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Screening of Analgesics
(Central Analgesic Activity) In Vitro Methods Binding Assay 3H-Dihydromorphine Binding to Opiate Receptors in Rat Brain 3H-Bremazocine Binding to Opiate Receptors in Guinea Pig Cerebellum Inhibition of Enkephalinase Nociceptin Receptor Binding of Nociceptin Bioassays for Nociceptin Vasoactive Intestinal Polypeptide (VIP) and Pituitary Adenylate CyclaseActivating Peptide (PACAP) Cannabinoid Activity Receptor Binding of Cannabinoids Vanilloid (Capsaicin) Activity Vanilloid Receptor Binding Evaluation of Vanilloid Receptor Antagonists In Vivo Methods Haffners Tail Clip Method Radiant Heat Method Hot Plate Method Tail Immersion Test Electrical Stimulation of the Tail Grid Shock Test Tooth Pulp Stimulation Monkey Shock Titration Test Formalin Test in Rats Neuropathic Pain Chronic Nerve Constriction Injury Peripheral Nerve Injury Model Spared Nerve Injury Model Spinal Cord Injury Chemotherapy-Induced Pain Trigeminal Neuropathic Pain Model Migraine Model in Cats
Sumanth Dept of Pharmacology
3H-Naloxone

In Vitro Methods for Central Analgesic Activity

These methods are based on high-affinity stereospecific binding of radio-labelled opiate compounds by CNS membrane preparations. The in vivo pharmacological potency of opiate agonists and antagonists parallels the in vitro displacement of 3Hnaloxone, a potent narcotic antagonist. Based on these findings, the 3H-naloxone binding assay was introduced for evaluation of potential analgesics with opiate-like properties. According to different pharmacological profiles of opiates, several receptor types have been identified designated as , , , and receptor ( for morphine = MOP receptor, for Ketocyclazocine = KOP receptor, for deferens because it was first identified in mouse vas deferens = DOP receptor). The receptor ( for SKF10047) was only initially classified as an opioid receptor. The opioid receptors were reclassified according to recommendations of the International Union of Physiological Sciences, the International Union of Pharmacology (IUPHAR). This nomenclature applies an abbreviation of the generic term for the family (OP for opioid) and a subscript number. OP1 stands for , OP2 for , and OP3 for receptor. For the receptor, subtypes named 1 and 2 have been described. Analgesia is thought to involve activation of receptors (largely at supraspinal sites) and receptors (principally within the spinal cord); receptors may also be involved at the spinal and supraspinal level. Other consequences of activation include respiratory depression, miosis, reduced gastrointestinal motility, and euphoria. The 1 receptors are postulated to mediate the supraspinal analgesic action and the 2 receptors to mediate respiratory depression and suppression of gastrointestinal motility. Two endogenous peptides were described, named Endomorphins, as agonists with high specific affinity for the -receptor. Endogenous ligands for receptors are Enkephalins. Endogenous ligands for receptors are Dynorphins. OTHER RECEPTORS There is some evidence that other opioid receptors may exist, such as a -endorphinsensitive receptor. The receptor and a high affinity binding site referred to as the site may also be part of the opioid receptor system.

The heterogeneity of opioid receptors has been studied in isolated tissue preparations in which neurotransmission is sensitive to inhibition by opioids. The relative potencies of opioid agonists are assessed by their ability to inhibit the electrically evoked contractions of isolated tissue preparations from five different species: the contractions of the mouse vas deferens are inhibited by -, -, and -agonists, those of the guineapig myenteric plexus-longitudinal muscle preparation by -and -agonists, those of the rabbit vas deferens by -agonists, and those of the hamster vas deferens by -agonists.
Sumanth Dept of Pharmacology

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3H-Naloxone

Binding Assay

PURPOSE AND RATIONALE A good correlation between the in vivo pharmacological potency of opiate agonists and antagonists with their ability to displace radiolabeled naloxone has been reported. The later discovery that Na+ (100mM) enhances the binding of antagonists and reduces the binding of agonists has led to the development of an as-say which is used to classify compounds as opiate agonists, mixed agonist-antagonists and antagonists by determining the IC50 values for 3H-Naloxone in the presence or absence of Na+.

