General Principles of Preclinical Screening

General Principles of Preclinical Screening Stages in New Drug Development  Synthesis/isolation of compound (1-2 years)  Preclinical studies: Screening, evaluation, pharmacokinetic and toxicity testing in animals (2-4 years)  Grant of permission for clinical trials (3-6 months)  Pharmaceutical formulation(0.5-1 year)  Clinical studies: Phase I, PhaseII, PhaseIII trials; long-term animal toxicity testing (3-10 years)  Review and grant of marketing permission (0.5-2 years)  Postmarketing Surveillance Phase IV studies

Preclinical Studies/Preclinical evaluation: A very large number of compounds from variety of sources such as Synthesised compounds, Natural product extracts, combinatorial chemistry libraries, Biotechnology products are tested on animals to expose the whole pharmacological profile. As the evaluation progresses unfavourable compounds get rejected at each step, so that only a few out of thousands reach the stage when administration to man is considered. Types of tests performed are 1. Screening tests 2. Tests on isolated organs, bacterial cultures, etc., 3. Tests on animal models of human disease 4. General observational test 5. Confirmatory tests and analogous activities 6. Mechanism of action 7. Systemic pharmacology 8. Quantitative tests 9. Pharmacokinetics 10.Toxicity tests

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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1.Screening tests: - Screening is a scanning procedure designed to distinguish useful from non-useful drugs as rapidly, comprehensively and inexpensively as possible. - It involves a test or group of tests which permits the detection of physiological activity. - Main purpose of screening is to determine whether the new drugs are worth for further attention & to indicate which among them have the most interesting pharmacological property. - In drug evaluation it is important to note the whole range of qualitative changes produced by the drug & the quantitative relation between them. 3 types of Screening  Simple Screening  Blind Screening  Programmed screening Simple Screening: - Screening is called simple when only one or two tests are used to find substances having a particular property E.g. Test for con. Of sugar in the blood might be used to screen compounds for hypoglycaemic activity. Blind screening: - This type of screening is for new series of chemical substances obtaind either through natural source or synthesis. - Contain techniques for detecting pharmacological activity in a group of substances without pharmacological history. - Requires considerable planning & skilful execution of the tests in order to be economical of time and money. - In this no assumptions are made about what the probable actions of a compound may be (except when an already carefully studied series of compounds having similar structures have been investigated).

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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Programmed Screening: - When a new drug of a specific type or when a series of compounds are to be investigated for some pharmacological activity, a program of testing is identified, this could limit the screening procedure for particular tests that could provide greater precision. - The program should also provide indications for potential side effects. Screening tests are designed & performed to identify agents having a certain set of characteristics that will either exclude them from further consideration or cause them to be selected for closer attention. Characteristics of screening tests:      

Sensitivity Specificity Positive Accuracy Negative Accuracy Capacity (no. of compounds that can be evaluated) Reproducibility (probability that a screening test will produce the same result at another time)

Considerations (General principles) for screening tests:  Screening tests should always focus on detecting a single point of effect (such as mutagenicity, lethality, neurotoxicity).  It should evaluate large number of compounds with ease and speed of performance.  Screening test must be very sensitive in its detection.  Screening test should use small amounts of compound.  Any screening system should be validated initially using a set of blind (positive & negative) controls. These blind controls should also be evaluated in the screening system to ensure continuing proper operation of the screen.  Proper dose selection is essential for effective and efficient screen design and conduct. 2. Tests On Isolated Organs, Bacterial Cultures, etc., - These are preliminary tests to detect specific activity, such as antihistaminic, antisecretory, vasodilator, antibacterial etc., Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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3. Tests on Animal Models of Human Disease: - These tests are conducted on animal models of human disease. - The disease may be induced experimentally e.g. Alloxan induced diabetes in rats, Pentylene tetrazole induced convulsions 4. General Observational Test: - Performed either in the beginning (in case of totally novel compounds) or after detecting useful activity in screening test, the drug is injected in tripling doses to small group of mice which are observed for overt effects. - Preliminary clues are drawn from the profile of effects observed. 5. Confirmatory tests and analogues activities: - Compounds found active are taken up for detailed study by more elaborate tests which confirm and characterise the activity. - Other related activities e.g. Antipyretic and anti-inflammatory activity in an analgesic are tested. 6. Mechanism of action - Attempts are made to find out the mechanism of action. E.g. whether an antihypertensive is an α-blocker/β-blocker/Calcium channel blocker/ACE Inhibitor/Centrally acting etc. 7. Systemic Pharmacology - Effects on major organ systems such as nervous, cardiovascular, respiratory, renal, GIT by the drug under development are worked out. 8. Quantitative tests - Dose-response relationship - Maximal effect & - Comparative efficacy with other existing drugs are ascertained.

