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Pharmacological screening of analgesic activity

1. The document describes several screening methods used to evaluate potential analgesic agents, including in vivo models using thermal, electrical, or chemical stimuli to induce pain states in animals, as well as in vitro receptor binding assays. 2. Common in vivo models discussed are the hot plate test, tail flick test, writhing test induced by acetic acid injection, and formalin test, which create persistent pain. 3. Details are provided on procedures for these tests, which involve administering a potential analgesic and measuring its ability to delay pain-induced responses like paw licking or jumping.

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SCREENING METHODS OF ANALGESIC AGENTS SUBMITTED BY: BISWASH SAPKOTA FIRST M.PHARM PHARMACOLOGY SUBMITTED TO: RAJESH KOWTI ASSISTANT PROFESSOR DEPT of PHARMACOLOGY
Introduction  Pain is an unpleasant sensory and emotional experience associated with actual and potential tissue damage.  Various types of pain are seen in humans for example somatic pain, visceral pain, referred pain, neuropathic pain, cancer pain etc.  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.  from pain.
Classification Analgesic can be classified as 1. Narcotics : Morphine, Pethadine, fentanyl 2. Non-Narcotics: NSAIDs
Mechanism of action  Analgesic drugs act in various ways on the peripheral and central nervous systems.  Opioids produce analgesia by binding to specific G – protein coupled receptors ( µ, ƙ, δ) in brain and spinal cord which decrease the release of neurotransmitter.  NSAIDs inhibit the activity of both cyclooxygenase-1 (COX- 1) and cyclooxygenase-2 (COX-2) and thereby the synthesis of prostaglandins and thromboxane.  Inhibition of COX-2 leads to the anti-inflammatory, analgesic and antipyretic effects.
Screening models A. Invivo methods  Pain - state models using Thermal stimuli * Tail - flick model using radiant heat. * Hot - plate test. * Paw - withdrawal test.  Pain - state models using Electrical stimuli * Electrical stimulation of the tail. * Grid - shock test. * Tooth pulp test  Pain - state models using Chemical stimuli * Formalin test. * Acetic acid induced writhing test.
Contd.. B. Invitro method 3H-Naloxone binding assay. 3H-Dihydromorphine binding to 𝜇 opiate receptors in rat brain.  Receptor binding of nociceptin. Bioassays for nociceptin.  Receptor binding of cannabinoids.  Vanilloid receptor binding.
Hot plate method Purpose and 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 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 Groups of 10 mice (18-22g) are selected and divided into standard, test & control group respectively 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 20, 60 and 90 min after the administration of standard or test compound
Evaluation  The latency time of the test, standard and control animals are recorded and compared with values before administration  Using various doses ED50 values can be calculated.
Tail flick method Purpose and rationale  The tail flick test with radiant heat is an simplified method.  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.
Procedure Wistar rats (170-210g) are selected and divided into standard, test & control group respectively Appropriate temperature is maintained on the radiant source The tail of the rat is placed on the radiant source & time taken for the rat 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.
Evaluation  The tail flick latency in the test, standard and control animals are compared.  Using various doses ED50 values can be calculated
Writhing test Purpose and rationale  Pain is induced by injecting irritants like acetic acid into peritoneal cavity of mice.  The animals react with characteristic stretching behavior which is writhing.  The test is suitable to detect analgesic activity of peripherally acting drugs.
Procedure Mice (20-25g) are selected and divided into standard, test & control group respectively re Appropriate volume of acetic acid solution is administered to the mice (control group) and placed individually in the glass jar. 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. n.
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 acetic acid treated control
Tooth Pulp Test Procedure Rabbit weighing 2-3 kg are take and thiopental sodium of 15mg/kg I.V body weight is given. Using dental drill , tooth pulp chambers are exposed close to upper incisors. Clamping electrode are placed into drilled holes After 30min electrical stimulus is applied by rectangular current of frequency 50Hz upto 1sec. Current is started 0.2mA and increased until animal starts licking and a threshold is determined
Animal serves as its own control. Test compound is administered orally or intravenously, After 15, 30, 60 and 120 min threshold current is measured and compared with the threshold current prior to drug administration.
Formalin test Purpose and rationale The formalin test uses a 10% formalin solution as a chemical noxious stimulus. By injecting the formalin solution into the paw of a rat or a mouse, a model of persistent (chronic) pain caused by peripheral tissue injuries and inflammation is created. This model is highly sensitive for opioids like drugs.
Procedure Male Wistar rats weighing between 180-300 g are used In the dorsum of front paw of the animal 0.05 ml of 10% formalin is injected subcutaneously. Each animal is placed into separate cage for observation of pain responses in early and late phases. These responses are elevation or favoring of the paw or excessive licking and biting of the paw Scoring of these pain responses is done according to a pain scale.
After administration of the test drug again scoring is done after 30, 60 min for comparison. If both paws of the animal are allowed to rest on the floor with no obvious favoring of the injected paw, this is taken as a positive analgesic response.
Bioassay for Nociceptin Purpose and Rationale  Nociceptin receptors in the periphery 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).
Procedure Tissues are taken from male Swiss mice, guinea pigs, Sprague Dawley rats &New Zealand albino rabbit. Suspended in 10 ml organ baths containing Krebs solution oxygenated with 95% O2 & 5% CO2. Temperature is set around 33°-35°C & a resting tension of 0.3-1g is applied. The tissues are stimulated through two platinum ring electrodes The electrically evoked contractions are measured isotonically with a strain gauge transducer and recorded on a multichannel chart recorder.
After equilibration period of about 60 min the contractions induced by electrical field stimulation are stable; at this time, cumulative concentration response curves to nociception or opioid peptides are performed. Four electrical field stimulation are performed with each tissue at 30 min intervals. Agonists & Antagonists are added to the bath. Contractile responses to electrical field stimulation are expressed as % increment to the spontaneous activity of the tissue. The biological effects of the application of agonists or antagonists are expressed as % inhibition of electrical filed stimulation- induced contraction.
Evaluation The agonists and antagonists potencies are recorded and expressed as means of ±SEM.
References  Patel PK, Sahu J, Chandel SS. A detailed review on nociceptive models for the screening of analgesic activity in experimental animals. Int J Neurol Phys Ther. 2016;2:44-50.  Milind P, Monu Y. Laboratory models for screening analgesics. Int Res J Pharm. 2013;4(1):15-9.
Pharmacological screening of analgesic activity