PROCEDURE Reagents [N-allyl-2,3-3H] Naloxone (3858 Ci/mmol) is obtained from New England Nuclear. For IC50 determinations 3H-naloxone is made up to a concentration of 100nM and 50l is added to each tube yielding a final concentration 5 nM in the assay. Levorphanol tartrate is obtained from Hoffmann LaRoche. A stock solution of 1mM levorphanol is made up in distilled water. This stock is diluted 1:200 in distilled water and 20l is added to 3 tubes to determine stereospecific binding yielding a final concentration of 0.1M in the assay. Dextrorphan tartrate is obtained from Hoffmann LaRoche. A stock solution of 1mM dextrorphan is made up in distilled water. This stock is diluted 1:200 in distilled water and 20l is added to the tubes containing the various concentrations of test drug and the tubes for total binding. Test compounds: For most assays, a 1mM stock solution is made up in a suitable solvent and serially diluted, such that the final concentration in the assay ranges from105 to 108 M. At least 7 concentrations are used for each assay. Higher or lower concentrations may be used, depending on the potency of the drug. Tissue Preparation Male Wistar rats are decapitated and their brains rapidly removed. Whole brains minus cerebella are weighed and homogenized in 50 volumes of ice-cold 0.05M Tris buffer with a Tekmar tissue homogenizer. The homogenate is centrifuged at 40,000 g for 15min, the supernatant is decanted and the pellet resuspended in fresh buffer and recentrifuged at 40,000 g. The final pellet is resuspended in the original volume of fresh 0.05M Tris buffer. This yields a tissue concentration in the assay of 10mg/ml Assay 310l H2 O
Sumanth Dept of Pharmacology

20l 50l 50l 20l 50l 500l

5 M dextrorphan (total binding) 5 M levorphanol (non-specific binding) 2M NaCl or H2O 0.5M Tris buffer, pH 7.7 Drug or Vehicle 3H-Naloxone Tissue Suspension

The tubes are incubated for 30min at 37C. The assay is stopped by vacuum filtration through Whatman GF/B filters which are then washed 3 times with ice-cold 0.05M Tris buffer, pH7.7. The filters are then counted in 10ml of Liquiscint liquid scintillation cock-tail. Stereospecific binding is defined as the difference between binding in the presence of 0.1Mdextrorphan and 0.1M Levorphanol. Specific binding is roughly 1% of the total added ligand and 50% of the total bound in the absence of Na+ and 2% of the total added ligand and 65% of the total bound ligand in the presence of Na+ (100mM). The increase in binding is due to an increase in specific binding. EVALUATION Data are converted into % stereospecific 3H-naloxone binding displaced by the test drug. IC50 values are determined from computer-derived log-probit analysis. The sodium shift is calculated from IC50 values with and without NaCl. High sodium shifts are found with agonists, low values with antagonists and medium values with mixed agonists-antagonists.
3H-Dihydromorphine

Binding to Opiate Receptors in Rat Brain

PURPOSEAND RATIONALE Receptors are considered to mediate the supraspinal activity of opioids.3HDihydromorphine (3H-DHM) exhibits some selectivity for the receptor, a high affinity opiate binding site. The test is used to detect compounds that inhibit binding of 3H-DHM in a synaptic membrane preparation obtained from rat brain. PROCEDURE Reagents [1,7,8-3H]Dihydromorphine (3H-DHM) (specific activity 69 Ci/mmol) is obtained from Amersham. For IC50 determinations a 20nM stock solution is made up. Fifty l are added to each test tube to yield a final concentration of 0.5nM in the 2ml assay. Levallorphan tartrate is used for the determination of nonspecific binding. A 0.1mMstock solution is pre-pared in deionized water. Twenty l added to each of 3 tubes yields a final concentration of 0.1M in the 2ml assay.
Sumanth Dept of Pharmacology

A 1mM stock solution is made up of the test com-pounds in a suitable solvent and serially diluted, such that the final concentrations in the assay range from 106 to 109 M. At least 7 concentrations are used for each assay.