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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9. Pharmacokinetics -

Absorption Tissue distribution Metabolism Excretion Volume of distribution & Half -life of the drug are quantified.

10. Toxicological Evaluation Of New Drugs      

Acute Toxicity studies Dose-ranging Studies Sub-acute Toxicity Studies Chronic Toxicity Studies Carcinogenicity Studies Reproduction Studies  General Reproduction & Fertility  Teratogenicity  Perinatal & Postnatal Study  Genotoxicity Testing  Metabolic Activation  Special Studies  Mutagenicity  Immunological Toxicity  Nephrotoxicity  Behavioural Teratological Effects

Acute Toxicity Studies - Determination of acute toxicity is the initial experiment which a toxicologist undertakes with a compound under development. - These studies are normally carried out in rat and mice and often in animals of both the sexes. - Parameter associated with acute toxicity is the LD50 value. - Other parameters derived are Minimum Lethal Dose & Maximum No-effect Dose. Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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Objectives of Acute Toxicity Studies:  To give information that allows assessment of the toxic potential of the compound and hence to predict the likely hazard involved in its use.  To provide information on one half of the risk-benefit equation by considering the observed toxicity within the framework of likely clinical advantages.  To provide information on the mechanism of the toxic reaction.  To provide information of use in designing sub-acute toxicity studies.

- Routes of administration chosen for these studies must always be that intended for use in man. - In addition a parenteral route, preferably intravenous, is also chosen to provide information on toxicity by route which ensures complete absorption.(Comparison of toxicity between the IV and oral routes can also provide information on the degree of absorption) - In addition to studies in rodents some regulatory authorities also demand acute toxicity investigations in a non-rodent species.

Dose-ranging Studies What happens on repeated administration? Objectives Of Dose –ranging Studies:    

To determine the type of toxicity which is encountered on repeated dosing. To identify target organs. To provide information on the choice of doses for the formal studies to follow. Also provide information which can relate to the decision as to whether or not to proceed with the development of a particular compound.

Although two species are chosen, it is common to carry out dose ranging in three species (one rodent, dog and primate).

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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Sub-acute Toxicity Studies Length of sub-acute studies - 14, 28 or 90 days. Objectives of Sub-acute studies:        -

To determine the nature of toxic events To determine the target for toxicity, eg.specific organs(liver, kidney) To establish if a dose-response relationship exits. To identify species differences in toxic response. To identify differences in sensitivity between the sexes. To investigate accumulative effects. To investigate any tolerance. Three dose levels are normally chosen based on knowledge and experience gained previously during the dose ranging studies and also the Pharmacology. - At this stage of the development of a new compound, the objective is to define its toxicity. Administration of a compound in sufficiently high doses allows a number of criteria to be met:  The load of the drug within the animal will increase until a steady state is reached.  The animal may adapt to this load by altering its way of handling the drug, either in pharmacological response, metabolic response or excretory mechanisms.  Continued high doses may overload the body systems such that different metabolic pathways are exposed , possibly leading to the formation of new metabolites which may be toxic.  Failure of physiologic functions may occur, leading ultimately to pathological change. Done on two species (Rodent & Nonrodent) Route of administration according to intended route of use.

