Pre-clinical studies or pre-clinical trials are very important stage of research in drug development process. Pre-clinical trials are also known as non-clinical safety assessment. The main purpose of carrying out these studies is to ensure that the test compound is safe before testing in humans (clinical trials), characterise toxicity and target organs, to fulfil regulatory requirements and to protect employees in manufacturing. In other words, quality, safety and efficacy of a test compound are determined during pre-clinical studies. Animals such as rodents (mice, rat) and non-rodent (monkey, dog) are used in pre-clinical trials before administration to humans (clinical trial phase) so as to separate efficacy from toxicity in human volunteers. The international conferences on harmonisation (ICH) for drug development are regulatory authorities which provide instructions for development and registration of new chemical entities (NCE) likewise instructions for carrying out assessment in animals. The concept of the 3Rs (Reduction, Refinement and Replacement) is widely employed by ICH for pharmaceutical industries to comply. This concept is employed so as to reduce the numbers of animals and also to avoid prolonged testing period. Anti-arthritis drug are used to treat arthritis (inflammation of joints). Important pre-clinical tests required include general toxicology, safety pharmacology, reproductive toxicology, carcinogenicity studies and genetic toxicology .
Test for Carcinogenicity
The carcinogenicity study evaluates the carcinogenic potential of the compound. During pre-clinical studies, animals are used firstly so as to determine the potential risk of the anti-arthritis drug in humans. In order to evaluate carcinogenicity, two types of studies are conducted which are the short-term study and the long-term study. The short-term study involves using the four transgenic models. The four transgenic models used are inactivated tumour suppressor gene (p53+/- model), activated oncogene (Tg.Ac model and rasH2) and inactivated DNA repair gene (XPA-1-model). The genetic alterations of the four transgenic models are made in relation to carcinogenesis processes. The long-term study involves the use of mice or rats of both sexes and is usually a two years study. The correlation between rats to humans is about 70% and is more sensitive making them the major specie of animal used for the long-term carcinogenicity studies. Spragne-daweley has high chances of survival and as a result, these strains of rats are required for the 2years bioassay. Treated animals are divided into three groups each containing about 50-100 animals per sex. In rats, the treated groups are observed for 24months while 18months in mice. Large numbers of animal are used for this study because continuous dosing of the drug could induce tumour and also to achieve a strong statistical result. Furthermore, non-genotoxic carcinogens can cause some rodent strains to be susceptible to tumour induction and in order to distinguish rodent specific processes related to human, understanding carcinogenesis mechanisms based on the specificity of tissues is very vital.
Genotoxicity test is carried out so as to determine whether the anti-arthritis drug can cause genetic damage. The genotoxicity test required include; mouse lymphoma assay (MLA) otherwise known as in vitro mammalian cell clastogenecity, the Bruce Ames test (Ames test) and mouse micronuclei assay. These tests detects whether the anti-arthritis can cause alterations in chromosome and damage to DNA leading to genetic mutation and ultimately results in malignant tumour (cancerous cell).”The Ames test is commonly used for the genotoxicity test and it detects whether the drug is genotoxic. This occurs by causing back mutation in bacteria colonies and it takes up to about 48hours” . Gene mutation, clastogenecity of a genotoxic compound and chromosomal aberrations are determined via the mouse lymphoma assay (MLA) or in vitro mammalian cell clastogenecity. The MLA requires between 2-3weeks and can either give a negative result to the Ames test or not detected at all. An additional test used in genotoxicity testing is the mouse micronuclei assay which is an in vivo study. This is required because regulatory authorities (ICH) requires both in vitro and in vivo test. The process of ADME of the anti-arthritis drug is used to detect genotoxicity via the mouse micronuclei assay. After performing all the three tests discussed and the anti-arthritis drug remains positive to all, then the drug is probably carcinogenic to humans.
Test for Organ Toxicity and Biochemical Dysregulation
Safety pharmacology of the anti-arthritis drug is required to detect target organ toxicity such as cardiovascular, central nervous, respiratory, renal and gastrointestinal system. Rodents (such as mice and rats) and non-rodents (such as guinea pigs and dogs) are required or used for safety pharmacology test. Rats or mice are required for CNS and respiratory studies while dog is required for cardiovascular studies. The pharmacological activity of the anti-arthritis drug is determined by carrying out the ligand binding assay which makes in vitro studies preferable to in vivo studies. In safety pharmacology studies, the animals are divided into four groups, three treated group and one control group. The maximum number of rats required per group is fifteen and four dogs group. The duration of dosage in safety pharmacology studies is usually one month .” The ICH (S7A) perspective on cardiovascular safety pharmacology required core battery studies and follow up studies. Some of the core battery studies include heart rate, electrocardiogram and blood pressure must be evaluated. In vitro and in vivo evaluations, conductance abnormalities including methods for assessing repolarisation must be put into consideration” . “Some of the follow up studies include vascular resistance, cardiac output, the effects of exogenous and/or endogenous compound on the cardiovascular responses and so on” .
