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Sumanth,Dept Of Pharmacology,JSSCP,Mysore.
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.
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. 3
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 -blocker/-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.
9. Pharmacokinetics Absorption Tissue distribution Metabolism Excretion Volume of distribution & Half -life of the drug are quantified.
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.
Sumanth,Dept Of Pharmacology,JSSCP,Mysore.
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. 8
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. 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.
II.
III.
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. 9
- 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 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
In vitro In situ
In silico
Examples: Evaluation of Anti-epileptic activity: In vivo 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
In vitro
Evaluation of Anti-Parkinsonism activity: In vivo MPTP model of Parkinsons disease (N-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine) Transgenic animal models of Parkinsons disease Culture of Substantia nigra Inhibition of apoptosis in neuroblastoma SH-SY5Y cell
In vitro
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
Sumanth,Dept Of Pharmacology,JSSCP,Mysore.
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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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