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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Chronic Toxicity Studies - Chronic toxicity is the result of continuous or repeated exposure to the drug. - Chronic toxicity can also be produced by continuous exposure to low levels of drug. - The tests for chronic toxicity are usually run for 2 years or through the life of test animal. - The route chosen should be that intended for clinical use. The chemical being tested is either administered in the diet or given at least once daily. - Conducted preferably on 2 species rodent and nonrodent.

Carcinogenicity Studies - Carcinogen may be defined as any agent which significantly increases the incidence of malignant neoplasms. - The principle of carcinogenicity test is that it should involve lifetime exposure to the test compound. - Rodents are the species usually chosen for carcinogenicity studies. - In interpreting a carcinogenicity study, alterations in the incidence of tumours should be considered in the terms of whether the test compound is an Initiator or a Promoter. - Agents which are Initiators are capable, either directly or after metabolic activation, interact with cellular targets (usually DNA) and bring changes in cell leading to tumour growth. - Promoter is not itself capable of inducing tumour growth, but promotes by bringing about an alteration in endocrine function or unmasking the effect of a virus or of a concurrently present genotoxic initiation.

Reproduction Studies -

The aim of these studies is to investigate any effects a new drug may have on mating behaviour, foetal development seen as foetal loss and abnormalities, and any effect on the development of offspring in later life.

Objectives of the studies are to find out:  Changes in fertility or in the production of normal young due to damage to the male or female gametes. Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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 Interference with pre-implantation and implantation stages in the development of conceptus.  Toxic effects on the embryo.  Toxic effects on the foetus.  Changes in maternal physiology producing secondary effects on embryo or foetus.  Effects on uterine or placental growth or development.  Interference with parturition.  Effects with postnatal development and suckling of the progeny and on maternal lactation.  Late effects on progeny. These objectives are satisfied with 3 studies I.

II.

III.

General Reproduction and fertility involving commencement of dosing of both males and females before mating and observing effects on both offspring and parents. Teratogenicity/Teratology study where any effects on the embryo are investigated by dosing the pregnant dams during specific periods of their gestation. Perinatal and Postnatal study aims to investigate effects on the suckling and lactating dam and upon development of the offspring.

Genotoxicity Testing - Genotoxicity describes a deleterious action on a cell's genetic material affecting its integrity. - Assays must be conducted in the presence and absence of metabolic activation. Metabolic Activation: - Large proportion of genotoxic agents are detected in short term in vitro assays only after metabolic activation to the relevant active moiety. - Normal method of activation is by incorporation of S-9 mix normally generated from a microsomal preparation of the livers of rats pre-treated with an enzyme inducer (generally the detoxification enzymes are excluded). - It is of particular value in tests such as the Ames Test which employ bacteria. Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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- Although ‘S-9’ is also incorporated into assays involving mammalian cells, these cells have the metabolic capability of their own and can give rise to a different set of metabolites. Available methods for Genotoxicity testing: EEC asks for i) ii) iii)

Tests in both prokaryotes and eukaryotes (as the organisation of genetic material in these two types of organisms differ) One in vivo test should be included. Metabolic activation systems in in vitro systems should be included

To comply with these guidelines EEC suggests one test from each of the following    

Gene mutation in bacteria E.g. Ames Test Chromosome aberrations in mammalian cells in vitro Gene mutation in eukaryote systems An In vivo test

In vivo genotoxic test - Relies on microscopic examination of possible chromosome damage. - This is the micronucleus test which involves counting of micronuclei in polychromatic erythrocytes. Erythrocytes are obtained from rodents previously treated with the drug.

