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In vitro test for interaction with Tryptamine D-receptors in a rat fundus strip - Automated assay Introduction: Serotonin induces contractions in a rat fundus strip placed in an organ bath by interaction with specific receptors on the smooth muscle cells. Drugs with affinity for these receptors can induce similar contractions and/or inhibit Serotonin-induced contractions. This fully automated in vitro test is used to evaluate a test compound for interaction with tryptamine D-receptors in a rat fundus strip. Test medium: Fundus strip from male Cpb: WU rats (200 - 400 g), spf-bred by CPB-TNO, Zeist, The Netherlands. The fundus strips are placed in an organ bath containing modified Krebs solution, composition: NaCl 6,78 g 116 mmol KCl 0,33 g 4,4 mmol MgCl2 .6H 2O 0,24 g 1,2 mmol CaCl2 .2H 2O 0,37 g 2,5 mmol Na2 SO 4 0,16 g 1,1 mmol KH2 PO 4 0,21 g 1,5 mmol NaHCO3 1,03 g 12,3 mmol Glucose 2,00 g 11,1 mmol Made up to 1 litre with ultrapure water (Super-Q quality); the pH is approx. 7.4. This solution contains 130 mmol/l Na+ -ions, 5,9 mmol/l K + -ions and 128 mmol/l Cl- -ions. To reduce the tone of the strips, 5,6 nmol/l indomethacin are added (Frankhuijzen and Bonta). Reference compound: Serotonin creatinine sulphate : pD2 = 7,6 - 8,1. Methysergide hydrogen maleate : pA2 = 8,8 + 0,1 (n = 4); pD 2 ' = 8,5 + 0,1 (n = 7). Cyproheptadine HCl : pA 2 = 8,1 + 0,1 (n = 7); pD 2 ' = 6,9 + 0,3 (n = 3). Vehicle: Only solutions can be used in this test. The vehicle for the stock solutions is chosen from the following list, which is arranged in order of preference: 1. Water (pH range 1-10) 2. Poloxalene (Pluronic F 68, Wyandotte Chemicals Corp., Michigan, U.S.A.), less than 10% in water 3. Polyethylene glycol (Carbowax C 200), less than 25% in water 4. Propylene glycol, less than 25% in water For aqueous solutions, water for injection (USP) or ultrapure water (Super-Q quality) is used. The stock solutions of test compounds are diluted with Krebs solution before the start of the experiment. The diluted solutions are kept at room temperature. Technique: Apparatus The experimental set-up (constructed by the Organon Technical Department, see Figure 1 in Appendix) is fully automated and controlled by a computer programme. The set-up consists of: 1. a thermostated 10-ml organ bath with equipment for computer-controlled dispersions (inlet at the bottom and outlet at the top of the bath) of test medium and 3 test compounds and for computer-controlled aeration of the medium. 2. an isotonic transducer (position sensing detector) with equipment for automatic adjustment to a preset base-line level 3. a high-precision gradient pump for computer-controlled step-wise dispersion of solutions of agonist 4. a MINC-II computer (DEC, Maynard, Mass., U.S.A.) with VT 105 monitor controlling the experiment. The experimental data are collected using a system which eventually reports results alpha-numerically and/or graphically. Rat fundus strip preparation A male rat is killed by a blow on the head and exsanguinated. The abdomen is opened and the stomach is lifted. The stomach is cut at the pylorus and at the oesophagus and dissected free from other tissues. The stomach is opened along its larger curvature, rinsed with Krebs solution, placed in a dish and covered with Krebs solution. Two strips are cut from the fundus, each 3-4 mm thick and 3-4 cm long. The strips are handed with fingers and not with forceps to avoid damage to the tissue. A small hook, connected to a thread, is fixed in the lower end of the fundus strip and a second hook, fitted with an eye, is fixed in the other end. The fundus strip is fixed in the organ bath with Krebs solution and kept at 37 ° C. The thread is tied to the isotonic transducer (loaded with 1 g) and the eye is slipped over the fixed pin at the bottom of the organ bath. The organ bath is aerated with oxygen/carbon dioxide (95% O 2 + 5% CO2 ). After equilibration, the output of the transducer is set at 0 mm for the resting length of the fundus strip. Procedure Each experiment consists of 2 to 6 experimental cycles and each cycle of 2-5 determinations of a cumulative log-dose response curve with Serotonin creatinine sulphate (CRC-runs). Each CRC-run is followed by a washing procedure. The 1st experimental cycle is used to check the consistency of the response of the fundus strip and the other cycles to investigate test compounds. a. Washing procedure Each washing procedure consists of three 300-s washings (approx. 