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Screening of analgesics

This document describes various in vivo and in vitro screening methods used to evaluate potential analgesic compounds. Some key in vivo methods include the tail flick test, hot plate test, and acetic acid-induced writhing test, which assess analgesic effects using thermal, mechanical, and chemical pain stimuli, respectively. Important in vitro assays involve measuring the inhibition of nociceptin, binding to opioid receptors, and inhibition of enkephalinase enzyme. The screening methods aim to distinguish between narcotic and non-narcotic analgesics and help identify new compounds for treating different pain states.

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SCREENING OF ANALGESICS Prepared by: ASMA M A First M. Pharm Pharmacology
INTRODUCTION • Pain is an unpleasant sensory and emotional experience associated with actual and potential tissue damage. • Various types of pains seen in humans are: • Somatic pain (arising from skin, muscles, joints, ligaments and bones) • Visceral pain • Referred pain • Neuropathic pain • Cancer pain etc.
• The pain sensing neurons “nociceptors” are stimulated by physical (heat, cold and pressure) and chemical stimuli. • Pain can be classified as acute or chronic, • Acute pain: it is of soft tissue damage, infection or inflammation and will be short of duration. • Chronic pain: it lasts for 6 months or larger than that period (E.g. cancer pain, neuropathic pain and arthritic pain). • Analgesics may be Narcotic or Non-narcotic.
Narcotic Analgesics Non-Narcotic Analgesics Act centrally Act peripherally Cause addiction Do not cause addiction Produce CNS depression Do not produce CNS depression Do not produce gastric irritation Produce gastric irritation Show no anti-inflammatory effect Show anti-inflammatory effect e.g. Morphine, Tramadol Pethidine etc. e.g. Diclofenac, Ibuprofen, Aspirin etc.
SCREENING METHODS • Screening of analgesic agents include - • IN-VIVO METHOD A) Pain-state models using Thermal stimuli i. The tail-flick model using radiant heat/ Immersion of the tail in hot water. ii. Paw-withdrawal test. iii. Hot-plate test. iv. Pain-state models using Cold- stimuli
B) Pain-state models using Mechanical stimuli i. Strain gauges. ii. Von-Frey filaments. iii. Inclined-plane test. C) Pain-state models using Electrical stimuli i. Electrical stimulation of the tail. ii. Grid-shock test. iii. Stimulation of the tooth pulp. iv. Monkey-shock titration test. v. Stimulation of the limbs.
D) Pain-state models using Chemical stimuli i. Formalin test. ii. Acetic acid induced writhing test. iii. Stimulation of hollow organs. • IN-VITRO METHOD A. Bioassay of nociceptin. B. Recetptor binding of nociceptin. C. 3H- naloxone binding assay. D. 3H- bremazocine binding assay. E. Cannabinoids binding assay. F. Inhibition of Enkephalinase.
PAIN-STATE MODELS USING THERMAL STIMULI 1. THE TAIL-FLICK MODELS (using radiant heat) AIM • Tail flick method is widely and reliably used for the testing of opioid analgesics. REQUIREMENTS Animals required: Albino mice (18-22g) Chemicals required: Test compound Equipment required: cages leaving tail exposed out
PRINCIPLE AND ROCEDURE • The application of thermal radiation to the tail of an animal provokes the withdrawal of the tail by a brief vigorous movement. • The withdraw of the tail from the heat source is referred to as tail-flick latency. • In this model a timer is started at the same time as the application of the heat source and time taken by the rat to withdraw its tail is recorded. • Usually withdrawal time is within 2 to 10s. • The lengthening of this reaction time by the animal seen after the administration of a drug is interpreted as an analgesic action.
• It is advisable not to prolong the exposure to radiant heat beyond 20s because the skin of the tail may be burnt. • A rheostat is inserted in the apparatus so as to control the intensity of the current passing through the filament, which further controls the intensity of radiant heat. • This test is more sensitive to morphine. EVALUATION • At each time interval those animals that show higher reaction time than the time before drug administration are regarded as positive. • Percentage of positive animals are counted for each time interval and each dose and ED50 values of test compound can be calculated
MERITS • This method is very effective for screening morphine- like analgesics. • The technique is simple and does not require any special skill. • The results of experiments are quite accurate and less time consuming. DEMERITS • The tail-flick response is prone to habituation. • The tail- flick response is not consistent upon repetitive stimulation. • Habituation is observed with a shortening of the inter- stimulus interval and increased intensity of heat. • This test is truly efficient only for revealing the activity of opioid analgesics (but not of opioid partial agonists.
