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. INTRODUCTION 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 CLASSIFICATION Analgesics are classified as : Narcotics Ex. Morphine, Pethadine, Fentanyl Non-narcotics Ex. NSAID MECHANISM OF ACTION 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. SCREENING MODELS IN VIVO MODELS 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 VITRO 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 VIVO MODELS HOT – PLATE 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, whereas peripheral analgesics of the acetylsalicylic acid or phenylacetic acid type do not generally affect these 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. Procedure Wistar rats (170-210g) are selected and divided into standard, test & control group respectively temperature is Appropriate 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 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 The second). 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. 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. 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 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 ele The electrically evoked contractions are measured isotonically with a strain gauge transducer and recorded on a 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. Contractile responses to electrical field stimulation are expressed as % increment to the spontaneous activity of tissue. effects of the application of agonists or Thethe biological antagonists are expressed as % inhibition of electrical filed stimulation-induced contraction. Evaluation The agonists and antagonists potencies are recorded and expressed as means of ±SEM. REFERENCES Drug screening methods – SK Gupta. Essentials of Medical Pharmacology - Tripathi K.D. Drug Discovery and Evaluation - H. Gerhard Vogel. International Research Journal Of Pharmacy.
