Pharmacological Screening methods for Analgesics

Pharmacological Screening methods for Analgesics

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  PRESENTED BY : Mr.Devkar Sumit Uddhav Roll No. – 05 M.Pharm (Pharmacology) Sem-01 DR. VITTHALRAO VIKHE PATIL FOUNDATION’S COLLEGE OF PHARMACY, VILAD GHAT, AHILYANAGAR (2024-25) GUIDED BY : Prof. Hemant J. Pagar Professor & HOD (Dept. of Pharmacology) Pharmacological And Toxicological Screening methods of Analgesics Identifying effective Pain relief agents
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  Content  Introduction  Classificationof Pain  Pain Pathways  Mediators of Pain and inflammation  Analgesics  Classification of Analgesics  Classification of Screening models  In-Vivo Models  Models using Thermal stimuli  Models using Electrical stimuli  Models using Chemical Stimuli  Models using Mechanical Stimuli  In-Vitro Models  References
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  INTRODUCTION  Pain isthe most important symptom that brings the patient to the doctor and demands immediate relief.  Pain also called “Algesia”, is a component of virtually all clinical pathologies.  Pain can sometimes causes discomfort and suffering ; may even be unbearable and incapacitating.  To screen the analgesic drugs that reduce pain or increase the threshold of pain perception, various animal models are developed that generate pain using different sources of stimuli like thermal stimuli, chemical stimuli, mechanical stimuli and electrical stimuli.  This presentation describes commonly employed models used for screening the antinociceptive or analgesic agents.
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  Classification of Pain I.Somatic Pain : Pain arising from the skin and integumental structures, muscles, bones and joints is known as somatic pain. e.g. Wounds , Burns II. visceral pain : Pain arising from the viscera which is difficult to pinpoint to a site is known as visceral pain. It is dull (not much intense) Pain. E.g. spasm, ischemia or inflammation. III. Referred Pain : When pain is referred to a cutaneous area which receives nerve supply from the same spinal segment as that of the affected viscera, it is known as referred pain, e.g. cardiac pain referred to the left arm.  Pain is defined as unpleasant bodily sensation perceived as suffering, usually evoked by an external or internal noxious stimulus.  Pain is subjective experience(6).  Nociception is Neural processes of encoding and processing noxious stimuli. Pain (Algesia) : Classification :
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  Pain Pathway Peripheral Nerve ending releaseof Prostaglandins Cell membrane rupture Na+ Impulse flow Sensory neuron (1st order neuron) Substantia gelatinosa Nociceptor (2nd order neuron)
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  When any chemical, mechanical or electrical stimuli stimulates cell membrane, enzyme Membrane Phospholipase A2 readily convert Phospholipids present in Cell membrane into Prostaglandins. These Prostaglandins binds to prostanoid receptors present on cell body of 1st order Neuron (sensory Neuron).Action Potential is generated in the sensory neuron and impulse is conducted to nerve ending through axon. Substance P(Neurotransmittor of pain)is released from presynaptic cleft into synapse in response to generated action potential. This Substance P further binds to Noceceptors which are present on 2nd order neuron in substantia gelatinosa region of pain pathway. Noceceptors are specialized receptors for Pain sensation. Further Pain impulse is transmitted to somatosensory cortex in cerebrum by passing through Medulla , Pons and Midbrain which is collectively called as Spinothalamic tract. Pain is sensitized.
