Problems of drug selectivity and dose – pharmacology

Abstract

In a recent study entitled ‘Midline serotonergic neurones contribute to widespread synchronized activity in embryonic mouse hindbrain’ by Hunt et al. (2005) published in The Journal of Physiology, the authors used 5-HT receptor agonists and antagonists to mimic and inhibit these hindbrain effects to show that serotonergic neurones were involved. This approach can be considered the standard method in determining the nature of the chemical transmitter system(s) involved in the production of a physiological phenomenon. However, the use of drugs has certain pitfalls which are usually related to the fact that no drug is specific for any receptor subtype and that so-called specificity of a drug for a receptor subtype is related to the dose used. In addition, to overcome other pitfalls such as drug-specific effects, antagonists and agonists with similar affinities for the receptor subtype under investigation but with different chemical structures are often used. Further, the different selectivity profiles of such drugs also eliminates other possible receptors. In this paper the authors have carried out a study to determine the transmitter and receptor involved in a physiological phenomenon. The experimental protocols they have used to do this are, in fact, appropriate. However, I am writing this letter because of one very interesting and surprising observation that they reported: that the non-selective 5-HT receptor antagonist methiothepin did not block or interfere with the phenomenon they were studying. Because of an incorrect understanding of the selectivity of this drug for 5-HT2 receptor subtypes, I do not think the authors can conclude that the effects are mediated by 5-HT2A receptors and thus 5-HT. The authors state in their paper that ‘Methiothepin, a 5-HT1B/1D/2C/7 antagonist, at 100 μm, did not block activity (n = 4).’ This is not correct. If you look at the data from Bonhaus et al. (1997) on recombinant rat 5-HT2A and 5-HT2C receptors, the pKi for methiothepin is 9.6 and 8.61, respectively. So it would seem that methiothepin is a 2A antagonist as well as having activity at the receptors mentioned above. In addition, methiothepin is also an α-adrenoceptor and dopamine receptor antagonist. However, the authors do use other drugs that are known to also block 5-HT receptors: ketanserin (pKi of 9.05 at 5-HT2A and 7.3 at 5-HT2C, respectively) and spiperone (pKi of 9.37 at 5-HT2A and 6.06 at 5-HT2C, respectively, data from Bonhaus et al. 1997). In fact, if only these compounds had been used it would be, even in my opinion, appropriate to conclude that the effects observed in this paper could be mediated by 5-HT2A receptors, as α-adrenoceptor antagonists were reported to be ineffective (it would have been interesting to know what dopamine antagonists do in this preparation). It should be pointed out that both compounds are α-adrenoceptor antagonists and spiperone is also a potent dopamine antagonist as well as interfering with other 5-HT receptor subtypes. However, from the pKi data both ketanserin and spiperone should be equi-effective in attenuating spontaneous activity, but in Fig. 6 spiperone is between 1000 and 100 × more potent. The other important piece of evidence for determining if an effect is mediated by a transmitter is to determine if it can be mimicked by the transmitter itself or a selective agonist for the particular receptor suspected to initiate the phenomenon. However, such experiments do not prove physiological, but only pharmacological involvement. The authors have in fact carried out such experiments but the data are, I think, inconclusive; to quote, ‘Serotonin, either alone or after inhibition of the serotonin transporter with citalopram (5 nM, n = 8), at concentrations of 1.0 μm (n = 2) or at 10–50 μm (n = 16) did not modulate the activity.’ At least the authors made a very good attempt but the result is puzzling although explainable. The authors also went on to use DOI, a so-called selective 5-HT2A receptor agonist, which I would prefer to call just a 5-HT2 receptor agonist; again the results were not clear-cut as the authors point out in the discussion: ‘Application of the serotonergic agonist DOI did not consistently activate or increase activity in isolated lateral regions: in one experiment, DOI (50 μm) caused one transient in quiescent lateral tissue; an average increase in frequency of 1.66 (to an average rate of 1.45 events min−1, n = 2, 20 μm DOI) in two experiments; and no effect in another (20 μm).’ From the data presented there is not enough evidence to conclude that the effect is mediated by 5-HT2A receptors or even 5-HT.