Analgesic Screening Models

Analgesic Screening Models

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  DR. A.P.J. ABDULKALAM TECHNICAL UNIVERSITY NOIDA INSTITUTE OF ENGINEERING AND TECHNOLOGY (PHARMACY INSTITUTE), GREATER NOIDA Topic: Pharmacological Screening of Analgesic Agents Submitted to : Submitted by: Dr. Saumya Das Madhurima Professor, M.Pharma HOD 1st Sem (Pharmacology) (Pharmacology)
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  ANALGESIA According to theInternational Association for the Study of Pain , updated in 2019, Pain is defined as an unpleasant sensory and emotional experience associated with potential or actual tissue damage. Analgesia is the absence of pain in response to stimuli that would normally be painful. Reasons to minimize pain and distress: Ethical Reasons Legal Reasons Scientific Reasons Minimize the suffering of animals and provide quality environment and quality life to them. THE PREVENTION OF CRUELTY TO ANIMALS ACT, 1960 Good science and animal welfare goes hand in hand. Researchers should use and handle the experimental animals with kindness, care and concern & cause minimum discomfort, distress or pain Laws that regulate animal experimentation require that suffering is reduced to a minimum. Pain, discomfort and distress can alter various physiological, biochemical and neuroendocrine parameters in animals which in turn has significant effect on experimental results.
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  Signs of Painin Laboratory Rodents •Decreased activity, piloerection, and an ungroomed appearance. •There can be excessive licking and scratching, which can progress to self-mutilation. •They might adopt an abnormal stance or a hunched posture. •Respiration can be rapid and shallow with grunting or chattering on expiration. •In albinos, porphyrin secretion ("red tears") can be seen around the eyes. • Rats and mice in acute pain might vocalize and become unusually aggressive when handled. •Inappetance or a change in feeding activity can be noted. •If housed with others, normal group behavior might change.
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  Analgesics Opioids NSAIDS Local Anesthetics Morphine Buprenorphine Butorphanol AIbuprofen Carprofen Diclofenac Acetaminophen Aspirin Ibuprofen Lidocaine Xylocaine Opioidagonists bind to G-protein coupled receptors to cause cellular hyperpolarisation Mechanism of action of NSAIDs is the inhibition of the enzyme cyclooxygenase (COX). LAs reversibly inhibit nerve transmission by binding voltage- gated sodium channels (Nav) in the nerve plasma membrane.
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  In-Vivo Models 1.Pain statemodels using thermal stimuli:  Hot plate method.  The tail flick model using radiant heat/immersion of the tail in hot water.  Paw withdrawal test. 2. Models using electrical stimuli:  Stimulation of the tooth pulp  Electrical stimulation of the tail  Monkey shock titration test 3. Models using chemical stimuli: • Formalin test • Writhing test • Rat sigmoid colon model • Inflammatory uterine pain model 4. Models using mechanical stimuli: • Haffner’s tail clip method • Randall Selitto Test
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  In-Vitro Models  3H-naloxonebinding assay.  𝜇 opiate receptor binding assay.  Receptor binding of cannabinoids.  Receptor binding to nociceptin.  Vanilloid receptor binding.
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  HOT- PLATE METHOD Purposeand 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 are 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.
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  Procedure: Mice (18-22g) areselected and divided into standard, test & control. 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 30, 60 and 90 min after the administration of standard or test compound.
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  Evaluation: The prolongation oflatency time between the test, standard and control animals are compared. Using various doses ED50 values can be calculated.
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  TAIL FLICK MODEL Purposeand rationale: • 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.
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  Procedure: Wistar rats (170-210gm)are selected and divided into standard, test & control groups respectively. Appropriate temperature is maintained on the radiant source. The tail of the mice is placed on the radiant source & time taken by the mice 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.
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  Evaluation: • The tailflick latency in the test, standard and control animals are compared. • Using various doses ED50 values can be calculated. Modification: • Cold tail Flick test • Cold ethanol Tail flick test
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  Grid Shock Test Describedby Blake et al. (1963) Approach to measure the analgesic properties by the “Flinch- jump” procedure.
