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In vitro test for interaction
with Histaminergic H1-receptors in isolated guinea-pig ileum Introduction: Histamine induces contractions in a piece of guinea-pig ileum placed in an organ bath by interaction with (H1) receptors on the smooth muscle cells. Drugs with affinity for these receptors can induce similar contractions and/or inhibit histamine-induced contractions. This in vitro test is used to evaluate a test compound for interaction with histaminergic H 1 -receptors in isolated guinea-pig ileum. Test medium: Pieces of ileum from male albino guinea-pigs (300 - 500 g), SPF-bred by CPB-TNO, Zeist, The Netherlands. The pieces of ileum are placed in an organ bath containing modified Tyrode solution, composition: NaCl 8,0 g (137 mmol) KCl 0,2 g ( 2,7 mmol) CaCl 2 0,2 g ( 1,8 mmol) NaHPO 4 0,05 g ( 0,4 mmol) NaHCO3 1,0 g (11,9 mmol) Glucose 1,0 g ( 5,6 mmol) Atropine sulphate 2x10-6 g Made up to 1 litre with distilled water. Ref. compound: Dexchlorpheniramine maleate : pA>2 approx. 8,9 Cyproheptadine HCl: pA2 approx. 8,5 Vehicle: Only solutions can be used in this test. The vehicle for the stock solutions is preferably chosen from the following list, which is arranged in order of preference: 1. Modified Tyrode solution 2. NaCl, 0,9% in distilled water 3. Distilled water 4. Poloxalene (Pluronic F68, Wyandotte Chemicals Corp., Michigan 48192, U.S.A.) less than 10% in distilled water 5. Polyethylene glycol (Carbowax C200), less than 25% in distilled water 6. Propylene glycol, less than 25% in distilled water Technique: Guinea-pig ileum preparation A male guinea-pig is killed by a blow on the head and exsanguinated. The abdomen is opened and the caecum is lifted. A part of the ileum, attached to the rear of the caecum, is removed, placed in a dish and covered with modified Tyrode solution. The ileum should be handled with fingers, and not with forceps, to avoid damage to the tissue. The mesentery is trimmed away and the ileum is cut into pieces of 2 - 3 cm (in relaxed condition). If a piece of ileum is not clean, the contents must be washed out with modified Tyrode solution. Excessive pressure must be avoided to prevent irreparable damage of tissue. A thread is attached to each end of the piece of ileum by inserting a needle from the inside of the ileum to the outside. Care should be taken that the lumen of the ileum is not closed off. The piece of ileum is firmed in a vertical position in a 10 ml organ bath filled with modified Tyrode solution, kept at 37C 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 isotonic transducer loaded with 1 g. Procedure: a. Preliminary investigation The ileum preparation is equilibrated in the bath fluid for at least 10 minutes to stabilize the base-line value. Every 5 minutes it is washed. All washes consist of 3 replacements of bath fluid. To determine a log dose-response curve, histamine is pipetted into the organ bath in a cumulative way, starting with a bath concentration of 10-8 mol/l. After equilibration to stabilize the response the concentration is raised by a factor of 3,2. This is continued until the maximum contraction is obtained. The ileum preparation is washed with bath fluid and this wash is repeated every 5 minutes until the zero-level is stabilized. Then 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 ileum preparation is washed with bath fluid every 5 minutes until the zero-level is stabilized. Test compound is pipetted into the organ bath and the ileum preparation is equilibrated for at least 15 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 ileum preparation is washed with bath fluid and equilibrated for at least 10 minutes (a wash every 5 minutes). Then another log dose-response curve for histamine is made. If the last curve is identical with the 2 initial curves the ileum preparation 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 ileum preparations. To check that the effect is mediated via the H1-receptors a cumulative log dose-response curve is made for the test compound in the presence of dexchlorpheniramine maleate, a specific H1-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 after equilibration for at least 15 minutes the curves for histamine are made again. The curves for each dose of test compound are made in at least 2 different ileum preparations. 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 ileum preparations. Compound doses: If no relevant data obtained in other tests are available, the usual starting concentration is 3 x 10-5 mol/l bath fluid. Evaluation of responses: The heights of deflection on the recording paper are measured in mm, using the response just before addition of test compound or histamine as zero level. Each response is expressed as a percentage based on the maximum response of the preceding curve for histamine (= 100%). Cumulative log dose-response curves are made for each series of measurements by plotting the percentage change (ordinate) against the log concentration (abcissa), as described by Van Rossum Agonistic 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: H1-receptor - agonist. It is the negative logarithm of the concentration of agonist which induces a contraction of 50% of the maximum response. This value can be read from the cumulative log dose-response curve. The final result for a test compound is expressed as the mean pD2 -value of all corresponding curves, together with the s.e. mean. The pD 2-value of histamine is approx. 6 - 7. The intrinsic activity α is a measure for the potency of an agonist in comparison to histamine, defined as the ratio of the maximum responses induced by the test compound and histamine. 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 α -value 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). pA 2 is a measure for the dissociation constant of the equilibrium: H1-receptor - antagonist. It is the negative logarithm of the concentration of antagonist which produces a log dose-response 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: pA 2 = pAx + log (x - 1) where: pAx = negative logarithm of the concentration of antagonist 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 pA 2 -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 induces 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). pD 2' is a measure for the blocking of H1-receptors for interaction with histamine, 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: 100 - Δ pD 2' = pA x + log --------------- D where: pAx = 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. The final result for a test compound is expressed as the mean pD2'-value of all curves made in the presence of test compound, together with the s.e. mean. Mixed competitive and non-competitive (dualistic) antagonistic activity of a test compound (it induces a shift of the log dose-response curve for histamine and all responses are decreased by the same ratio) is characterized by the pA2 - and pD 2 '-values (figure 4). These values can be calculated using the formulas: pA2 = pAx + log (x-1) and 100 - Δ pD 2' = pA x + log --------------- D The final result for a test compound is expressed as the mean pA2 - and pD 2 '-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 dexchlorpheniramine,, have histamine-like activity; they are histaminergic compounds. Compounds having a low intrinsic activity and an antagonistic activity are called "partial agonists". The pA2 -value and the pD 2 '-value are measures for the competitive and the non-competitive antihistaminergic 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 ileum. This effect is difficult to interpret. Other tests are required for its evaluation. Quantities required: 20 mg. Attached: Appendix with figures. Reference : Rossum, J.M. van, Archives Internationales de Pharmacodynamie, 143(1963) 299 - 330.  |