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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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A procedure by which compds are tested for biological activity. • The lead compounds are compounds showing a desired pharmacological property which provides a start for the drug design & development process.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. Page 4 . • SCREENING :.

SCREENING METHODS • SCREENING BY NMR • AFFINITY SCREENING • HIGH THROUGHPUT SCREENING • SCINTILLATION PROXIMITY ASSAY • BIOASSAY Page 5 .

SCREENING BY NMR • NMR Spectroscopy -determine molecular stucture.C or N2 • On stopping radiation excited nuclei relax back to ground state giving off energy. • 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 . Page 6 .

Page 7 .. • Size of the molecule plays an important role in the length of relaxation time. • 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.Contd………….

Page 8 .PROCEDURE 1. Protein is added & spectrum is run. 2. 3. NMR spectrum of drug is taken. 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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 If the extract lost activity active compound has bound to the resin. • The dipeptide was linked to sepharose resin & resin was mixed with extracts from various microbes having antibacterial activity.AFFINITY SCREENING • Eg:.Vancomycin family of antibacterial agents have a strong binding affinity for dipeptide D-AlaD-Ala. Page 10 .

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HIGH THROUGH PUT SCREENING Page 12 .

of targets.• Involves the automated testing of large no. of compd vs a large no. Page 13 . • Test should produce an easy measurable effect which can be detected & measured automatically. • Results may be seen as an easily identifiable colour changes.

Page 14 .PROCEDURE Automatic & quick process to carry out biological testing of structures produced by comb. Miniaturisation using closed system is on horizon.syn. Fluorescence & chemiluminescence aiiow the identification of active wells. Compounds are automatically tested & analysed on a plate containing 1536 wells & test volume of 1 -10 µl. Microfluidics can be used.

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SCINTILLATION PROXIMITY ASSAY Page 16 .

• Target is immobilised by linking covalently to beads coated with a scintillant.Detects whether a ligand binds to a target.125I donates energy to bead results in emission of light.Binding by novel compd reduces binding by labelled ligand resulting in reduction of emission of light.• SPA –Visual method . • A soln of known ligand labelled with 125I is added to beads. • Novel compd is added to soln of labelled ligand & the mixture is added to beads. Page 17 .

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enzymes.BIOASSAY • DEFINITION Estimation of the potency of an active principle in a unit quantity of preparation.(invivo) Page 19 .tissues.receptors (invitro) or in animals. (or) Observation of pharmacological effects on isolated cells.

• Bronchodilator activity can be tested by observing how well compds inhibit contraction of isolated tracheal smooth muscle.INVITRO TESTS • Cheaper. automated. easier. Page 20 . • Receptor agonists & antagonst can be tested on isolated tisssues or cells which expreess the target receptors on the surface.

INVIVO TESTS • Induce a clinical condition in animal.Development of NSAIDs • Animals whose genetic code is altered -Transgenic animals. • Treated to see whether the drug alleviates the problem. • Eg:. • Eg:.Replacing mouse genes with human genes in mouse produces human enzyme or receptor Page 21 .

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. Page 22 .

) or quantal (max.METHODS OF BIOASSAY • Agonist produce graded (response α to dose. response. Matching(Bracketing method) 2. Graphical method Page 23 .1.) • Quantal – End point methods • Graded .

(one receiving std & other the test) • Eg:-Bioassay of Digitalis in cats. results of two grps of animals. Conc.END POINT METHOD • Threshold dose producing a positive effect is measured on each animal & compare between the avg. Of unknown = Threshold dose of Std * Threshold dose of Test Conc. Of Std Page 24 .

Bioassay of Histamine on Guinea pig ileum Page 25 . Of unknown = Dose of Std * Conc of Std Dose of Test Eg:.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.

• Simple method & chances of error are less. Page 26 . 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.GRAPHICAL METHOD • Based on the relationship.

OTHER ASSAY METHODS • 3 POINT ASSAY • 4 POINT ASSAY • 5 POINT ASSAY • 6 POINT ASSAY • 8 POINT ASSAY Page 27 .

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 Page 28 Acetylcholine Histamine Adrenaline Oxytocin . Guinea pig BP.DRUGS Digitalis PREPARATION Cat BP.

• 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.BIOASSAY OF ACETYLCHOLINE • USING RECTUS ABDOMINIS MUSCLE OF FROG • Ach produces a dose dependent contraction of rectus abdominis muscle through stimulation of nicotinic receptors. Page 29 .

• Bath is maintained of temp-320c . • Before 18-24 hours of expt 100µ gm/kg of oestradiol is injected im • Rat is sacrified . Page 30 .BIOASSAY OF OXYTOCIN • Female rats are separated from males.uterine horns are isolated & mounted in organbath.Uterine horn contractions are recorded by frontal lever.wt-120-200mg.

This causes contraction & on completion of contraction soln is drained out..Contd………….a dose of 0. Page 31 . • Std Oxytocin .05.0. • The potency of the sample is calculated by comparing the height of contraction with that produced by the Std sample. • Repeated addition of oxytocin may be made at regular itervals.1 unit is added in to the inner bath.

The jugular vein is dissected out & cannulated by venous canula.BIOASSAY OF DIGITALIS • PRINCIPLE Potency of the test sample is compared with that of Std preparation by determining the action on the cardiac muscle. Page 32 . • GUINEA PIG METHOD Guinea pig is anaesthetised with a suitable anaesthetic.A pin is inserted in the heart so we can observe the heart beats by up & down movements of the pin.

lethal dose of the sample to the test.Contd……………… • Inj.Amount of extract required to produce this effect is lethal dose • Another 19 animals of same species are used & avg. • Lethal dose of test sample is determined in a similar way by using 6 guinea pigs. is continued through venous canula until the heart arrests. • Potency of test sample is calculated in relation to that of Std prep by dividing avg. lethal dose is determined. Page 33 .

It produces a dose dependent relaxation of rabbit ileum • PROCEDURE • Animal kept for overnight fasting is sacrificed.Adrenaline stimulate α & β receptors except in GI muscles.Abdominal cavity is opened & a piece of ileum is isolated & placed in Tyrode soln maintained at 370C Page 34 .BIOASSAY OF ADRENALINE ON RABBIT ILEUM • Graphical Method • PRINCIPLE :.

• Graded doses of Adrenaline are added to the bath & relaxant effect is recorded. • 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.Contd……………. • Conc. Of unknown is determined. Page 35 .

• Through NMR Spectroscopy compounds can be tested for their affinity to a target.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. • A suitable bioassay can demostrate whether a drug has activity against a particular target. Page 36 .

REFERENCES An Introduction To Medicinal chemistry3rd Edition – Graham.K.R.L.Patrick  Practicals In Pharmacology -4th Edition Dr.Goyal Page 37 .

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