40 s for replacement and approx. 260 s for equilibration). At the end of each washing, the length of the fundus strip is estimated. The length estimated at the end of the last washing should be within the range of -5,0 to +3,0 mm. If this requirement is not met, additional washings are performed until this requirement is fulfilled. If after the 10th washing the requirement is not yet met, the experiment is stopped. b. Control and test CRC-runs A control CRC-run starts with two 600-s washings and at the end of each washing the length of the fundus strip is estimated. If the length estimated at the end of the last washing meets the (washing procedure) requirements, the actual CRC-run is performed; if not , the experiment is stopped. A test CRC-run starts with three 600-s replacements of bath fluid by a solution of test compound in Krebs solution (approx. 60 s for replacement and approx. 540 s for equilibration). At the end of each replacement, the length of the fundus strip is estimated. As no requirements for these lengths are defined, the actual CRC-run is always performed after the last measurement. The actual CRC-run consists of a step-wise cumulative addition of Serotonin at 20-sec intervals. In the last 10-sec period of each interval, the Serotonin induced contraction is determined. For the cumulative additions, 3 solutions of Serotonin creatinine sulphate, 1 x 10-8, 1 x 10-6 and 1 x 10-4 mol/l NaCl, 0,9% in water, are used. These solutions are kept at 6 °-8°C. The usual starting concentration is 3 x 10-10 mol/l bath fluid and the concentration is increased step-wise by a logarithmic gradient factor of 100,1. The actual CRC-run is terminated if the increase in contraction per step is less than approx. 0,3 mm over 3 steps and if the E max (see c.1.) is at least 10 mm. If the E max is less than this value, the increase in contraction per step should be less than 0,3 mm over 30 steps in a control CRC-run and 40 steps in a test CRC-run. c. Characterization of a CRC The responses of each CRC-run can be expressed as a CRC by plotting the agonist induced contraction in mm (ordinate) against the negative logarithm of the concentration of agonist (pDx ) (abscissa) as described by Van Rossum. The resting length of the observed CRC is adapted to determine the base-line value using an iterative fitting procedure. This CRC can be characterized by the following 4 variables: 1. Maximum effect E max : The maximum effect is calculated using the formula: E max = Lb - Lmin where: L b = base-line value of the CRC expressed in mm L min = minimum length (maximum contraction) of the CRC expressed in mm. Accepted maximum difference for identical CRC's: 9%. 2. pD 2 : The pD2 is the negative logarithm of the concentration of agonist which induces a contraction of 50% of its Emax . Accepted maximum difference for identical CRC's: 0,15. 3. Relative position of the inflexion point: The relative position of the inflexion point in the CRC is calculated using the formula : Inf = L inf - Lmin        E max where: L inf = length at the inflexion point in the CRC expressed in mm. Accepted maximum difference for identical CRC's: 0,25. 4. Normalized maximum slope: The normalized maximum slope is the slope through the inflexion point corrected for the E max -value, expressed as its arctangent. Accepted maximum difference for identical CRC's: 12%. Two CRC's are considered to be identical, if the differences in all 4 variables are less than the accepted maximum differences mentioned above. A control CRC is considered to be anomalous if does not meet one of the following requirements: 1. The difference between the observed resting length and the fitted base-line value should be less than 0,2 x Emax mm. 2. The relative position of the inflexion point should be within the range 0,25 - 0,75. Anomalous control CRC's are not used for comparisons and evaluations. If 2 anomalous control CRC's are observed within one cycle, the experiment is