SCREENING METHODS FOR

ANALGESICS

SCREENING METHODS FOR

ANALGESICS

PRESENTED BY: AMEENA MEHABOOB

Pain is a symptom of many diseases

requiring treatment with analgesics.

Analgesics are drugs that selectively

inhibit the perception (sensation) of

the pain.

INTRODUCTION

ANALGESICS

PERIPHERAL

CAUSAL

(Atropine)NON CAUSAL

LOCAL ANESTHETICS

COUNTER IRRITANTS

NARCOTICS

NATURAL (codeine)

SYNTHETIC

(heroin)

SEMI SYNTHETIC

(pethidine)

Endgenousopiates

(enkephalins)

NON NARCOTICS

NSAIDs

(Aspirin, Diclofenac)

CLASSIFICATION OF ANALGESICS

SCREENING METHODS

IN VITRO

• 3H-Naloxone binding assay

• 3H-Bremazocine binding to κ opiate receptors in guinea pig cerebellum

IN VIVO

• HAFFNER’s tail clip method

• Radiant heat method

• Hot plate method

• Tail immersion test

• Electrical stimulation of the tail

• Grid shock test

• Tooth pulp stimulation

• Monkey shock titration test

• Formalin test in rats

IN VITRO TESTS

1) 3H-Naloxone binding assay

PURPOSE AND RATIONALE-

There is a correlation between the in

vivo pharmacological potency of

opiate agonists and antagonists with

their ability to displace radiolabeled

naloxone.

TISSUE PREPARATION

Male wistar rats decapitated and brains removed

Whole brain minus cerebella- weighed, homogenised in 50 vol of ice cold 0.05M Trisbuffer

Homogenate is centrifuged at 40000g, 15min

Supernatent decanted, pellet resuspended in fresh 0.05M Tris buffer

Yields tissue concentration of 10mg/ml

ASSAY

1

• Tubes containing H2O dextrorphan (total binding) or 5 μMlevorphanol (non-specific binding), 2 M NaCl or H2O, 0.5 M Trisbuffer, pH 7.7, drug or vehicle, 3H-naloxone tissue suspension are incubated for 30 min at 37 °C.

2

• The assay is stopped by vacuum filtration through Whatman GF/B filters.

• Washed 3 times with icecold 0.05 M Tris buffer, pH 7

3

• Specific binding is roughly 1% of the total added ligand and 50% of the total bound in the absence of Na+ and 2% of the total added ligand and 65% of the total bound ligand in the presence of Na+ (100 mM).

• The increase in binding is due to an increase in specific binding.

EVALUATION

Data are converted into % stereospecific3H-naloxone binding displaced by the test drug. IC50 values are determined from computer-derived analysis.

The sodium shift is calculated from IC50 values with and without NaCl. High sodium shifts are found with agonists, low values with antagonists and medium values with mixed agonists-antagonists.

IN VIVO TESTS

1) HAFFNER’s tail clip method

PURPOSE AND RATIONALE

The raised tail (Straub phenomenon)

in mice treated with morphine or

similar opioid drugs and found the tail

after drug treatment to be less

sensitive to noxious stimuli.

PROCEDURE

The animals were divided into four groups of sixmice. An artery clip is applied to the root of thetail of mice and the reaction time is noted. Thetest compounds are administered orally to fastedanimals.

The drug is administered 15, 30 or 60 min priortesting. An artery clip is applied to the root of thetail (approximately 1 cm from the body) to inducepain.

The animal quickly responds to this noxiousstimuli by biting the clip or the tail near thelocation of the clip. The time between stimulationonset and response is measured by a stopwatch.

2) EDDY’S HOT PLATE METHOD

PROCEDURE

The animals were divided into four

groups of six mice. They are placed on

Eddy’s hot plate heated to 55°C.

The animal responds by either jumping

or licking the paw. The response time

prior to and after 30, 60, 120 minutes

are taken. The cut off time is 15

seconds.

3) TAIL IMMERSION

METHODPROCEDURE

The animals are divided into four groups

of six mice. The tail is immersed into hot

water of 55°C.

Within a few seconds, the mouse

responds by withdrawing its tail. The

reaction time is noted prior to and after

30, 60, 120, 180 minutes of drug

administration

REFERENCES

H. Gerhard Vogel, Drug Discovery and

Evaluation, Pharmacological Assays,

Second edition, Pages 385-693.