PHARMACOLOGICAL SCREENING OF ANALGESIC AGENTS

Presented by :

CHAITRA. N

1st year M-Pharm

Dept.of Pharmacology

PESCP,Bangalore

CONTENTS

Introduction.

Mechanism of Action.

Classification.

Screening techniques.

In vivo.

In vitro.

References.

Pain is a complex unpleasant phenomenon composed of sensory

experiences that include time, space, intensity, emotion, cognition and

motivation.

An analgesic or painkiller is any member of the group of drugs used to

achieve analgesia — relief from pain.

Analgesics is defined as the are agents which selectively relieve pain by

acting in the CNS or by peripheral pain mechanisms without significantly

altering consciousness.

INTRODUCTION

Narcotics

Ex. Morphine, Pethadine, Fentanyl

Non-narcotics

Ex. NSAID

Analgesics are classified as :

CLASSIFICATION

Analgesic drugs act in various ways on the peripheral and central

nervous systems.

Opioids produce analgesia by binding to specific G – protein

coupled receptors in brain and spinal cord.

NSAIDs inhibit the activity of both cyclooxygenase-1 (COX-1)

and cyclooxygenase-2 (COX-2) and thereby the synthesis

of prostaglandins and thromboxanes.

Inhibition of COX-2 leads to the anti-inflammatory, analgesic and

antipyretic effects.

MECHANISM OF ACTION

SCREENINGMODELS

Pain - state models using Thermal stimuli.

* Tail - flick model using radiant heat.

* Hot - plate test.

* Paw - withdrawal test.

Pain - state models using Electrical stimuli.

* Electrical stimulation of the tail.

* Grid - shock test.

* Stimulation of the limbs.

Pain - state models using Chemical stimuli.

* Formalin test.

* Acetic acid induced writhing test.

IN VIVO MODELS

3H-Naloxone binding assay.

3H-Dihydromorphine binding to 𝜇 opiate receptors in rat brain.

Receptor binding of nociceptin.

Bioassays for nociceptin.

Receptor binding of cannabinoids.

Vanilloid receptor binding.

IN VITRO MODELS

IN VIVO MODELS

HOT – PLATE TEST

The paws of mice and rats are sensitive to heat at temperatures

which are not damaging to skin.

The responses are jumping, withdrawal of the paws and licking of

the paws.

The responses is prolonged after administration of centrally acting

analgesics, whereas peripheral analgesics of the acetylsalicylic acid

or phenyl-acetic acid type do not generally affect these responses.

Purpose and rationale

Procedure

Groups of 10 mice (18-22g) are selected and divided into standard, test & control group respectively

The temperature of the hot plate is maintained at 55° to 56°C.

The animals are placed on the hot plate & time until either licking or jumping occurs is recorded.

The latency is recorded before & after 20, 60 and 90 min after the administration of standard or test compound.

Evaluation

The prolongation of latency time between the test, standard

and control animals are compared.

Using various doses ED50 values can be calculated.

TAIL FLICK MODEL

Purpose and rationale

The tail flick test with radiant heat is an simplified method.

The application of thermal radiation to the tail of an animal

provokes the withdrawal of tail.

The morphine like drugs are capable of prolonging the reaction

time.

Wistar rats (170-210g) are selected and divided into standard, test & control group respectively

Procedure

Appropriate temperature is maintained on the radiant source

The tail of the rat is placed on the radiant source & time taken for the rat to withdraw its tail is recorded.

Usually withdrawal time is within 2-10s

The Tail-flick latency is recorded before & after the administration of standard or test compound.

Evaluation

The tail flick latency in the test, standard and control

animals are compared.

Using various doses ED50 values can be calculated.

GRID - SHOCK TEST

Purpose and rationale

The electric grid shock test in mice has been described by

Blake et al.

The analgesic properties of drugs like Morphine,

Acetylsalicylic acid can be measured by the Flinch – jump

response of rats.

Male mice (18-20g) are selected and placed individually in plastic chamber.

Procedure

The floor of the box is wired with stainless steel wire.

The stimulus is given in the form of square wave pulses ( 30 cycles per second).

The output of stimulator is connected to alternate wires of grid.

The fixed resistance is placed with the grid & parallel to an oscilloscope to allow calibration in milliamperes.

With increase in shock intensities the mice flinch, exhibit startling reaction & increase locomotion or

attempt to jump.

The behavior is accurately reflected on the oscilloscope by marked fluctuations of the

displayed pulse.

Pain thresholds are determined in each individual mouse twice before & after the administration of

the test drug.

Evaluation

The current measured in milliamperes is recorded for each

animal before and after administration of the drug.

The average pain threshold values for each group at each time

interval are calculated and statistically compared with the

control values.

WRITHING TESTS

Purpose and rationale

Pain is induced by injecting irritants like acetic acid into

peritoneal cavity of mice.

The animals react with characteristic stretching behavior

which is writhing.

The test is suitable to detect analgesic activity of

peripherally acting drugs.

Mice (20-25g) are selected and divided into standard, test & control group respectively

Procedure

Appropriate volume of acetic acid solution is administered to the mice (control group) and placed individually in the glass jar.

The onset of writhing, abdominal contractions &trunk twist response are recorded for 10 min.

The test and standard drug is administered 15 min prior to the acetic acid administration.

Evaluation

The writhing period is recorded and compared with

the control group.

Writhing response in the drug treated must be less

when compared to the acetic acid treated control.

IN VITRO MODELS

BIOASSAYS FOR NOCICEPTIN

Purpose and rationale

Nociceptin receptors in the periphery can be characterized by studies in

isolated organs (Guerrini et al. 1998; Bigoni et al. 1999): the guinea pig

ileum according to Paton (1957), the mouse vas deferens according

to Hughes et al. (1975), the rabbit vas deferens according to Oka et al.

(1980), the guinea pig renal pelvis (Giuliani and Maggi 1996).

Procedure

Tissues are taken from male Swiss mice, guinea pigs, Sprague Dawley rats & New Zealand albino rabbit.

Suspended in 10 ml organ baths containing Krebs solution oxygenated with 95% O2 & 5% CO2.

Temperature is set around 33°-35°C & a resting tension of 0.3-1g is applied.

The tissues are stimulated through two platinum ring electrodes.

The electrically evoked contractions are measured isotonically with a strain gauge transducer and recorded on a multichannel chart recorder.

After equilibration period of about 60 min the contractions induced by electrical field stimulation are stable; at this time, cumulative concentration response curves

to nociceptin or opioid peptides are performed.

Four electrical field stimulation are performed with each tissue at 30 min intervals.

Agonists & Antagonists are added to the bath.

The biological effects of the application of agonists or antagonists are expressed as % inhibition of electrical filed stimulation-induced contraction.

Contractile responses to electrical field stimulation are expressed as % increment to the spontaneous activity of the tissue.

Evaluation

The agonists and antagonists potencies are recorded

and expressed as means of ±SEM.

Drug screening methods – SK Gupta.

Essentials of Medical Pharmacology - Tripathi

K.D.

Drug Discovery and Evaluation - H. Gerhard

Vogel.

International Research Journal Of Pharmacy.

REFERENCES