The in vitro anti-5-hydroxytryptamine activity of compounds was investigated using the isolated oestrus rat uterus preparation. Dose-response curves to 5-hydroxytryptamine (5HT) were obtained in the presence or absence of varying concentrations of antagonist and the concentration of antagonist required to reduce the sensitivity of the preparation by a factor of 10 was calculated. Compounds were also investigated for their ability to antagonize phenylquinone-induced writhing, essentially by the method of Hendershot and Forsaith3. Compounds were administered orally, dissolved or suspended in 5 per cent gum acacia, to groups of 6 starved male albino mice (Tuck strain) weighing 1822 g. One hour later, unless otherwise stated, each animal received an intraperitoneal injection of phenylquinone, 2 mg/kg. The total number of writhes in a group of 6 animals was counted for the 20-min period immediately following the phenylquinone injection. A new aqueous solution of phenylquinone was prepared using gentle heat before individual experiments. The solution was kept in an amber glass bottle, stoppered when not in use, in order to avoid decomposition of the phenylquinone. The ED50 of a compound (the dose required to reduce by 50 per cent the number of writhes induced by phenylquinone treatment alone) was calculated by the method of Litchfield and Wilcoxon4. To improve the precision of the determinations, duplicate experiments were performed. Table 1.COMPARATIVE in into ANTI-5TH AND in vivo ANTI-PHENYL-QUINONE ACTIVITIES OF LSD, HOMOCHLOROCYCLIZINE, PROMETHAZINE AND MEPYRAMINEAnti-5HT i Concentration to ictivity Anti-phenylquinone activity Compound reduce uterine sensitivity 10 times (g/ml.) Relative potency EDSO (95 per cent Relative confidence limits) potency (mg/kg) LSD 0-01 1 0-019 (0-012-0-030) 1Homochlor-cyclizine 0-25 0-04 6-80 (4-50-10-9) 0-0028 Promethazine 0-1-0-2 0-05-0-1 12-0 (8-60-16-8) 0-0016Mepyramine > 2-5<0-004 133 (80-6-220) 0-0001 Table 2. EFFECT OF REPEATED INTRAPERITONEAL INJECTIONS OF PHENYL-QUINONE ON THE WRITHING RESPONSE IN MICEDay 1 2 3 4 5 Writhes to phenylquinone (percentage of control) 100 54 11 3 02 mg/kg phenylquinone was injected once daily on each of five successive days to a single group of mice; on each day a fresh group of animals was injected with phenylquinone to serve as control. d-Lysergic acid diethylamide (LSD), homochlorcyclizine and promethazine are known to antagonize the effects of injected or released 5HT; mepyramine is less active in this respect. The comparative activities of these compounds in antagonizing phenylquinone-induced writhing in mice together with their in vitro anti-5HT activities are summarized in Table 1. Eckhardt, Cheplovitz, Lipo and Govier5 have suggested that the release of 5HT may be a factor in the phenylquinone writhing response. It was thought that if the release of 5HT was implicated, then repeated intraperitoneal injections of phenylquinone to an animal would deplete the intestinal tissues of 5HT and decrease the number of writhes obtained at each injection. A group of 6 mice was injected with phenylquinone on each of 5 successive days; each day a fresh group of animals was injected with phenylquinone to serve as controls. The results of this experiment, summarized in Table 2, show that repeated injections of phenylquinone caused the expected reduction in the number of writhes. We also found that the 5HT content of the small intestine of animals given 5 injections of phenylquinone was only 60.7 per cent that of untreated animals; in a further experiment a single injection of phenylquinone reduced the 5HT content of intestinal tissue to 76.3 per cent that of untreated animals in 1 h. The 5HT was determined by a spectrofluorometric method6. It is not known whether the depletion of 5HT by phenylquinone is a specific drug effect, but although treated animals exhibited diarrhoea, autopsy did not reveal obvious irritation or damage to the intestinal tract.The effect of phenylquinone was next investigated in mice sensitized to 5HT by adrenalectomy since Higgin-botha.m7 has shown that there is up to a 25-fold increase in sensitivity to 5HT in these animals. We have found that the sensitivity of mice, adrenalectomized 5 days previously, to injected phenylquinone was markedly increased, a 69 per cent increase in the writhes being observed. Next, the effects of hydrazine and non-hydrazine monoamine oxidase inhibitors (MAOI) were investigated in the phenylquinone test, since these compounds are known to increase tissue concentrations of 5HT. In view of the results obtained with 5HT antagonists, MAOI's were expected to potentiate the writhing response. Surprisingly, tranylcypromine, phenelzine and iproniazid administered orally 4 h prior to the phenylquinone injection showed definite antagonistic activity to the writhing response (Table 3), and their potencies in this respect closely followed their activities as inhibitors of monoamine oxidase. Harmine and pargyline also inhibited the writhing response. This result need not necessarily preclude involvement of 5HT in the writhing response since MAOI's might inhibit its release from peripheral stores.Table 3. ANTAGONISTIC EFFECTS OF MONOAMINE OXIDASE INHIBITORS ON THE PHENYLQUINONE WRITHING RESPONSE IN MICE Compound ED50 (95 per cent confidence limits) (mg/kg)Tranylcypromine 3-70 (2-21-6-18) Phenelzine 7-70 (3-50-16-9)Iproniazid 112 (62-2-201) As a result of these tests two additional classes of compounds, 5HT antagonists and MAOI's, have been shown to be potent inhibitors of phenylquinone-induced writhing in mice. This adds to the type of compounds already known to be active in this test. One might, therefore, ask if the test has any predictive value in the pharmacological evaluation of new compounds. Certainly, a positive result can no longer be regarded as indicating a high probability of weak analgesic or anti-inflammatory activity. However, we believe the test is of value as a simple primary screening test to detect a diversity of known pharmacological types. These have to be characterized more fully by other tests.