2. HOT PLATE MODEL AIM • Hot plate method has been widely used to evaluate opioid analgesics. REQUIREMENTS Animals required: Albino mice Chemicals required: Test compound Equipment requirement: Hot plate at 55-66 C (electrically heated late)
PRINCIPLE AND ROCEDURE • This test consists of introducing a rat or mouse into an open-ended cylindrical space with a floor consisting of a metallic plate that is heated by a thermode or a boiling liquid. • A plate heated to a constant temperature produces two behavioural components that can be measured in terms of their reaction times, namely paw licking and jumping. • Animals are placed on the hot plate, which consist of electrically heated surface. • Temperature on the hot plate is maintained at 55-56 degree celcius. • Responses such as jumping ,withdrawal of the paws and licking of the paws are seen.
• Both are considered to be supra-spinally integrated responses. • The time period (latency period), when animals are placed and until responses occur, is recorded by a stopwatch. • After administration of analgesic substances, the paw licking behavior time and the jumping reaction time is increased. EVALUATION • Test compounds are administered orally or subcutaneously and latency or latency period is recorded after 20,60.90 min. • These values are compared with the values before administration of the drug b using T-test.
DEMERIT • The sedatives, muscle relaxants or psychomimetics yield false positive results, while partial opiate agonist- antagonists produce unreliable results.
PAIN- STATE MODELS USING ELECTRICAL STIMULI 3. TOOTH PULP TEST AIM • This test is used of the screening of analgesics using electrical stimuli. REQUIREMENTS Animals required: Rabbits (2-3 kg) Chemicals required: Thiopental(15 mg /kg) Equipment required: Dental drill tooth pulp, clamping electrodes
PRINCIPLE AND PROCEDURE • This method is based on the stimulation of the tooth-pulp of the animal by applying electric current. • Stimulation of the tooth-pulp produces characteristic reactions such as licking, biting, chewing and head flick due to induction of pain, which can be observed easily. • Rabbits of either sex are anaesthetized with 15 mg/kg thiopental or 0.2 mg/kg fentanyl-citrate i.v. • Pulp chambers are exposed close to the gingival line in the lateral margins of the two front upper incisors with a high- speed dental drill. • On the day of the experiment, clamping electrodes are placed into the drilled holes.
• After an accommodation period of 30min, electrical stimulation is started to determine the threshold value. • The stimulus is applied by rectangular current with a frequency of 50 Hz for duration of 1 s. • The electrical current is started with 0.2 mA and increased until the phenomenon of licking occurs. • In some cases, the current has to be increased and then decreased again in order to find the appropriate threshold. EVALUATION • Test compound is administered orally or i.v . • After 15,30 60 and 120 min threshold current is measured and compared with the threshold current prior to drug administration.
PAIN-STATE MODELS USING CHEMICAL STIMULI 4. ACETIC ACID INDUCED WRITHING TEST AIM • This test is used to screen analgesics by using chemical stimulus. REQUIREMENTS Animals required: Albino mice Chemicals required: Acetic acid 0.7% v/v (1ml/100g) Aspirin (100mg/kg)
PRINCIPLE AND PROCEDURE • Pain is often induced in rats or mice by injecting certain irritants such as phenyl quinone or acetic acid into the peritoneal cavity. • The animal reacts with a characteristic stretching behavior which is called writhing. • The intra-peritoneal administration of agents that irritate serous membranes elicits a stereotyped behavior in the mouse/rat, which is characterized by abdominal contractions, movements of the body as a whole (particularly of the hind paws), twisting of dorso-abdominal muscles, and motor in- coordination. • Divide the animals into 2 groups each consisting of 5 animals. • The first group serves as control and the second group is the test group
• Administer the appropriate volume of acetic acid solution to the first group and place individually under glass jar for observation. • Note the onset of writhing and number of writhing for a period of 10 minutes. • Administer Aspirin 100mg/kg orally to the second group of animals and 30 minutes later administer acetic acid solution. • Note the onset and number of writhing responses. EVALUATION • Calculating the mean writhing response of control and test group. • From this value the percentage protection of analgesics can be calculated.