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  Mediators of Painand inflammation Prostaglandins and Leukotrienes(6) LTA2 LTB4 Membrane Phospholipids Arachidonic Acid Chemical and mechanical stimuli Membrane Phospholipase A2 Activation Cycloxygenase Lipoxygenase PGG2 PGH2 PGE2 PGD2 PGF2α LTC4 LTD4 Thromboxane synthase Isomerase Prostacyclin synthase TXA2 PGI2 Fever Pain Hyperalgesia Vasodilaton Allergic reaction Uterine Contraction Vasoconstriction Platelet aggregation Bronchoconstriction Vasodilation Prevent Platelet aggregation Hyperalgesia bronchoconstrictor
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  Whenever any Chemical,electricalor mechanical stimuli triggers enzyme Membrane Phospholipase A2 it readily convert Phospholipids present in Cell membrane into Arachidonic acid. Two types of enzymes acts on arachidonic acid viz. Cycloxygenase and Lipoxygenase. There are two types of Cycloxygenase enzyme namely COX1 and COX2. COX1 enzyme is involved in wide range of physiological functions, Hence it is called Houskeeping enzyme. COX2 enzyme is involved in pathological functions. Cycloxygenase acts on arachidonic acid to convert it into PGG2 further it isomerizes to PGH2. Enzyme Isomerase acts on PGH2 to produce three different types of prostaglandins viz. PGE2, PGD2, PGF2α. Enzyme thromboxane synthase acts on PGH2 to produce TXA2.it is called as Thromboxane A2. Enzyme Prostacyclin synthase acts on PGH2 to produce PGI2.It is also called as Prostacyclin. Enzyme LipoxygenaseActs on Arachidonic acid to convert it into
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  Analgesics (Antinociceptive agents) A. Analgesics(referred as Painkillers) are agents that selectively relieves pain by acting in the CNS or on peripheral pain mechanisms, without significantly altering consciousness. B. These are the agents which are used to achieve analgesia – relief of pain. C. Analgesics relieve pain as a symptom, without affecting its cause. D. Analgesics are conceptually distinct from anesthetics. E. recent research has suggested that classes of drugs that are not normally considered analgesics, such as tricyclic antidepressants and anticonvulsants may be considered as an alternative
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  Classification of Analgesics Analgesics A. Opioid/narcotic/morp hine-likeanalgesics. B. Nonopioid/non- narcotic/aspirin- like/antipyretic or antiinflammatory analgesics C. Adjuvant analgesics: Anticonvulsants, viz. gabapentin/pregabalin, carbamazepine, larnotrigine; antidepressants, viz. amitryptyline, duloxetine.
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  Classification of Pharmacologicalscreening models  In vivo method  Models using thermal stimulus • Hot plate method/ Paw withdrawal test • Tail flick method o Tail flick model : radiant heat method o Tail flick model : Immersion of tail method  Cold ethanol tail flick test  Models using electrical stimulus • Tooth pulp stimulation test • Grid shock test • Electrical stimulation of tail • Monkey shock titration test  Models using chemical stimulus • Formalin test • Writhing test  Model using mechanical stimulus • Haffner's tail clip method • Randall selitto test • Von frey method  Animal models of chronic pain • Neuropathic pain models o Vincristine induced neuropathy model o Diabetic neuropathy model • Persistent post thoracotomy pain • Cat model of incisional pain • Rat model of bone cancer pain  Distension of hollow organs using chemical stimulus • Rat sigmoid colon model • Inflammatory uterine pain model  In vitro methods  3 H- Dihydromorphine binding to µ- opiate receptor binding assay  3 H- naloxone binding assay  Assay to study cannabinoid activity  Assay to study nociceptin activity  Inhibition of enkephalinase  Assay to study Vanilloid ( capsaicin) activity  Evaluation of Vanilloid receptor antagonists  Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase – activating peptide (PACAP)
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  In-Vivo Models
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  Models using Thermal Stimulus
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  1. Hot PlateMethod
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  Hot plate method The Hot plate method is widely used for screening of Opoid Analgesics  Method is also known as Paw withdrawal test. Requirements : Animal: Albino mice Chemicals : Test compound,Morphine, Codeine Equipment used: Eddy’s Hot plate (electrically heated plate at 55-56 °C)(8). Demerit: Sedatives , muscle relaxants or psychomimetics can give false positive results, while partial opiate agonist-antagonist produce unreliable results
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  Procedure: * Animal isplaced on the hot plate, which consists of electrically heated surface. Temperature of the hot plate is maintained at 55-56 °C. * Responses such as jumping, withdrawal of the paws and licking of the paws are seen. * The time period (latency period), when animal is placed and until responses occur, is recorded by a stopwatch(8).
