15screening model of analgesic

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Evaluation of Analgesic Drugs Guided by: Dr. Pankaj Tripathi M.Pharm, Ph.D Vishal Patel M.Pharm,Sem II Department of Pharmacology S.K. Patel College of Pharmaceutical Education and Research Ganpat Vidhyanagar

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“ Pain is an unpleasant sensory or emotional experience associated with actual or potential tissue damage” PAIN Types of Pain 1.Acute pain : It is temporary; instantaneous it may be treated by drug e.g. headache 2 chronic pain: Continuous and gradual pain is referred as chronic pain.e.g. colic. 3.Psychogenic pain: no anatomic or physiologic reason is available and relief may be obtain by the use of the placebo or sedative

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* CLASSIFICATION OF NARCOTIC AND NON NARCOTIC ANALGESIC DRUGS: * NARCOTIC ANALGESIC phenanthren derivatives : morphin , codeine, levorphenol , oxymorphin , oxycodone etc. phenylpiperidin : meperidin , fentanayl , etc diphenylheptan : methadone , propoxyphene * NON NARCOTIC ANALGESIC salicylate and their derivate; aspirin pyrazolon derivative; phenylbutazone Indol and related drugs; indomethacin , sulindac arulacetic acid derivative : diclofenac , ketorolac propionic acid derivatives : ibuprofen ; naproxen, pirofen . fenamates : mefenamic acid oxicam : piroxicam sulphonamide : nimesulide Analgesics

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Methods for evaluation In vivo methods In vitro methods Thermal methods Electrical methods Mechanical method Chemical method 3 H-Naloxone binding assay 3 H-Dihydromorphine binding to µ opiate receptors in rat brain 3 H-Bremazocine binding to κ opiate receptors in guinea pig cerebellum Isolated tissue preparation

Classification of methods according to activity:

Classification of methods according to activity Method for central analgesic agents Hot plate method Tail immersion method Radiant heat method Tail clip method Tooth pulp stimulation Pododolorimeter Rectodolorimeter Method for peripheral analgesic agents Writhing method Formalin test in rats Pain in inflamed tissue (RANDALL-SELITTO-test) Mechanical visceral pain model in the rat Pododolorimeter Rectodolorimeter

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In vivo techniques Principle Pain is induced to a suitable animal and the response of the animal to the painful stimuli is recorded before and after administration. Thermal methods Hot plate method Tail immersion method Radiant heat method Mechanical method Tail Clip Method Electrical methods Pododolorimeter Rectodolorimeter Tooth pulp stimulation Chemical method Writhing method

In vivo methods:

In vivo methods Criteria for selection of methods: Methods must permits quantitative determination of threshold value of nociceptive stimulus. It should yield quantitative information on difference in intensity of stimuli. Applicable to both animal and man .

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Material Animal: Rats or mice Instrument: Hot plate analgesiometer Painful stimulus: Heat (55 ± 1 °C) Drug used: Morphine Hot – plate Purpose and rationale Evaluation of opioid analgesic Stimulus is easy to control & applied to moving subject To determine Analgesic potency ,Analgesic peak time and Duration of analgesic drug.

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The method originally described by Woolfe and Mac Groups of 10 mice of either sex with an initial weight of 18 to 22 g are used for each dose. The hot plate, which is commercially available, consists of a electrically heated surface. The temperature is controlled for 55° to 56 °C. This can be achieve by a copper plate or a heated glass surface. The animals are placed on the hot plate and the time until either licking or jumping occurs is recorded by a stop-watch. The latency is recorded before and after 20, 60 and 90 min following oral or subcutaneous administration of the standard or the test compound

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Response observed - licking of fore paw and/or hind paws, jumping. Evaluation (Thompson,1990) The prolongation of the latency times comparing the values before and after administration of the test compounds or the values of the control with the experimental groups can be used for statistical comparison using the t -test. Find out the % maximum possible effect and find out ED 50 { post drug latency-pre drug latency} MPE = {cut off time(60s) -pre drug latency} ED 50 : Plot graph of %Maximum Possible Effect (MPE) Vs Dose Peak analgesic time: Plot graph of mean group latency (s) Vs time (min) Duration of analgesic action: Plot graph of mean group latency (s) Vs time (min) and find out AUC