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  Procedure: • Male mice(18 and 20 g) are individually placed into clear plastic chambers. • The floor of the box is wired with tightly strung stainless steel wire, spaced about 1 mm apart. • The stimulus is given in the form of square wave pulses, 30 cycles per second with a duration of 2 ms per pulse. • The output of the stimulator has to be connected to alternate wires of the grid. • A fixed resistance is placed in series with the grid and in parallel to an oscilloscope to allow calibration in mill amperes. • With increasing shock intensities the mice flinch, exhibit a startling reaction, increase locomotion or attempt to jump. • Pain thresholds are determined in each individual mouse twice before administration of the test drug and 15, 30, 60, 90 and 120 min after dosing. • The current as measured in mill amperes is recorded for each animal before and after administration of the drug.
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  Evaluation: • The currentmeasured in milliampheres 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 control values.
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  WRITHING TEST Purpose andrationale : • Pain is induced by injecting irritants like 0.6% acetic acid, Phenylquinone 0.02%, 4% NaCl into peritoneal cavity of mice. • The animals react with characteristic stereotyped behavior which is writhing. • A writhe is indicated by stretching of the abdomen with simultaneous stretching of atleast 1 hind limb. • The test is suitable to detect analgesic activity of peripherally acting drugs.
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  Procedure: Mice of eithersex (20-25 g) are selected and divided into standard, test & control groups respectively. Pain is induced by intraperitoneal injection of chemicals that irritate serous membranes. 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.
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  Trunk-Twist in Mice 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 control group.
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  HAFFNER’S TAIL CLIPMETHOD Purpose and rationale: • Preferred sites for applying nociceptive mechanical stimuli are the hind paw and the tail • Highly sensitive for centrally acting drugs • Tests using constant pressure have been abandoned progressively for those applying gradually increasing pressures
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  Procedure: An artery clipis placed at the root of tail of mice. A quick response is seen as biting the clip or tail, where clip has been placed. Then after 15, 30, and 60 minutes, the same procedure is repeated and the reaction time is measured.
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  Bioassay of Nociceptin Nociceptinreceptors is a G-Protein coupled receptor located in the periphery and 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 according to Oka et al. (1980), the guinea pig renal pelvis (Giuliani and Maggi 1996).
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  PROCEDURE: • Take thetissues from either 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.
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  •After equilibration periodof 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. Agonistic and antagonistic activity of drugs evaluated .
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  References 1)Tjølsen, A., Lund,A., Berge, O. G., & Hole, K. (1989). An improved method for tail- flick testing with adjustment for tail-skin temperature. Journal of neuroscience methods, 26(3), 259-265. 2)O'Callaghan, J. P., & Holtzman, S. G. (1975). Quantification of the analgesic activity of narcotic antagonists by a modified hot-plate procedure. Journal of Pharmacology and Experimental Therapeutics, 192(3), 497-505. 3)Gawade, S. (2012). Acetic acid induced painful endogenous infliction in writhing test on mice. Journal of Pharmacology and Pharmacotherapeutics, 3(4), 348. 4) Parimaladevi, B., Boominathan, R., & Mandal, S. C. (2003). Studies on analgesic activity of Cleome viscosa in mice. Fitoterapia, 74(3), 262-266. 5) Yaksh, T. L., Ozaki, G., McCumber, D., Rathbun, M., Svensson, C., Malkmus, S., & Yaksh, M. C. (2001). An automated flinch detecting system for use in the formalin nociceptive bioassay. Journal of applied physiology, 90(6), 2386-2402. 6) Blake, L., Graeme, M. L., & Sigg, E. B. (1963). Grid shock test for analgesic assay in mice. Pharmacology, 9(3), 146-150. 7) Sharma Shubha (2018) , Slideshare- Pharmacological Screening of Analgesic Agents. 8) Kulkarni S. K. (1987). Hand book of experimental pharmacology. Vallabh prakashan.