INVITRO METHOD 1. BIOASSAY OF NOCICEPTIN AIM • Nociceptin receptors in the periphery can be characterized by studies in isolated organs such as the guinea pig ileum, the mouse vas deferens, the rabbit vas deferens, the guinea pig renal pelvis. REQUIREMENTS Tissue : Guinea pig ileum, mouse vas deferens, rabbit vas deferens, guinea pig renal pelvis. Physiological salt solution: Kreb’s solution.
PROCEDURE • Take the tissues from male Swiss mice, guinea pigs, Sprague Dawley rats & New Zealand albino rabbit. • Suspend in 10 ml organ baths containing Krebs solution oxygenated with 95% O2 & 5% CO2. • Set the temperature around 33°-37°C & apply 0.3-1g of resting tension. • Stimulate the tissue with two platinum ring electrodes. • Measure the electrically evoked contractions isotonically with a strain gauge transducer and record on a multichannel chart recorder.
• After equilibration period of about 60 min the contractions induced by electrical field stimulation become stable; at this time, perform the cumulative concentration response curves to nociceptin or opioid peptides. • Perform four electrical field stimulation with each tissue at 30 min intervals. • Add Agonists & Antagonists 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 • Data are expressed as means of n experiments and statistically analyzed and recorded.
2. 3H- NALOXONE BINDING ASSAY AIM This test is used for the study of opiate agonist and antagonist. REQUIREMENTS Animal required: Wistar rat Physiological salt solution: Tris buffer
PROCEDURE • Decapitate the male Wistar rat and rapidly remove their brain. • Weigh whole brain except cerebella and homogenize in 50ml of 0.05M Tris buffer with a Tekmar tissue homogenizer. • Centrifuge the homogenate for 15 minutes, decant the supernatant and re-suspend the pellet in fresh buffer and centrifuge. • Then suspend the final pellet in original volume of fresh 0.05M Tris buffer. • Incubate the tubes for 30 minutes at 37 degree Celsius.
• The assay is stopped by vacuum filtration through Whatman GF filters which are then washed three times with ice-cold 0.05M Tris buffer of pH 7.7. • The filters are then counted in 10ml of Liquiscint liquid scintillation cocktail. • Stereospecific binding is defined as the difference between binding in presence of 0.1μM dextrorphan and 0.1μM levorphanol. EVALUATION • Data are converted into % stereospecific naloxone binding displaced by the test drug. • Data can be analyzed by using a computer program as described by McPherson in 1985.
3. INHIBITION OF ENKEPHALINASE AIM • It is found that the enkephalinase inhibitor thiorphan shows anti-nociceptive activity in mice. • So inhibition of enkephalinase is used as the invitro method for the screening of analgesics. REQUIREMENTS Animal required: Rat Physiological salt solution: Tris-HCl buffer
PROCEDURE • Fresh rat kidney is homogenized in 10 vol of cold 0.05M Tris-HCl buffer of pH 7.4 using a Polytron homogenizer. • The homogenate is centrifuged for 5 minutes. • The pellet is discarded and the supernatant centrifuged for 60 minutes. • The resulting pellet is resuspended in 50mM Tris-HCl buffer and used as the enzyme source . • The assay is carried at 37 C in hemolysis tubes. • A 0.1ml amount of 50mM Tris-HCl buffer is preincubated 15 minutes at 37 C. • The reaction is initiated by addition of 50μl of the enzyme preparation together with 0.5 μM Captopril.
• The tubes are incubated for 30 minutes in a water bath with constant shaking • The enzymatic reaction is stopped by boiling at 100 C for 5 minutes. • The samples are then diluted with 1.35ml of Tris-HCl buffer and centrifuged for 30 minutes. • 1ml of supernatant is transferred to thermostat cells of a spectrofluorometer. • Reading are performed at 562nm with an excitation wavelength of 342nm. • A calibration curve is prepared. EVALUATION • The inhibitory potencies of test compounds are compared with the standard.