Screening of analgesic activity and dependence ability 1) Narcotic analgesics Q used to relieve sever visceral pain ex. renal colic, post operative pain and cancer pain O narcotic analaesic are either: a) natural opioid + obtained from opium ex. morphine, codeine b) semi-synthetic opioid ex. heroin ) synthetic opioid ex. meperidine analgesics are drugs used to treatment of pain without loss of consciousness Q analvesics are 2 types: 1) narcotic analgesic “morphine like drugs; opioid” 2) non narcotic analgesic “aspirin like drugs; non- opioid analgesics” O Pharmacological effects of narcotic analgesic: a) produce analgesic effect b) produce sedative effect ©) produce euphoria 4) produce constipation e) causes vomiting ) produce tolerance + all those effects antagonized by 1raluxone * narcotic analgesic are screened for both analgesic and dependence liabilityScreening procedure for analgesic drugs methods used for inducing pain in animals: 1) thermal method + vosd for screening of nunenarcotic analgesics a) Hot plate metnod a) use IP mice or 18 rato ‘ere divided into 3 groups control group (n=) group 2.. injected with standard drug ‘ex morphine (n=6) 3) group 3 ...injected with test drug (n=6) c) use hot plate: a = | 5 control test dr ‘hermal method there are 4 techniques of thermal method a) hot plate method b) tail immersion method c) tail flick method 4) radiant heat method | Procedure: a) set the temperature of hot plate at 55 +1°C ') experimental time (off time is 30 sec) * to avoid tissue damage" ‘c) response seen “licking of fore limb or jumping” 4) put the animals one by one on hot plate e) calculate the reaction time + Reaviiun ine is tine between placing Ure animal on hot plate to the appearance of response (tt) 1) inject NS. standard drugs (morphine) and test drug and wait 30 min|) again put the animals one by one on hot plate and calculate the reaction time (t1 * before injection of, + after 30 min of |) calculate the % % analgesia = (tt -t2)/tt]x 100 P by 2x2 assay ... see 2 move 2 Procedure 4) fix the temperature of water at 5 11°C fesic Effect by the formula: ) Tail immersion method a) use 18 mice or 18 rats, b) mice are divided into 3 groups 1) group 1 ...contrg¥froup (n=6) ted with standard drug injected with test drug (n=6) fot water in a beaker #) immerse, the tal of each animal in the beaker one by ong’nd calculate the reaction time . time is time between immersing the tail ot animal in hot water to the appearance of response (t1) e) inject NS, standard drugs (codeine) and test drug and wait 30 min f) again immerse the tail of each animal in the beaker one by one and calculate the reaction time (2) 16fi. after 30 min of inj calculate the % Anal Q Procedure: a) response seen “movement (flicking) of tail” ») put the animal in the restraint holder ©) focus the light from heafadf tungsten on tail of it reaction time me from focusing the light ingsten on tail of animal to the NS, standard drugs (pethidine) and test iy and walt 30 min ) Tail flick method b) pri€ are divided into 3 groups 1) group 1 ...control group (n=6) 2) group 2.. injected with standard drug (pethidine) (n=6) 3) group 3 ...Injected with test drug (n=6) c) use heated tungsten wire directed to the tail o! rat at a constant point ) again focus the light from heated tunasten on il of animal and calculate the reaction time (t2) fi . reaction time is calculated twice a) before injection of drug (t1) b)after 30 min of injection of drugs (12) €) calculate the % Analgesic Fffect hy the formula: % analgesia effect = [(tt — t2) / tt] x 100 f) calculate the RP by 2x2 assayG Procedure: ) Radiant heat method a) response seen “twitching of the animal skin” fe) fuse ye mics Onde Kate ») put the animal in the restraint holder 2 eae mOS OOS ©) focus the light from heated lamp on shaved eae ee 6 area of animal and calculate the reaction time group 2 .. injected with standard dru Se : ; {morphine} (n=5) Reaction time is time from focusing of the igh 3) group 3 ...injected with test drug (n=6) im heated lamp on the shaved area to the ) shave the back of rats mn oe ot ee nine) ane 9) use electrical lamp “1000 watt Jamp” directed Standard drugs (morphine) and tes to the shaved area on Nibepaekor the rat Grug and wait 30 min gf) 29ain focus the lignt trom heated lamp on the shaved area on the back of the animal and calculate the reaction time (t2) a i.e., reaction calculated twice Advantage of radiant heat method Tomes rckere (tt) 