Tissue Preparation Male Wistar rats are sacrificed by decapitation. Whole brains minus cerebella are removed, weighed and homogenized in 30 volumes of ice-cold 0.05M Tris buffer, pH7.7. The homogenate is centrifuged at 48,000 g for 15min, the supernatant is decanted and the pellet resuspended in the same volume of buffer. This homogenate is then incubated for 30min at 37C to remove the endogenous opiate peptides and centrifuged again as before. The final pellet is resuspended in 50 volumes of 0.05M Tris buffer, pH7.7. Assay 1850l 80l 20l 50l Tissue Suspension Distilled water Vehicle, or Levallorphan, or appropriate concentration of drug [3H]DHM

Tubes are incubated for 30min at 25C. The assay is stopped by vacuum filtration through Whatman GF/B filters which are washed twice with 5ml of 0.05M Tris buffer. The filters are then placed into scintillation vials with 10ml Liquiscint scintillation cocktail and counted. EVALUATION Specific binding is defined as the difference between total binding and binding in the presence of 0.1mM Levallorphan. IC50values are calculated from the percent specific binding at each drug concentration. The KD value for [3H] DHM binding was found to be 0.38nM by Scatchard analysis of a receptor saturation experiment. The Ki value may be calculated from the IC50 by the ChengPrusoff equation: Ki =IC50/1 + L/KD

In Vivo Methods for Testing Central Analgesic Activity

Haffners Tail Clip Method

Sumanth Dept of Pharmacology

PURPOSEAND 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. Since then, the method has been used and modified by many authors. 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 25g 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 710mice. The drug is administered 15, 30 or 60min prior testing. 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 in 1/10 seconds increments. EVALUATION A cut-off time is determined by taking the average reaction time plus 3 times the standard deviation of the combined latencies of the control mice at all time periods. 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 1.5mg/kg s.c. for morphine and 7,5mg/kg for codeine s.c.

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 acetyl salicylic acid or phenyl-acetic acid type do not generally affect these responses. PROCEDURE Groups of 10 mice of either sex with an initial weight of 18 to 22g are used for each dose. The hot plate, which is commercially available, consists of an electrically heated surface. The temperature is con-trolled for 55 to 56C. 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
Sumanth Dept of Pharmacology

and after 20, 60 and 90min following oral or subcutaneous administration of the standard or the test compound. EVALUATION The prolongation of the latency times comparing the values before and after administration of the test com-pounds 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 7.5mg/kg s.c. morphine hydrochloride, 30mg/kg s.c. codeine hydrochloride, 30mg/kg s.c. pethidine hydrochloride and 400mg/kg s.c. phenazone were found to be effective, whereas aspirin showed no effect even at high doses.

Tail Immersion Test

PURPOSEAND 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 55C. PROCEDURE Young female Wistar rats (170210g bodyweight) 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 30min 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 55C. 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 deter-mined before and periodically after either oral or subcutaneous administration of the test substance, e. g., after 0.5, 1, 2, 3, 4 and 6h. 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. 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 behavioural changes. For example, an ED50 of 3.5mg/kg s.c. for morphine and an ED50 of 1.7mg/kg s.c. methadone was found. Acetylsalicylic acid at a dose of 640mg/kg p.o., phenylbutazone at a dose of 160mg/kg s.c. as well as nalorphine at a dose of 40mg/kg s.c. were inactive.
Sumanth Dept of Pharmacology

Electrical Stimulation of the Tail

PURPOSEAND RATIONALE Since the tail of mice is known to be sensitive to any stimulus, a method of electrical stimulation has been described as early as 1950 by Burn et al. The stimulus can be varied either by the duration of the electric shock or by an increase in the electric current. PROCEDURE As described by Kakunaga et al. (1966), male mice a weight of 20g are placed into special cages. A pair of alligator clips is attached to the tail whereby the positive electrode is placed at the proximal end of the tail. Rectangular wave pulses from a constant volt-age stimulator at an intensity of 4050V are applied. The frequency of the stimulation is 1 shock/s, and the pulse duration 2.5ms. The normal response time range of the stimuli is 34 s. Following administration of the drug, the response time is registered at 15min intervals until the reaction time returns to control levels. EVALUATION The data for each animal are plotted with reaction times on the ordinate and time intervals following ad-ministration on the abscissa. The area under the time response curve is calculated. In control animals the re-action time remains fairly constant and the area under the curve is approximately zero. Effects of morphine at 5mg/kg s.c. and meperidine 30mg/kg s.c. could easily be demonstrated.

Sumanth Dept of Pharmacology