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Screening of New Drugs

Presented by

Silpa P Sankar M.Pharm Part-1


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CONTENTS
INTRODUCTION SCREENING METHODS Screening By NMR Affinity Screening High Throughput Screening Scintillation Proximity Assay Bioassay

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INTRODUCTION
Drugs are designed to interact with a particular target Once a target have been chosen the next stage is to find a lead compound.
The lead compounds are compounds showing a desired pharmacological property which provides a start for the drug design & development process. SCREENING :- A procedure by which compds are tested for biological activity.

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SCREENING METHODS
SCREENING BY NMR

AFFINITY SCREENING
HIGH THROUGHPUT SCREENING SCINTILLATION PROXIMITY ASSAY BIOASSAY

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SCREENING BY NMR
NMR Spectroscopy -determine molecular stucture. Recently NMR used to detect whether a compound binds to protein target. A compd is radiated with a short pulse of energy which excites the nuclei of H2 ,C or N2 On stopping radiation excited nuclei relax back to ground state giving off energy.

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Contd..

RELAXATION TIME Time taken by different nuclei to give off energy. A different signal will be obtained for each atom in the molecule & a spectrum is obtained. Size of the molecule plays an important role in the length of relaxation time.

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PROCEDURE
1. NMR spectrum of drug is taken. 2. Protein is added & spectrum is run. 3. If the drug bind to the protein its nuclei have a short relaxation time & no NMR spectrum will be detected. 4. If the drug fails to bind to the protein NMR Spectrum will be still detected.

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AFFINITY SCREENING
Eg:- Vancomycin family of antibacterial agents have a strong binding affinity for dipeptide D-AlaD-Ala.
The dipeptide was linked to sepharose resin & resin was mixed with extracts from various microbes having antibacterial activity. If the extract lost activity active compound has bound to the resin.

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HIGH THROUGH PUT SCREENING

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Involves the automated testing of large no. of compd vs a large no. of targets. Test should produce an easy measurable effect which can be detected & measured automatically. Results may be seen as an easily identifiable colour changes.

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PROCEDURE
Automatic & quick process to carry out biological testing of structures produced by comb.syn. Compounds are automatically tested & analysed on a plate containing 1536 wells & test volume of 1 -10 l. Fluorescence & chemiluminescence aiiow the identification of active wells. Miniaturisation using closed system is on horizon. Microfluidics can be used.

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SCINTILLATION PROXIMITY ASSAY

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SPA Visual method .Detects whether a ligand binds to a target. Target is immobilised by linking covalently to beads coated with a scintillant. A soln of known ligand labelled with 125I is added to beads.125I donates energy to bead results in emission of light. Novel compd is added to soln of labelled ligand & the mixture is added to beads.Binding by novel compd reduces binding by labelled ligand resulting in reduction of emission of light.

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BIOASSAY
DEFINITION
Estimation of the potency of an active principle in a unit quantity of preparation. (or) Observation of pharmacological effects on isolated cells,tissues,enzymes,receptors (invitro) or in animals.(invivo)

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INVITRO TESTS
Cheaper, easier, automated.
Receptor agonists & antagonst can be tested on isolated tisssues or cells which expreess the target receptors on the surface. Bronchodilator activity can be tested by observing how well compds inhibit contraction of isolated tracheal smooth muscle.

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INVIVO TESTS
Induce a clinical condition in animal. Treated to see whether the drug alleviates the problem. Eg:- Development of NSAIDs Animals whose genetic code is altered -Transgenic animals. Eg:- Replacing mouse genes with human genes in mouse produces human enzyme or receptor

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PRINCIPLE INVOLVED IN BIOASSAY

Compare the test substance with the International std preparation of the same & to find out how much test substance is required to produce the same biological effect as produced by the std.

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METHODS OF BIOASSAY Agonist produce graded (response to dose.) or quantal (max. response.) Quantal End point methods Graded - 1. Matching(Bracketing method) 2. Graphical method
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END POINT METHOD Threshold dose producing a positive effect is measured on each animal & compare between the avg. results of two grps of animals.(one receiving std & other the test) Eg:-Bioassay of Digitalis in cats.