1) it canX6 used for human . head b)atter 30 min of injection of drugs (12) ‘fe black towel on the foreheat 5) calculate the % Analgesic Effect hy the formula % analgesia effect = =)/ ti} x 100 I when feel pain gives sign to put (a 9) calculate the RP by 2x2 assaytage of radiant heat method ‘an be used for human use black towel on the forehead when feel pain gives sign to put lamp off / a1 Procedure: a) response seen “squeaking” b) put the the animal in the restraint holder jure on the tail of animal a1 time is time from applying pressu ie clip on the tail of animal to t pearance of response (t1) ) inject NS, standard drugs (morphine) and t drug and wait 20 min 2) Physical “mechanical” method a) Tail clip method a) use 18 mice or 18 rats b) rats are divided into 3 groups 41) group 1 ...control group (n=6) 2)group 2 .. injected with standard druc (morphine) (n=6) 3) group 3 ...injected with test drug (n=6) ) use pressure at the base of tail 28 of drugs (t1) b)atter gO min of injection of drugs (12) 5 ealeulayfne % Analgesic Effect by the formula: (gesia effect = [(t1 — 12) / ti] x 100 late the RP by 2x2 assay|. Screening of dependence liability Q drugs that show analgesic activity in physical or thermal thod should be screened for use jumping method d) inject morphine and test drug at doses 8, 16, 25, (0 and 100 mg/kg fnject drugs i.p for 2 days = onday (1) > 5 injections every 2 hours: ie., inject at 9, 11, 1, 3 and 5 o'clock + on day (2) > 2 injections every 2 hours ie., inject at 9 and 11 o'clock and on 1 o'clocl inject naloxone f) response seen “jumping of animal” a) Mouse jumping method a) use 18 mice b) mice are divided into 3 groups. 1) group 1 ...control group (n=6) 2) group 2 .. injected with morphine (n=6) 3) group 3 ...injected with test drug (n=6) ©) drugs are given in 5 increasing doses 3) count the number of jumping during 10 min period after the injection of naloxone h) also observe the withd symptoms like tremor, and salivation i) calculate the RP usij assay2) Non narcotic analgesics (non-opioid) * they are used to relieve peripheral pain ex. headache, toothache Q they are divided into: a) NSAIDs “aspirin like drugs” Screening of non narcotic analgesics b) paracetamol like drugs a) NSAIDs “aspirin like drugs” screening procedure for analgesic drugs Q this group is scregned for Q methods used tor inducing pain In animals: i. analgesic gétivity }) thermal method ipyret activity A) physical method . mmatory activity * used for screening of narcotic analgesic >) Paracetgpfiol like drugs 3) chemical method Q this/group has analgesic and antipyre’ 4) electrical method ities but no anti-inflammatory effect «used for screening of non-narcotic screened for analgesics i. analgesic activity ii. antipyretic activity _3) Chemical method 4 Screening methods of non narcotic analgesics a) use 18 mice or 18 rats * there are 2 methods i eee ae 3) chemical method G 2) r- 2. injected with standard drug (aspirin) lectrical method (n=8) tes 3) group 3 ...injected with test drug (n=6) ») rectodoirimeter aap eniiaal ©) tooth pulp method ») parabenzoquinone ©) phenyl quinone U Procedure 8) experimental time “20 mint” 4) Electrical method ©) neat NS, stra crugs(aspiin) and test drug a) olorimeter ©) Inject pain iaucer* ex. acetic acid” b) rgétodolorimeter seen “writhing or stretching” C) th pulp method fithing is @ contraction of abdominal muscle followed by extension of hind limb ©) Start count writhing after 5 min of injection of acetic acid for a period of 20 min ..See move4) Pododolorimeter a) use 18 mice or 18 rats ) mice are divided into 3 groups +. group 1 ..cuniuol group (N=6) 2. group 2.. injocted with standard drug (diclofenac sodium) (n=6) 3. group 3... inject ©) Use cage with, electrical, test drug (n=6) F of metallic rod connected with “voltage” g) record the amount of voltage applied that make the animal squeak (v2) i.e., amount of voltage was recorded twice 1. before injection of drugs (v1) 2. after 30 min of injection of drugs (v2) h) calculate the % Analgesic Effect by the formula: % analgesia effect = [(v2 — v1) / v2] x 100 i) calrulate the RP by 2x2 assay O Procedure: a) response seen “squeak or struggle ” b) put the animal in the cage ©) gives shock in the feet d) record the amount the animal squeal @) inject NS, stz fard drugs (diclofenac sodiuin) and wait for 30 min ‘the animal in the cage and gives the feet for 2 sec %ectodolorimeter a) use 18 mice or 18 rats b) mice gre divided into 