Experimental models for Antipyretic, analgesic and anti inflammatory activity

Evaluation of Antipyretic analgesic and anti inflammatory activity

1. Experimental Models For The Evaluation Of Antipyretic, Analgesic And Anti-Inflammatory Activity By : Dr. Anand P. V. 2 nd Year Pg Scholor Dept. Of Dravyaguna Under The Guidance Of Dr.Shashidhar Naik Dr.Darshan Dhari Dr. Vidya Pujari Dr. Gurudevi Biradar

2. Antipyretic activity General considerations Pyrexia, also known as fever, is an increase in the body temperature of an individual beyond the normal range. An antipyretic is a substance that reduces fever. Antipyretics are the agents which reduce the elevated body temperature. Yeast-induced pyrexia is called pathogenic fever and its etiology involves production of prostaglandins, which set the thermoregulatory centre at a lower temperature.  For anti-inflammatory compounds, an antipyretic activity is regarded as a positive side effect. To evaluate these properties, fever is induced in rabbits or rats by injection of lipo polysaccharides or Brewer’s yeast.

3. Antipyretic testing in rats PURPOSE AND RATIONALE The subcutaneous injection of Brewer’s yeast suspension is known to produce fever in rats. A decrease in temperature can be achieved by administration of compounds with antipyretic activity. PROCEDURE A 15% suspension of Brewer’s yeast in 0.9% saline is prepared. Groups of 6 male or female Wistar rats with a body weight of 150 g are used. By insertion of a thermocouple to a depth of 2 cm into the rectum the initial rectal temperatures are recorded. The animals are fevered by injection of 10 ml/kg of Brewer’s yeast suspension subcutaneously in the back below the nape of the neck. The site of injection is massaged in order to spread the suspension beneath the skin. The room temperature is kept at 22–24 °C. Immediately after yeast administration, food is withdrawn. 18 h post challenge, the rise in rectal temperature is recorded. The measurement is repeated after 30 min. Only animals with a body temperature of at least 38 °C are taken into the test. The animals receive the test compound or the standard drug by oral administration. Rectal temperatures are recorded again 30, 60, 120 and 180 min post dosing EVALUATION The differences between the actual values and the starting values are registered for each time interval. The maximum reduction in rectal temperature in comparison to the control group is calculated. The results are compared with the effect of standard drugs, e.g. Paracetamol

4. Antipyretic testing in rabbits PURPOSE AND RATIONALE Lipo polysaccharides from Gram-negative bacteria, e.g. E. coli, induce fever in rabbits after intravenous injection. Only lipo polysaccharide fractions are suitable which cause after 60 min an increase of body temperature of 1 °C or more at a dose between 0.1 and 0.2 µg/ kg. In the rabbit 2 maxima of temperature increases are observed. The first maximum occurs after 70 min, the second after 3 h. PROCEDURE Rabbits of both sexes and of various strains with a body weight between 3 and 5 kg can be used. The animals are placed into suitable cages and thermocouples connected with an automatic recorder are introduced into the rectum. The animals are allowed to adapt to the cages for 60 min. Then 0.2 ml/kg containing 0.2 µg lipo polysaccharide are injected intravenously into the rabbit ear. Sixty min later the test compound is administered either subcutaneously or orally. Body temperature is monitored for at least 3 h. EVALUATION A decrease of body temperature for at least 0.5 °C for more than 30 min as compared with the temperature value before administration of the test compound is regarded as positive effect. This result has been found after 45 mg/kg phenylbutazone s.c . or 2.5 mg/kg indomethacin s.c .

5. ANALGESIC ACTIVITY Pain is an unpleasant sensory and emotional experience associated with actual and potential tissue damage An analgesic or painkiller is any member of the group of drugs used to achieve analgesia - relief from pain. Analgesics is defined as the agents which selectively relieve pain by acting in the CNS or by peripheral pain mechanisms without significantly altering consciousness

6. Analgesic Activity Although the in vivo methods have been used more extensively in the past, they are still necessary in present research analgesic tests in animals before a compound can be given to man. Mostly, rodents, such as mice or rats, are used for analgesic tests, but in some instances experiments in higher animals such as monkeys are necessary. Several methods are available for testing central analgesic activity, such as • HAFFNER’s tail clip method in mice, • tail immersion tests, • hot plate methods in mice or rats, • electrical stimulation (grid shock, stimulation of tooth pulp or tail), • monkey shock titration, • formalin test in rats.

7. • HAFFNER’s tail clip method in mice PURPOSE AND RATIONALE The method was described as early as 1929 by Haffner who observed 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. He already described the high sensitivity of this method to morphine. Procedure An artery clip is applied to the root of the tail of mice and the reaction time is noted. Male mice (Charles River strain or other strains) 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.  An artery clip is applied to the root of the tail (approximately 1 cm 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 .