stopped. d. Experimental cycles The 1st experimental cycle used for checking the consistency of the response of the fundus strip preparation and establishing the base-CRC for the 1st test CRC, consists of 2-5 control CRC-runs. The 1st control CRC is stored in the "actual" memory for comparison with the 2nd control CRC. After this comparison the 2nd control CRC replaces the 1st one in the "actual" memory. If the 2nd control CRC is identical to the 1st one, the 2nd experimental cycle can be performed; if not, a 3rd control CRC-run is performed. The evaluation of this 3rd control CRC is similar to that of the 2nd one. If necessary, this procedure is continued until 2 consecutive identical control CRC's have been obtained. If after the 5th control CRC-run this requirement has not yet been met, the experiment is stopped. The last control CRC of this experimental cycle is used as the base-CRC for the test CRC obtained in the 2nd experimental cycle. The 2nd experimental cycle used for investigating a test compound and establishing the base-CRC for the 2nd test CRC, consists of one test CRC-run and 1-3 control CRC-runs. This cycle starts with the test CRC-run, which is followed by the 1st control CRC-run. This control CRC is compared with the last control CRC of the 1st experimental cycle. If both control CRC's are identical, the control CRC of the 2nd cycle replaces that of the 1st cycle in the "actual" memory and the 3rd experimental cycle can be performed. If they are not identical, no replacement occurs and a 2nd control CRC-run is performed. The evaluation of this 2nd control CRC-run is the same as that of the 1st one. If necessary, a third control CRC-run is performed. If the 3rd control CRC is not identical to the last control CRC of the 1st experimental cycle, the experiment is stopped. The 3rd and 4th experimental cycles are similar to the 2nd one. After the 4th experimental cycle, the experiment is stopped or, if required, the same test compounds (in the same sequence) are tested again. Each test compound is investigated in at least 2 different fundus strips. 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: Each test CRC is evaluated using the preceding control CRC as base-CRC. Using the CRC characteristics, the following variables are calculated: 1. A change in base-line value induced by the test compound (measured during the 2nd interval for equilibration in the test CRC-run) with respect to the pre-set base-line value (during the first 2 intervals of the equilibration in the test CRC-run the automatic correction of the base-line value interrupted). A change of more than 4 mm is considered to be significant. An increase in Lmin indicates that the test compound may have agonistic been changed. This can be confirmed by performing a similar 2nd experiment in which the test compound is used as agonist and Methysergide hydrogen maleate as tryptamine D-antagonist. Agonistic activity is characterized by the pD 2-value and the intrinsic factor α of the test compound. The pD 2-value is a measure dissociation constant of the equilibrium: receptor-agonist and the intrinsic factor for the potency of the agonist, defined as the ratio of the E max induced by the agonist (determined in the 2nd experiment) and Serotonin (usually the mean value of the last 10 base-CRC's). 2. Δ pD2 = change in negative logarithm of the concentration of agonist (which induces a contraction of 50% of the Emax ) of the test CRC with respect to the base-CRC. A change of more than 0,15 is considered to be significant. If Δ pD2 < 0 (the test CRC is shifted to the right), the test compound has competitive antagonistic activity. This activity can be characterized by the pA 2 -value, defined as the negative logarithm of the concentration of antagonist which produces a CRC for Serotonin, identical to the original one when twice as much Serotonin is used (a shift: Δ pD2 = log 0,5 = -0,301). This value can be calculated using the formula: pA 2 = pAx + log (x-1- 1) where pA = negative logarithm of the concentration of test compound x = antilogarithm of Δ pD2 . lf Δ pD 2 > 0 (the test CRC is shifted to the left), the test compound induces a sensitisation of the fundus strip. This activity can be characterized by the pSt -value, defined as the negative logarithm of the lowest concentration of sensitizer (threshold concentration) which produces a CRC for Serotonin, identical to the original one when a significant lower amount of Serotonin is used (a shift in Δ pD 2 of at least 0,16) 3. R = ratio of the Emax -values for the test CRC and the base CRC. A change of more than 9% is considered to be significant. A decrease in E max combined with no or only a weak change in the relative position of the inflexion point and/or normalized slope, indicates that the test compound has non-competitive antagonistic activity. This activity can be characterized by the pD 2 '-value, defined as the negative logarithm of the concentration of antagonist which decreases the Emax of the agonist Serotonin by 50%. This value can be calculated using the formula: pD 2' = pAx + log (R-1 - 1). An increase in E max combined with no or only a weak change in the relative position of the inflexion point and/or normalized slope, indicates that the test compound potentiates the efficacy of Serotonin. This activity can be characterized by the pPt -value, defined as the negative logarithm of the lowest concentration of potentiator (threshold concentration) which increases the E max of the agonist Serotonin by at least 10%. If possible, for each test compound, the final result per concentration range is expressed as the mean value of all corresponding curves, together with the s.e. mean. Interpretation of results: Usually experiments with control CRC's with an Emax -value of less than 10 mm are discarded. If the relative position of the inflexion point and/or normalized maximum slope of test CRC differ considerably from the base-CRC or if, on visible inspection, the shape of the test CRC shows abnormalities, the results should be interpreted with care or no interpretation should be given. A significant change in the base-line value usually indicates a direct effect of the test compound on the fundus strip. An increase in this length suggests the presence of agonistic activity, which can be characterized by the pD 2 -value and intrinsic factor α of the test compound (Fig. 2 in Appendix). An increase in base-line length is difficult to explain in terms of a direct interaction with tryptamine D-receptors. Test compounds inducing a parallel shift of the CRC to the right show competitive antagonistic activity, which is characterized by its pA2 -value. The pA 2 is a measure for the dissociation constant of the equilibrium: tryptamine D-receptor-antagonist (Fig. 3 in Appendix). Test compounds inducing a parallel shift of the CRC to the left, sensitize the fundus strip. This activity is characterized by its pS t -value. The pSt is difficult to explain in terms of a direct interaction with the tryptamine D-receptors. It can be related to an inhibition of the uptake or metabolism of Serotonin by the fundus strip. Test compounds inducing no shift of the CRC, but only a decrease in all responses by the same ratio show non-competitive antagonistic activity, which is characterized by its pD2-value. The pD2 is a measure for the blocking of tryptamine D-receptors for interaction with Serotonin, by irreversible binding of antagonists or by any other process (Fig. 4 in Appendix). Test compounds inducing no shift of the CRC, but only an increase in all responses by the same ratio potentiate the effect of Serotonin. This activity is characterized by its pPt -value. The pP t is difficult to explain in terms of a direct interaction with tryptamine D-receptors. Test compounds inducing both a parallel shift of the CRC to the right and a decrease of all responses by the same ratio have mixed competitive and non competitive (dualistic) antagonistic activity, which is characterized by its pA2 - and pD 2 ' -values (Fig. 5 in Appendix). Compounds showing agonistic (usually with a low intrinsic factor) and antagonistic activity are called "partial (ant)agonists". Quantities required: 20 mg Reference: Rossum, J.M. van, Archives Internationales de Pharmacodynamie, 143 (1963) 299-330. Frankhuijzen, A.L. and Bonta, I.L., European Journal of Pharmacology, 31,(1975) 44-52 Graaf, J.S. de, Vos, C.J. de, Steenbergen, H.J., Journal of Pharmacological Methods, 10 (1983) 113-135.