3 groups / group 2.. injected with standard drug ‘3. group 3 ....injected with test drug (n=6) ‘¢) use cage with a floor made of single copper plate connected to electrical supply while the other side is connected to a probe inserted into the animal rectum2 Procedure: a) response seen “squeak or struggle ” b) put the animal in the cage ¢) insert the probe in the animal rectum and then gives shack far 2 sec d) record the amount of voltage applied that make the animal squeak (v1) ®) inject NS, standard drugs (diclofenac sodium) and test drug ip and wait for 30 min ‘) again insert the probe in the animal rectum and then gives shock for 2 sec 2) Tooth pulp method a) use dogs or human ) dogs are divided into 3 groups 1) group 1 .... control group (n=6) 2) group 2 ... injected with standard drug (ibuprofen) (n-6) 3) group 3 ...injected with test drug (n~6) 6) filLin the tooth of animal with metal ‘we choose tooth because it gives rapid conduction af pain record the amount of voltage applied that mak the animal squeak (v2) i.e., amount of voltage was recorded twice 1) before injection of drug (v1) 2) after 30 min of injgZtion of drugs (v2) ) calculate esic Effect by the formul ct = [(v2 -— v1) / v2] x 100 the RP by 2x2 assay O Provedure: 2) response seen “sudden pull of head or tremors of lower jaw” the holder . standard drugs (ibuprofen) and test Tp and wait for 30 minf) again do electrical stimulation of the metal filled in the tooth @)again record the amount of voltage applied that make sudden pull of head or tremors of h) calculate the % Analgesic Effect by the formula: cere % analgesia etiect = |(V2 — V1) / v2] x 100 ite., amount of was recorded twice calculate assay 1)before injection of drug (v1) ) whe RPM 2)after 30 min of injection of drugs (v2) ‘narcotic gA@ non narcotic analgesic 53 How can differentiate between narcotic analgesic one . ‘and non narcotic analgesics? a) response seen “licking of hind paw = by the use of formalin test ») inject NS, standard drugs and test drug i.p. li and wait 30 min "Dye mice or 18 rls «) meas omaln sind pow 3 — are divided into 3 groups d) record the time spent in licking the injected saw in 1)group 1 ...control group (n=6) : = early phase (0-6 min after injection) 2)group 2.. injected with standard drug (n=6) = late phase (15-30 min after injection) 3)group 3 ...injected with tet drug (n~6) 568) decrease in number of licking in the early phase * it means that the test drug act like narcotic drug (morphine). of anti-inflammatory and antipyretic f) decrease in number of licking in the late phase activities * it means that the teet drug act like nur narcotje-drug (aspirin). 2 Inflammation: | Treatment of inflammation. * Inflammation is a local response of tissues to + ingnmatin weaedby he in ical or chemical 9 a. muy nas iby pryseal r = agent hoy act by inition the synthesis and release ne Oe ee eae ‘of PG due to inhibition of COX enzyme * different inflammatory mediators are released in ex., aspirin, ibuprofen, diclofenac sodium b) steroidal drugs = they act by inhibition of PG synthesis due to inflammation such as histamine, PG, 5HT, BK. * these inflammatory mediators are responsible for changes during inflammation such as. pain suppression of phospholipase A2 “an enzyme edema and increased vascular permeability, : fesponsible for the Iberation of PG precursor etc . arachidonic acid” ex,, dexamethasone, prednisolone, betamethasoneA. Screening of anti-inflammatory activity 4) cereening of ant-intammatory activity using acute inflammation ) ultraviolet erythema 'D) dye leakage ©) carrageenan induced foot oedema 2) screening of antiinflammatory activ chronic inflammation aa ~ using 8) chronic adjuvant arthritis, Q Procedure: a) response seen sprythema: it is a red area due to vascular, b) inj standard drug (diclofenac suuiuin) fest drug and wait 30 min ‘expose shaved skin area of each animal to UV light 4d) after 2 hr estimate the erythema visually (score 1/4) or measure the diaitteler of inflammation 4) Screening of antiinflammatory ectivity using acute inflammation: ) ultraviolet erythema i. use 18 guinea pig ji. guinea pig are divided into 3 groups - group (n=6) “injected with standard druy sodium) (n=6) 1) caleuilate the % inhibi formula: ‘ inhibition of inflammation = [(c - tW/c] x 100 C= number of score (diameter) of control group {= number of score (diameter) of treated group 9) calculate the relative Potency by 2x2 assay ition of inflammation by the