General Toxicity studies
General toxicology test is required for dose determination for No observed effect level (NOEL). The acute toxicity test is also known single dose toxicity and is required to assess biochemical dysregulation and also to determine the levels at which the anti-arthritis drug could cause an adverse reaction. The animals used for general toxicity test are rats and dogs and are usually dosed between 14-28days. Change in organ weight, histopathology, mortality rate, clinical pathology and necropsy are the parameters required to assess toxicity.General toxicology must be done before one month of reproductive studies.
Reproductive Toxicity Studies
The aim of reproductive toxicity studies is to reveal any effect of the anti-arthritis drug on mammalian reproduction. Rats and rabbits are the most commonly used and widely accepted animal. Rabbits are used because semen is easily collected. The ICH study design for reproductive toxicity studies include fertility and early embryonic development to implantation (rabbits dosed from day 6-18, rats dosed from day 6-15), organogenesis otherwise known as embryo-foetal development and pre and post-natal development (treatment last for 15gestation days and 21lactation days).The study design for fertility and early embryonic development studies requires four groups of 20males and 20females animal. Also, the study design for embryo-foetal development generally have four groups of 20rats or 20rabbits and ICH require evaluation of 16 to 20litters to provide a degree of consistency between studies [8; 9; 12; 13; 14]. Information derived from acute and repeated dose toxicity studies of at least one month are required before reproductive toxicology.
Pre-clinical studies must be carried out before clinical trials so as to protect human volunteers. All the studies described above determine how competent the anti-arthritis drug is before proceeding to clinical trials. All the regulations provided by ICH are widely used most especially the concept of 3Rs. Safety, quality and efficacy are the main objectives for carrying out preclinical studies. Some of the test required during preclinical studies includes carcinogenicity test which involves the short term and long term studies, genotoxicity test, reproductive test, safety pharmacology, renal toxicity test, cardiovascular toxicity test, general toxicity test and neurotoxicity test (functional observation battery test).
ICH harmonised tripartite guideline, Dose selection for carcinogenicity studies of pharmaceuticals S1C(R2).
Available at: http://www.ema.europa.eu/pdfs/human/ich/038395en.pdf
ICH harmonised tripartite guideline, Guidance on specific aspects of regulatory genotoxicity tests for pharmaceuticals S2A
Available at: http://www.bcg-usa.com/regulatory/docs/ich/ICHS2A.pdf
ICH harmonised tripartite guideline, Safety pharmacology studies for human pharmaceuticals S7A.
Available at: http://www.tga.gov.au/docs/pdf/euguide/ich/053900en.pdf
G.B. Jena et al., 2001, Genotoxicity testing, a regulatory requirement for drug discovery and development: impact of ICH guidelines, Indian Journal of Pharmacology.
David J. Tweats 1998, Impact of ICH guidelines on genotoxicity testing, PSTT Vol 1, No. 5.
ICH harmonised tripartite guideline, Guideline on the need for carcinogenicity studies of pharmaceuticals S1A.
Available at: http://www.bcg-usa.com/regulatory/docs/ich/ICHS1A.pdf
Guideline for industry, The need for long-term rodent carcinogenicity studies of pharmaceuticals.
ICH M3; Timing of pre-clinical studies in relation to clinical trials (see safety topics). Available at: http://www.ich.org/cache/compo/276-254-1.html
Preclinical toxicology: Points to consider in programme design.
Available at: http://www.pacificbiolabs.com/preclinical
James L. Stevens, (2006). Future of toxicology mechanisms of toxicity and drug safety: where do we go from here? Chem. Res. Toxicol., 19, 1393-1401.
Yasuo Ohno, (2002). ICH Guidelines-Implementation of the 3Rs: Incorporating Best Scientific Practices into the regulatory Process. Regulatory Testing and Animal Welfare. ILAR Journal V43 Supplement 2002.
Lecture notes by Dr Jean-Pierre Valentin, Director Safety Pharmacology, Safety Assessment UK, AstraZeneca.
Lecture notes by Dr Lorna M. Burns, Sequani limited, Ledbury, Herefordshire
Lecture notes by Dr M. Kelly.