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Tail immersion technique Purpose and rationale Evaluation of centrally acting analgesic drug To determine Analgesic potency ,Analgesic peak time and Duration of analgesic drug. In contrast to hot plate, this test is useful in repeated nociceptive evaluation using the same animal Material Animal: Rat Instrument: Tail-flick analgesiometer Painful stimulus: Hot water(55±1°C) Drug used: Morphine

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Young female Wistar rats (170 – 210 g body weight) are used. They are placed into individual restraining cages leaving the tail hanging out freely. The animals are allowed to adapt to the cages for 30 min before testing. The lower 5 cm portion of the tail is marked. This part of the tail is 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 by a stopwatch. Response observed – Violent jerk of tail , tail flicking.

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Modification of Method- Pizziketti et al. (1985) modified the tail immersion test in rats in this way that they used a 1 : 1 mixture of ethylene-glycol and water cooled to a temperature of minus 10 °C as noxious stimulus Evaluation ED 50 values can be calculated for each compound and time response curves (onset, peak and duration of the effect) be measured.

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Radiant heat method Purpose and rationale The test is very useful centrally acting morphine-like analgesics. To determine Analgesic potency ,Analgesic peak time and Duration of analgesic drug. Response observed  Flicking of tail . Critical assessment of the test The radiant heat test on the tail of mice is very effective to estimate the efficacy and potency of central acting analgesic drugs.

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Procedure The method was described by Lindner and Vogel (1963) as a modification of earlier publications Groups of 10 mice of both sexes with a weight between 18 and 22 g are used for each dose. Before administration of the test compound or the standard the normal reaction time is determined . The animal is put into a small cage with an opening for the tail at the rear wall. The tail is held gently by the investigator. By opening of a shutter, a light beam exerting radiant heat is directed to the tail. For about 6 s the reaction of the animal is observed by the investigator. The mouse tries to pull the tail away and turns the head. With a switch the shutter is closed as soon as the investigator notices this reaction. Mice with a reaction time of more than 6 s are not used in the test.

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In contrast, the simple tail flick as an endpoint of this test may be mediated as a spinal reflex. Therefore the observation of the escape reaction can be regarded as a true assessment of the influence of the drug on the brain. The test compounds and the standard are administered either orally or subcutaneously. The animals are submitted to the same testing procedure after 30, 60 and eventually 120 min. For each individual animal the reaction time is noted. Other time intervals can be used according to the question to be investigated Evaluation Calculate the % maximum possible effect and find out ED 50

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Tail clip method Purpose and rationale The tail in mice treated with morphine or similar opioid drugs and found the tail after drug treatment to be less sensitive to noxious stimuli. To described the high sensitivity of this method to morphine .

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PROCEDURE An artery clip is applied to the root of the tail of mice and the reaction time is noted. Male mice with a weight between 18 and 25 g are used. The control group consists of 10 mice.The test compounds are administered subcutaneously to fed mice or orally to fasted animals. The test groups and the control group consist of 7 – 10 mice. The drug is administered 15, 30 or 60 min prior testing. An artery clip is applied to the root of the tail (approximately1cm from the body) to induce pain. The animal quickly responds to this noxious stimuli by biting the clip or the tail near the location of the clip. The time between stimulation onset and response is measured by a stopwatch .

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Modifications of the method ( Bartoszyk and Wild, 1989) described a modification of the original Haffner clip test using pressure on the tail of rats instead of mice. Additionally hyperalgesia was induced by injection of carrageenan suspension into the tail. Response observed  biting the clip or the tail near the location of the clip.

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Writhing test Writhing : Stretching of the body, withdrawing of the limb, retraction of the abdomen & the stomach touches the ground Material Animal: Mice Painful stimulus: Chemicals e.g. acetic acid Drug used: NSAID e.g. Na salicylate Principle: The painful stimulus is induced by IP injection of an irritant substance (e.g. acetic acid) Response observed  writhing

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Procedure Mice of either sex with a weight between 20 and 25 g are used. Acetic acid in a concentration of 0.6% is in 1ml/100g body weight. 0.25 ml of this suspension is injected intraperitoneally . Groups of 6 animals are used for controls and treated mice. Preferably, two groups of 6 mice are used as controls. Test animals are administered the drug pretreatment times prior to acetic acid administration. The mice are placed individually into glass beakers and five min are allowed to elapse. The mice are then observed for a period of ten min and the number of writhes is recorded for each animal.