Special Studies    

Mutagenicity Immunological Toxicity Nephrotoxicity Behavioural Teratological Effects etc.,

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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Type Of Experiments Type of experiment In vivo

In vitro In situ

In silico

Meaning Within the living experimentation using a whole, living organism Within the glass i.e., in a test tube or petri dish In position in situ means to examine the phenomenon exactly in place where it occurs (i.e. without moving it to some special medium) performed on computer or via computer simulation

Examples: Evaluation of Anti-epileptic activity: In vivo

In vitro

Electroshock in mice (grandmal epilepsy) Pentylene tetrazole(Metrazole) induced convulsions 4-aminopyridine induced seizures in mice Bicuculline Test in rats (GABAA antagonist) Electrical recordings from Hippocampal slices Electrical recordings from isolated nerve cells

Evaluation of Anti-Parkinsonism activity: In vivo

MPTP model of Parkinson’s disease (N-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine) Transgenic animal models of Parkinson’s disease

In vitro

Culture of Substantia nigra Inhibition of apoptosis in neuroblastoma SH-SY5Y cell

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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Evaluation of Anti-depressant activity: In vivo In vitro

Despair Swim Test(forced swim) Tail Suspension Test Inhibition of Norepinephrine uptake in rat brain synaptosomes Inhibition of Dopamine uptake in rat striatal synaptosomes Inhibition of Serotonin uptake

List of reasons for pursuing the development of in vitro test systems:  They avoid the complications of animal and tissue/organ in vivo evaluation.  In vivo systems may assess only short term sites of application or immediate structural alterations produced by agents. However those tests may be intended to evaluate only local effects.  Technician training & monitoring are critical in in vivo testing.  If our objective is either the total exclusion of a particular type of agent or the identification of truly severe acting agents on an absolute basis, then in vivo tests in animals do not perfectly predict results in humans.  Structural and biochemical differences exist between test animals & humans.  In vivo systems are not standardized.  In vitro tests provide variable correlation with human results.  Small quantity of test substance is sufficient.  Results are quickly obtained compared to in vivo models.  The chemical is not absorbed at all or is poorly absorbed in in vivo studies.  The chemical is well absorbed but is subjected to first pass effect in the liver.  The chemical is distributed so that less or more reaches the target tissues that would be produced on the basis of its absorption.  The chemical is rapidly metabolised to an active or inactive metabolite that has a different profile of activity or different action than the parent drug.  The chemical is rapidly eliminated.

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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3R principle behind the development of In vitro systems:

 Reduction refers to the use of fewer animals  Refinement entails the modification of existing procedures, so that animals are subjected to less pain and distress.  Replacement means utilizing methods that do not use intact animals E.g. cell cultures replacing rats and mice. In vitro pyrogen test (in rabbits) replaced by LAL test and MAT. Draize rabbit eye test (screening of chemicals for eye irritation potential) replaced by Neutral red uptake assay. In vitro systems also have a number of ‘limitations’ that can contribute their not being acceptance models  In vitro data cannot predict the volume of distribution.  In vitro data cannot predict the rate constants for drug movements between compartments.  In vitro data cannot predict rate constants of chemical elimination.  In vitro data cannot predict whether linear or nonlinear kinetics will occur.  Pharmacokinetic parameters - Bioavailability - Peak plasma concentration - Half-life cannot be predicted based solely on in vitro studies.

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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References:  Robert A.Turner. Screening Methods in Pharmacology, Academic press an imprint of Elsevier Pg no.22-25  PK Gupta. Modern Toxicology, volume I, Basis of Organ & Reproduction Toxicity, Pharmamed Press Pg no.42-55  Shayne Cox Gad. Drug Safety Evaluation, Second Edition, Wiley Publication Pg no. 103-108, 788-803  M.N.Ghosh. Fundamentals Of Experimental Pharmacology, Fourth Edition, Hilton & company Pg no. 184-188  Hans Gerhard Vogel. Drug Discovery & Evaluation:Pharmacological Assays, Third Edition, volume 1&2, Springer  Corwin Hansch. Comprehensive Medicinal Chemistry-The rational design, Mechanistic Study and Therapeutic Application of Chemical compounds, Volume 1-General Principles, Pg no. 569-589  KD Tripathi. Essentials of Medical Pharmacology, Sixth Edition, Jaypee Brothers Medical Publishers(p)Ltd, new Delhi,2009, Pg no. 74-76

Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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Sumanth,Dept Of Pharmacology,JSSCP,Mysore.

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