Conc. Of unknown = Threshold dose of Std *


Threshold dose of Test

Conc. Of Std

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MATCHING METHOD A const. dose of test is bracketted by varying doses of Std till the exact match is obtained between test dose & std dose.
Conc. Of unknown = Dose of Std * Conc of Std Dose of Test

Eg:- Bioassay of Histamine on Guinea pig ileum

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GRAPHICAL METHOD
Based on the relationship. assumption of dose response

Log dose response curve is plotted & the dose of Std producing the same response as produced by the test sample is directly read from the graph. Simple method & chances of error are less.

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OTHER ASSAY METHODS


3 POINT ASSAY 4 POINT ASSAY

5 POINT ASSAY
6 POINT ASSAY 8 POINT ASSAY
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DRUGS
Digitalis

PREPARATION
Cat BP, Guinea pig BP, Pigeon Rectus abdominis muscle of frog Rat/Cat BP Anaesthetised & atropinized cat Rat uterus BP of spinal cat Rat uterus

ACTIVITY ASSAYED
Fall in BP &death Stopage of heart & death Emesis Contractile effect Fall in BP Fall BP Inhibition of tone Rise in BP Contractile effect
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Acetylcholine

Histamine Adrenaline Oxytocin

BIOASSAY OF ACETYLCHOLINE
USING RECTUS ABDOMINIS MUSCLE OF FROG Ach produces a dose dependent contraction of rectus abdominis muscle through stimulation of nicotinic receptors. In Graphical method 4-6 responses to graded responses of Ach are obtained & then 2 equipotent responses to unknown sample are taken.
The responses can be plotted against logdose of Std & the amount of Std producing the same response as produced by unknown is directly read from the graph & the conc. Of unknown is determined by the formula.

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BIOASSAY OF OXYTOCIN Female rats are separated from males.wt-120-200mg. Before 18-24 hours of expt 100 gm/kg of oestradiol is injected im Rat is sacrified ,uterine horns are isolated & mounted in organbath. Bath is maintained of temp-320c .Uterine horn contractions are recorded by frontal lever.

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Contd..

Std Oxytocin ,a dose of 0.05- 0.1 unit is added in to the inner bath.This causes contraction & on completion of contraction soln is drained out. Repeated addition of oxytocin may be made at regular itervals.
The potency of the sample is calculated by comparing the height of contraction with that produced by the Std sample.

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BIOASSAY OF DIGITALIS
PRINCIPLE Potency of the test sample is compared with that of Std preparation by determining the action on the cardiac muscle. GUINEA PIG METHOD Guinea pig is anaesthetised with a suitable anaesthetic.The jugular vein is dissected out & cannulated by venous canula.A pin is inserted in the heart so we can observe the heart beats by up & down movements of the pin.

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Contd Inj. is continued through venous canula until the heart arrests.Amount of extract required to produce this effect is lethal dose

Another 19 animals of same species are used & avg. lethal dose is determined. Lethal dose of test sample is determined in a similar way by using 6 guinea pigs.

Potency of test sample is calculated in relation to that of Std prep by dividing avg.lethal dose of the sample to the test.

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BIOASSAY OF ADRENALINE ON RABBIT ILEUM

Graphical Method PRINCIPLE :- Adrenaline stimulate &


receptors except in GI muscles.It produces a dose dependent relaxation of rabbit ileum

PROCEDURE Animal kept

for overnight fasting is sacrificed.Abdominal cavity is opened & a piece of ileum is isolated & placed in Tyrode soln maintained at 370C

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Contd.

The tissue is mounted in organ bath & connected to isotonic frontal lever It is allowed to stabilize till the spontaneous contractions become uniform for 5 minutes. Graded doses of Adrenaline are added to the bath & relaxant effect is recorded. Conc. Of unknown is determined.

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CONCLUSION
Recently Pharmaceutical companies tend to concentrate on developing drugs for diseases which are prevalent in developed countries & aim to produce compounds with better properties than existing drugs. A molecular target is chosen which is believed to influence a particular disease. Through NMR Spectroscopy compounds can be tested for their affinity to a target. A suitable bioassay can demostrate whether a drug has activity against a particular target.

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REFERENCES
An Introduction To Medicinal chemistry3rd Edition Graham.L.Patrick

Practicals In Pharmacology -4th Edition Dr.R.K.Goyal

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