8. A cut-off time is determined by taking the average reaction time. Any reaction time of the test animals which is greater than the cut-off time is called a positive response indicative of analgesic activity. The length of time until response indicates the period of greatest activity after dosing. An ED50 value is calculated at the peak time of drug activity. ED50 values found by this method were 1.5 mg/kg s.c . for morphine and 7,5 mg/kg for codeine s.c . EVALUATION There are two possibilities for evaluation: • The average values of reaction time after each time interval are calculated and compared with the pretest value by analysis of significance. • At each time interval only those animals which show a reaction time twice as high or higher as the pretest value are regarded as positive. Percentages of positive animals are counted for each time interval and each dose and ED50 values are calculated according to LITCHFIELD and WILCOXON. As standards codeine, pethidine and morphine can be used. The ED50 values of these drugs are • Codeine 12 mg/kg s.c . • Pethidine 12 mg/kg s.c . • Morphine 2 mg/kg s.c .

9. Hot plate method PURPOSE AND RATIONALE The paws of mice and rats are very sensitive to heat at temperatures which are not damaging the skin. The responses are jumping, withdrawal of the paws and licking of the paws. The time until these responses occur is prolonged after administration of centrally acting analgesics, whereas peripheral analgesics of the acetylsalicylic acid or phenyl-acetic acid type do not generally affect these responses. PROCEDURE The method originally described by Woolfe and Mac Donald (1944). 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 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.

10. EVALUATION 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. Alternatively, the values which exceed the value before administration for 50% or 100% can be regarded as positive and ED50 values can be calculated. Doses of 7.5 mg/kg s.c . morphine hydrochloride, 30 mg/kg s.c . codeine hydrochloride, 30 mg/kg s.c . pethidine hydrochloride and 400 mg/kg s.c . phenazone were found to be effective, whereas aspirin showed no effect even at high doses.

11. Tail immersion test PURPOSE AND RATIONALE The method has been developed to be selective for morphine-like compounds. The procedure is based on the observation that morphine-like drugs are selectively capable of prolonging the reaction time of the typical tail-withdrawal reflex in rats induced by immersing the end of the tail in warm water of 55 °C. PROCEDURE 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 in 0.5 s units by a stopwatch. After each determination the tail is carefully dried. The reaction time is determined before and periodically after either oral or subcutaneous administration of the test substance, e.g., after 0.5, 1, 2, 3, 4 and 6 h. The cut off time of the immersion is 15 s. The withdrawal time of untreated animals is between 1 and 5.5 s. A withdrawal time of more than 6 s therefore is regarded as a positive response.

12. 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. For example, an ED50 of 3.5 mg/kg s.c . for morphine and an ED50 of 1.7 mg/kg s.c . methadone was found. Acetylsalicylic acid at a dose of 640 mg/kg p.o ., phenylbutazone at a dose of 160 mg/kg s.c . as well as nalorphine at a dose of 40 mg/kg s.c . were inactive

13. Monkey shock titration test PURPOSE AND RATIONALE Generally, analgesic tests in rats and mice result in correlation with the analgesic activity of a drug in man. To clarify the mode of action in more detail and to find a suitable dose for therapy in man, experiments in monkeys may be necessary. PROCEDURE This test has been recommended by Weiss and Laties (1958) and later developed further by several authors. The monkeys are seated in restraining chairs. Electrical current is delivered by a Coulbourn Instrument Programmable Shocker through electrodes coupled to two test tube clamps which are attached to a shaved portion of the tail. The current ranges from 0 to 4 mA through 29 progressive steps. The monkey presses a bar to interrupt the shock. A stable baseline shock level is established for each monkey on the day prior to drug administration. After drug administration shock titration activity is rated according to the change in maximum level of median shock intensity attained for drug as compared to control levels. Doses of 3.0 mg/kg i.m . morphine, 1.7 mg/kg i.m . methadone and 10 mg/kg i.m . pentazocine were found to be effective.

