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In vitro test for interaction
with histaminergic H2
-receptors in an isolated guinea-pig
right atrium - Automated assay
. Introduction: Histamine increases the frequency of an isolated, spontaneously-beating guinea pig right atrium placed in an organ bath; this effect is brought about by interaction with specific H2 -receptors. Drugs with affinity for these receptors can induce similar increases in frequency and/or inhibit histamine-induced increases in frequency. This in vitro test is used to evaluate a test compound for interaction with histaminergic H2-receptors in an isolated guinea-pig right atrium. Test medium: Right atria from male albino guinea-pigs (300 - 500 g), spf-bred by CPB-TNO, Zeist, The Netherlands. The right atria are placed in an organ bath containing Krebs solution, composition: NaCl 6,78 g 117 mmol KCl 0,42 g 5,6mmol MgSO4 0,14 g 1,2 mmol CaCl2 0,28 g 2,5 mmol NaH2P O 4 0,18 g 1,3 mmol NaHCO 3 2,1 g 25 mmol Glucose 1,00 g 5,6 mmol Made up to 1 litre with distilled water. Ref. compound: Cimetidine : pA2 approx. 6 - 6,5. Vehicle: Only solutions can be used in this test. The vehicle is chosen from the following list, which is arranged in order of preference: 1. Krebs solution 2. NaCl, 0,9% in distilled water 3. Distilled water 4. Poloxalene (Pluronic F 68, Wyandotte Chemicals Corp., Michigan 48192, U.S.A.), less then 10% in distilled water 5. Polyethylene glycol (Carbowax C200), less then 25% in distilled water 6. Propylene glycol, less then 25% in distilled water Technique: Guinea-pig right atrium preparation A male guinea-pig is killed by a blow on the head. The chest is opened and the heart is dissected free from surrounding tissue as completely as possible. The main vessels are severed, the heart is removed and placed in a beaker with Krebs solution. The pericardium is removed and a small hook, connected to a thread, is fixed in the free end of the right atrium. The right atrium is lifted and cut at the other end as close to the ventricle as possible. A second hook, connected to a thread, is fixed in the other end. The right atrium is fixed in a 10 ml organ bath filled with Krebs solution, kept at 37 °C and bubbled through with oxygen/carbon dioxide (95% O2 + 5% CO2). One end is tied to a fixed pin and the other one to an isometric transducer loaded with 1g. Procedure: a. Preliminary investigation The right atrium is equilibrated in the bath fluid for at least 30 minutes to stabilize the frequency. Every 5 minutes it is washed. All washes consist of 3 replacements of bath fluid. To determine a log dose-response curve, histamine dihydrochloride is pipetted into the organ bath in a cumulative way, starting with a bath concentration of 10-8 mol/l. Two minutes after the addition of histamine the frequency is measured and the concentration is raised by a factor of 3,2. This is continued until the maximum frequency is obtained. The right atrium is washed with bath fluid and this wash is repeated every 5 minutes. When the frequency has returned to its pre-drug level a second curve for histamine is made. This procedure is repeated until 2 consecutive, identical cumulative log dose response curves for histamine have been obtained. After the last curve for histamine the right atrium is washed with bath fluid every 5 minutes until the pre-drug level is stabilized again. Test compound is pipetted into the organ bath and the right atrium is equilibrated for at least 30 minutes (the response must be stable) and the agonistic activity is evaluated. To evaluate antagonistic activity a log dose-response curve is made for histamine in the presence of test compound as described above. To check the reversibility of the interactions the right atrium is washed with bath fluid every 5 minutes and equilibrated for at least 30 minutes. Then another log dose-response curve for histamine is made. If the last curve is identical with the 2 initial curves the right atrium can be used for another experiment. b. Determination of agonistic activity For test compounds showing agonistic activity in the preliminary investigation a cumulative log dose-response curve is made. After the 2 consecutive, identical cumulative log dose-response curves for histamine, the curves for the test compound are made, alternated with one for histamine. The curves are made in at least 2 different right atria. To check that the effect is mediated via the H2-receptors a cumulative log dose-response curve is made for the test compound in the presence of cimetidine, a specific H2-receptor blocker. c. Determination of antagonistic activity For test compounds showing antagonistic activity in the preliminary investigation, cumulative log dose-response curves for histamine are made in the presence of at least 3 different doses of test compound (usually in the range 1:3,2:10). After the 2 consecutive, identical cumulative log dose-response curves for histamine test compound is added to the organ bath and the curves for histamine are made again. The curves for each dose of test compound are made in at least 2 different right atria. For test compounds showing potentiating activity in the preliminary investigation, cumulative log dose-response curves for histamine