SCREENING MODELS FOR CENTRAL AND PERIPHERAL ANALGESICS

by Bajgire Krushna bALIRAMDepartment of Pharmacology and Toxicology

National Institute of Pharmaceutical Education and ResearchMohali, 160062

FLOW OF PRESENTATION :

1. INTRODUCTION2. CLASSIFICATION3. MECHANISM OF ACTION4. SCREENING MODELS

CENTRAL ANALGESIC ACTIVITY

PERIPHERAL ANALGESIC ACTIVITY

INTRODUCTION

Pain is a symptom of many diseases which requires

analgesic treatment

Analgesics is defined as the are agents which selectively

relieve pain by acting in the CNS or by peripheral pain mechanisms

CLASSIFICATION OF ANALGESICS

Analgesics

Narcotics

Ex. Morphine, Codeine,

Pethidine, Dihydromorphine

, Diacetylmorphine

Non-narcotics

Ex. Aspirin Paracetamol,

Ibuprofen, Ketoprofen, Diclofenac,

Piroxicam

MECHANISM OF ACTION

Analgesic drugs act in various ways on the peripheral and central nervous system 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 thromboxane's Inhibition of COX1 and COX-2 leads to the anti- inflammatory, analgesic and antipyretic effects

SCREENING

MODELS

CENTRAL ANALGESIC ACTIVITY

1. IN VITRO MODELS: * 3H-Naloxone binding assay * 3H-Dihydromorphine binding to opiate𝜇 receptors in rat brain * Receptor binding of nociceptin * Bioassays for nociception * Receptor binding of cannabinoids * Vanilloid receptor binding

2.IN VIVO MODELS:

Haffner’s Tail Clip Method Hot - plate test Electrical stimulation of the tail Grid - shock test Formalin test in rats Chemotherapy-Induced Pain Spinal Cord Injury Radiant heat method Tail immersion test

Purpose and rationale: * 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

Hot plate method :

Procedure :

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

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.

Evaluation :

The prolongation of latency time between 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

Procedure :Male mice (18-20g) are selected and placed

individually in plastic chamber.

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.

Tail immersion test :

PURPOSE AND RATIONAL :

* The method has been developed to be selective for morphine-like compounds

* The procedure is based on the observation that morphine- like drugs are capable of prolonging the reaction time of the typical tail withdrawal reflex in rats induced by immersing end of tail in warm water at 55°C

Procedure :

Young female wistar rats ( 170-210 g) are placed into individual restraining cages leaving the tail hanging out freely

The animals are allowed to adapt to the cages 30 minutes prior to testing

The lower 5 cm portion of tail is marked and this portion is then immersed in a cup of freshly filled water of exactly 55°C

Within a few seconds the rat reacts by withdrawing the tail. The reaction time is recorded

The reaction time is determined before and periodically after either oral or subcutaneous administration of the test substance

Evaluation :

ED50 values can be calculated for each compound and time response curves (onset, peak and duration of the effect) be measured

All the morphine-like analgesics have been shown to be active at doses which do not produce gross behavioral changes

PERIPHERAL ANALGESIC ACTIVITY

Writhing test

Pain in Inflamed Tissue (RANDALL-SELITTO-Test)

Mechanical Visceral Pain Model in the Rat

Antagonism to Nerve Growth Factor

Effect of Analgesics on Spinal Neurons

Antagonism Against Local Effects of Bradykinin

Writhing test : 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

Procedure:

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

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

Pain in Inflamed Tissue (RANDALL- SELITTO-Test) :

This method for measuring analgesic activity is based on the principle that inflammation increases the sensitivity to pain and that this sensitivity is susceptible to modification by analgesics

Inflammation decreases the pain reaction threshold and this low pain reaction threshold is readily elevated by non-narcotic analgesics of the salicylate-amidopyrine type as well as by the narcotic analgesics

Purpose and rationale :

Procedure :

For a time response, groups of at least 7 animals are used, four groups for the agent to be tested and one for the vehicle control

Groups of male Wistar rats (130 to 175 g) are used

0.1ml of a 20% suspension of Brewer’s yeast in distilled water is injected subcutaneously into the plantar surface of the left hind paw of the rat to induce

inflammation

Three hours later, pressure is applied through a tip to the plantar surface of the rat’s foot at a constant rate by a special apparatus to the point at which the

animal struggles, squeals or attempts to bite

The tests are done at 15 min interval after subcutaneous administration and at 30 min intervals after oral administration for any change in

pain threshold

Evaluation :

The mean applied force is determined for each time interval tested

The percentage increase in pain threshold is calculated by subtracting the applied force of the vehicle control from the applied force of the drug group which is divided by the applied force of the vehicle control in order to give the percentage of increase in pain threshold of the drug group

The interval of time which indicates the greatest increase in pain threshold is regarded as the peak time