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  Responses to beobserved : Paw licking Jumping Paw withdrawal
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  Observation Table : Conclusion: Testcompounds are administered orally or subcutaneously and latency period is recorded after 20, 60, 90 min. These values are compared with the values before administration of the drug by using t-test. Sr. No. Animal weight Basal Reaction Time (Sec) Reaction time after Treatment (Sec) Paw licking Jump Response Paw licking Jump Responce 1. 2. 3. 4. 5.
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  2. Tail FlickModel : Radiant Heat Method
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  This test isreliably used test for revealing the potency of opioid analgesics. Tail Flick Model : Radiant Heat Method Requirements : Animal: Albino mice (18-22 gm) Chemicals : Test compound, Morphine ,Codeine Equipment used: Cages leaving tail exposed out , Radiant Heat Analgesiometer(8). Merit : Results are quite accurate and less time consuming. Demerit: Tail flick response is prone to habituation Response is not consistent upon repetitive stimulation
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  Procedure
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  Tail Deflection Sensor Responsesto be observed : Tail flicking Head rotation
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  Sr. No. Animal weight Basal Reaction Time (Sec) Reactiontime after Treatment (Sec) Tail flick Head Rotation Tail flick Head Rotation 1. 2. 3. 4. 5. Observation Table : Conclusion: 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 compounds can be calculated according to Litchfield and Wilcoxon method. Codeine, pethidine and morphine are used as standard. Usual tail flicking time ranges between 2 to 10 sec.
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  3. Tail FlickModel : Tail immersion method This test is Selective for morphine like analgesics. Procedure: • Rats are place in rat holder so that tail hangs freely. • Distal 5 cm portion of the tail is immersed(max.upto 15 sec) in a cup filled with warm water maintained at 55 ±1 °C • The time taken by the rat to withdraw the tail or attempt to escape or abrupt movement of tail and sometimes the recoiling of whole body is seen from hot water which is noted as reaction time. • reaction time is determined periodically after administration of test drug (0.5, 1, 2, 3, 4, and 6 hours) Modification methods: - Cold tail Flick test. - Cold ethanol tail flick test
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  Models using Electrical stimulus
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  4. Grid shocktest The method utilises specialised procedure - “Flinch-jump” procedure. Requirements : Animal: Albino mice (18-22 gm) Chemicals : Test compound, Morphine ,Acetyl salicylic acid Equipment used: Plastic chamber with grid shock facility.
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  Albino mice witha weight between 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. With increasing shock intensities the mice flinch, exhibit a startling reaction, increase locomotion or attempt to jump. The behavior is accurately reflected on the oscilloscope by marked fluctuations of the displayed pulse and defined as pain threshold response. 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. Groups of 10 animals are used for control and for the test drugs. Procedure
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  Evaluation : The currentas measured in milliamperes is recorded for each animal before and after administration of the drug. The average values for each group at each time interval are calculated and statistically compared with the control values. Placebo treated controls show a slightly less threshold over time. Morphine sulfate at dose of 10 mg/kg p.o. also acetylsalicylic acid in a dose of 200 mg/kg p.o. definitely increase the threshold. Sr. No. Animal weight Basal applied current(mA) Applied current after Treatment (mA) Flinch Reaction Jump Attempt Flinch Reaction Jump Attempt 1. 2. 3. 4. 5. Observation Table :
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  Responses to beobserved : Jumping flinch reaction
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  Models using Mechanical stimulus
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  Randall-Selitto-Test)
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   Before thetest, each animal handled for 5min so as to habituate.  Animal is hold with soft cotton cloth and carefully immobilized.  Apply an increasing mechanical force, so that the tip will produce pain onto the medial portion of the plantar or the dorsal surfaces of both fore and hind paws.  Observe and record the paw withdrawal response.  Note the applied pressure at the time of response generated by animal (animal struggles, squeals or attempts to bite).  The maximum force applied was limited to 250 g to avoid skin damage. Procedure: Sr. No. Animal weight Applied Presssure before treatment Applied Pressure after Treatment 1. 2. 3. 4. 5. Observation Table :
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  For your kind attentionand co- operation