen effect and also noticed that erythema reduces during the application of pressure to the arm during irradiation. The infringement of chemical link between molecules, cross-connection of molecules and disintegration and degradation of structure are radiation-induced changes of biomolecules ( Altman K I, Gerber G B, and Okada S 1970; EffHagen U, 1989). Biochemical behavior can be eradicated or modified through conformational and organizational transformation that opens and make prone interior organs to radical attack (George A M and Cramp W A, 1987)
THE NEW ANTI-ARTHRITIS DRUG TESTING In this study, following the 3Rââ‚¬â„¢s regulation for reduction, refinement and replacement; 40 rats in 4 groups of 10 rats each i.e 5 male and 5 female. The first group will be the control (normal) rats, 2nd group of rats injected with cancerous cells, 3rd group will be rats that are affected by arthritis and the 4th group will be exposed to radiation.
CONCLUSION Preclinical trials of chemical molecules in animals species has helped in reduction of toxicity to humans, provide information to employ different approach to testing of chemical molecules in animals, reduction of financial expenditure through the reduction of number of testing to be done and also time reduction for some assay testing through advancement and new regulation of 3Rââ‚¬â„¢s(replacement, refinement , reduction) although often difficult to interpret in respect to human relevance.