14. CRITICAL ASSESSMENT The monkey shock titration test may be used for final evaluation of a new compound before administration to man. For screening activities the procedure can not be recommended since the test is too time consuming and the apparatus too complicated. Furthermore, higher animals such as monkeys should only be used if absolutely necessary

15. Anti-inflammatory activity General considerations Inflammation was characterized two thousand years ago by Celsus by the four Latin words: Rubor , calor , tumor and dolor. Inflammation has different phases: the first phase is caused by an increase of vascular permeability resulting in exudation of fluid from the blood into the interstitial space, the second one by infiltration of leukocytes from the blood into the tissues and the third one by granuloma formation. Accordingly, anti-inflammatory tests have to be divided into those measuring acute inflammation, sub acute inflammation and chronic repair processes. In some cases, the screening is directed to test compounds for local application. Predominantly, however, these studies are aimed to find new drugs against poly arthritis and other rheumatic diseases. Since the etiology of poly arthritis is considered to be largely immunologically, special tests have been developed to investigate various immunological and allergic factors

16. Paw edema Method PURPOSE AND RATIONALE Among the many methods used for screening of anti inflammatory drugs, one of the most commonly employed techniques is based upon the ability of such agents to inhibit the edema produced in the hind paw of the rat after injection of a phlogistic agent. Many phlogistic agents (irritants) have been used, such asbrewer’s yeast, formaldehyde, dextran , egg albumin, kaolin, Aerosil ®, sulfated polysaccharides like carra-geenin or naphthoylheparamine . The effect can be measured in several ways. The hind limb can be dissected at the talocrural joint and weighed. Usually, the volume of the injected paw is measured before and after application of the irritant and the paw volume of the treated animals is compared to the controls. Many methods have been described how to measure the paw volume by simple and less accurate and by more sophisticated electronically devised methods. The value of the assess- ment is less dependent on the apparatus but much more on the irritant being chosen. Some irritants induce only a short lasting inflammation whereas other irritants cause the paw edema to continue over more than 24 h.

17. PROCEDURE Male or female Sprague- Dawley rats with a body weight between 100 and 150 g are used. The animals are starved overnight. To insure uniform hydration, the rats receive 5 ml of water by stomach tube (controls) or the test drug dissolved or suspended in the same volume. Thirty minutes later, the rats are challenged by a subcutaneous injection of 0.05 ml of 1% solution of carrageenan into the plantar side of the left hind paw. The paw is marked with ink at the level of the lateral malleolus and immersed in mercury up to this mark. The paw volume is measured plethysmo graphically immediately after injection, again 3 and 6 h, and eventually 24 h after challenge. EVALUATION The increase of paw volume after 3 or 6 h is calculated as percentage compared with the volume measured immediately after injection of the irritant for each animal. Effectively treated animals show much less edema. The difference of average values between treated animals and control groups is calculated for each time interval and statistically evaluated. The difference at the various time intervals give some hints for the duration of the anti-inflammatory effect. A dose- response curve is run for active drugs and ED50 values can be determined

18. Pleurisy test PURPOSE AND RATIONALE Pleurisy is a well known phenomenon of exudative inflammation in man. In experimental animals pleurisy can be induced by several irritants, such as histamine, bradykinin , prostaglandins, mast cell degranulators , dextran , enzymes, antigens, microbes, and nonspecific irritants, like turpentine and carrageenan (Survey by DeBrito 1989). Carrageenan -induced pleurisy in rats is considered to be an excellent acute inflammatory model in which fluid extravasation , leukocyte migration and the various biochemical parameters involved in the inflammatory response can be measured easily in the exudate .

19. PROCEDURE Male Sprague- Dawley rats weighing 220–260 g are used. The animal is lightly anaesthetized with ether, placed on its back and the hair from skin over the ribs of the right side is removed using animal clippers. The region is swabbed with alcohol. A small incision is made into the skin under the right arm between the seventh and eighth rib. The wound is opened and a further shallow incision is made into the exposed inter costal muscle. 0.1 ml of 2% carrageenin solution is injected into the pleural cavity through this incision. The injection needs to be made swiftly to avoid the risk of injuring the lung. The wound is closed with a Michel clip. 48 h thereafter, groups of 10 rats are treated with the standard or the test compound subcutaneously or orally. A control group receives only the vehicle of medication The animals are sacrificed 72 h after carrageenin injection by ether inhalation. The animal is pinned on a dissection board with the forelimbs fully extended .An incision in the skin over the xipho sternal cartilageis made to free the cartilage from overlying connective tissue. The cartilage is lifted with a forceps and a small cut is made with scissors in the body wall below to gain access into the pleural cavity. One ml of heparinized Hank’s solution is injected into the pleural cavity through this cut. The cavity is gently massaged to mix its contents. The fluid is aspirated out of the cavity using a pipette. This is made easier if the dissection board is raised to an angle of 45–60°; the contents then pool in the corners of the cavity. The aspirated exudate is collected in a graduated plastic tube.

20. EVALUATION One ml (the added Hank’s solution) is subtracted fromthe measured volume. The values of each experimentalgroup are averaged and compared with the controlgroup . ED50 values can be calculated using various doses. Several other parameters can be used: • Measuring the white blood cell number in the exudate using a Coulter counter or a hemato cytometer , • Determination of lysosomal enzyme activities, • Determination of fibronectin ,