are made in the presence of higher doses of test compound in order to detect a dose dependency or a change to an antagonistic effect. The curves are made in at least 2 different right atria. Compound doses: If no relevant data from other tests are available, the usual initial concentration is 10-6 mol/l bath fluid. Evaluation of responses: The lengths of 10 consecutive deflections on the recording paper are measured in mm and the frequencies are calculated. Each respons is expressed as a percentage based on the maximum response of the preceding curve for histamine (= 100%). (The frequency of the spontaneous beets before test compound is added is set at 0 %). Cumulative log dose-response curves are made for each series of measurements by plotting the percentage range (ordinate) against the log concentration (abscissa), as described by Van Rossum. Agonistc activity Agonistic activity of a test compound is characterized by the pD2-value and the intrinsic activity α (figure 1). pD2 is a measure for the dissociation constant of the equilibrium: receptor - agonist. It is the negative logarithm of the concentration of agonist which induces a frequency-increase of 50% of the maximum response. This value can be read from the cumulative log dose-respons curve. The final result for a test compound is expressed as the mean pD2-value of all corresponding curves, together with s.e. mean. The pD2-value of histamine is approx. 5,5-6. The intrinsic activity α is a measure for the potency of an agonist in omparison to histamine, defined as the ratio of the maximum responses induced by the agonist and histamine. It can be easily calculated with the help of the cumulative log dose-respons curves. The final result for a test compound is expressed as the mean α of all corresponding curves. Antagonistic activity Competitive antagonistic activity of a test compound (it induces a parallel shift of the log dose-response curve for histamine to the right) is characterized by the pA2-value (figure 2). pA2 is a measure of the dissociation constant of the equilibrium: recceptor - antagonist. It is the negative logarithm of the concetration of antagonist which produces a log dose-respons curve for histamine, identical to the original one when twice as much histamine is used (a shift of log 2 to the right measured at the 50% response level). This value can be calculated using the formula: pA2 = pA x + log (x - 1) where: pAx = negative logarithm of the concentration of test compound x = antilogarithm of the shift of the log dose response curve for histamine, measured at the 50% response level, due to the presence of antagonist (pAx). It can be easily calculated with the help of the cumulative log dose-response curves. The final result for a test compound is expressed as the mean pA2-value of all curves made in the presence of test compound, together with the s.e. mean. Non-competitive antagonistic activity of a test compound (it induced no shift of the log dose-response curve for histamine, but all responses are decreased by the same ratio) is characterized by the pD2'-value (figure 3). pD2' is a measure for the blocking of H2-receptors for interaction with hisamine, by irreversible binding of antagonist to the receptor or by any other process. It is the negative logarithm of the concentration of antagonist, which decreases the maximum response of histamine by 50%. This value can be calculated using the formula: pD 2' = pA 2 + log 100 - D D where: pA x = negative logarithm of the concentration of antagonist Δ = maximum response of histamine in the presence of antagonist (pAx), expressed as a percentage based on the maximum response of the preceding curve for histamine (= 100%). This value can be read from the log dose-response curve. Mixed competitive and non-competitive (dualistic) antagonistic activity of a test compound (it induces a shift of the log dose-response curve for histamine to the right and all responses are decreased by the same ratio) is characterized by the pA2 and pD2'-values (figure 4). These values can be calculated using the formulas: pA2 = pA x + log (x - 1) and pD2' = pA2 + log 100 - D D The final result for a test compound is expressed as the mean pA2- and pD2'-values of all curves made in the presence of test compound, together with the s.e. means. Potentiating activity of a test compound (it induces a parallel shift of the log dose-response curve for histamine to the left) is not evaluated. Interpretation of results: Test compounds showing agonistic activity, which is inhibited by cimetidine, have histaminergic H2-activity: they are H2-mimetic compounds. Compounds having a low intrincis activity and an antagonistic activity are called "partial agonist". The pA2-value and the pD2'-value are measures for the competitive and the non-competitive antihistaminergic H2-activities of the test compound, respectively. The interpretation of the results is as follows:
Test compounds showing potentiating activity may inhibit the uptake or the metabolism of histamine by the right atrium. This effect is difficult to interpret. Other tests are required for its evaluation. Quantities required: 20 mg. Reference: Rossum, J.M. van, Archives Internationales de Pharmacodynamie, 143 (1963) 299-330.  |