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For scoring purposes, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. The formula for computing percent inhibition is: average writhes in the control group minus writhes in the drug group divided by writhes in the control group The time period with the greatest percent of inhibition is considered the peak time. A dose range is reserved for interesting compounds or those, which inhibit writhing more than 70%. Compounds with less than 70% inhibition are considered to have minimal activity. Evaluation : The drugs are evaluated for their % inhibition in writhing and for peak time that gives highest analgesic activity.

In vitro methods:

In vitro methods 3 H-Dihydromorphine binding to µ opiate receptors in rat brain 3 H-Naloxone binding assay 3 H-Bremazocine binding to κ o piate receptors in guinea pig cerebellum

3H-Dihydromorphine binding to µ receptors :

3 H-Dihydromorphine binding to µ receptors Purpose and rationale µ Receptors are considered to mediate the supraspinal activity of opioids. 3 H-Dihydromorphine ( 3 H-DHM) exhibits some selectivity for the µ receptor, a high affinity opiate binding site. Tissue preparation Male Wistar rats are sacrificed brains are removed, weighed and homogenized in 30 volumes of ice-cold 0.05 M Tris buffer, pH 7.7. The homogenate is centrifuged at 48 000 g for 15 min, the supernatant is decanted and the pellet resuspended in the same volume of buffer. This homogenate is then incubated for 30 min at 37 °C to remove the endogenous opiate peptides and centrifuged again as before. The final pellet is resuspended in50 volumes of 0.05 M Tris buffer, pH 7.7 ..

3H-Dihydromorphine binding to µ receptors:

3 H-Dihydromorphine binding to µ receptors Assay 1 850 µl tissue suspension 80 µl distilled water 20 µl vehicle, or levallorphan, or appropriate concentration of drug 50 µl [3H]DHM. Tubes are incubated for 30 min at 25 °C. The assay is stopped by vacuum filtration through Whatman filters which are washed twice with 5 ml of 0.05 M Tris buffer. The filters are then placed into scintillation vials with 10 ml Liquiscint scintillation cocktail and counted. Evaluation Specific binding is defined as the difference between total binding and binding in the presence of 0.1 mM levallorphan. IC 50 values are calculated from the percent specific binding at each drug concentration. Determined from computer-derived log-probit analysis

3H Naloxone binding assay :

3 H Naloxone binding assay Purpose and rationale A good correlation between the in vivo pharmacological potency of opiate agonists and antagonists with their ability to displace radio labeled naloxone has been reported. Na+ (100 mM) enhances the binding of antagonists and reduces the binding of agonists. By determining the IC50 values for 3H-Naloxone in the presence or absence of Na+ used to classify compounds as opiate agonists and antagonists. Procedure Reagents Naloxone Levorphanol tartrate  to determine non-specific binding Dextrorphan tartrate  total binding Test compound

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Tissue preparation Rats brains rapidly removed. Whole brains minus cerebella are weighed and homogenized in 50 volumes of ice-cold 0.05 M Tris buffer with a Tekmar tissue homogenizer. The homogenate is centrifuged at 40 000 g for 15 min, the supernatant is decanted and the pellet resuspended in fresh buffer and recentrifuged at 40 000 g. The final pellet is resuspended in the original volume of fresh 0.05 M Tris buffer. Assay 310 µl H2O 20 µl 5 µM dextrorphan (total binding) or 5 µM levorphanol (non-specific binding) 50 µl 2 M NaCl or H2O 50 µl 0.5 M Tris buffer, pH 7.7 20 µl drug or vehicle 50 µl 3H-naloxone 500 µl tissue suspension

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The tubes are incubated for 30 min at 37 °C. The assay is stopped by vacuum filtration through Whatman filter which are then washed 3 times with icecold 0.05 M Tris buffer, pH 7,7. The filters are then counted in 10 ml of Liquiscint liquid scintillation cocktail. Evaluation: Data are converted into % stereospecific 3H-naloxone binding displaced by the test drug. IC50 values are determined from computer-derived log- probit analysis. Data can be analyzed using a computer program as described by McPhers on

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