AFFINITIES OF THE PROTONATED AND NON‐PROTONATED FORMS OF HYOSCINE AND HYOSCINE N‐OXIDE FOR MUSCARINIC RECEPTORS OF THE GUINEA‐PIG ILEUM AND A COMPARISON OF THEIR SIZE IN SOLUTION WITH THAT OF ATROPINE

Abstract

1 At 37°C in 0.1 M NaCl the pKa of hyoscine (10 mM) is 7.53; the non-protonated form has about one-tenth of the affinity (log K = 8.58) of the protonated form (log K = 9.58) for muscarine-sensitive receptors of the guinea-pig ileum at 37°C.2 In the same conditions the pKa of hyoscine N-oxide is 5.78 and the non-protonated form is inactive on the ileum whereas the protonated form is highly active with log K estimated to be 9.9, at least as active as hyoscine methobromide (log K = 9.85).3 Hyoscine methobromide appears to occupy less space in water than atropine methobromide; hyoscine hydrochloride occupies less space than hyoscyamine hydrochloride: the non-protonated forms are slightly bigger. Hyoscine N-oxide hydrobromide is slightly smaller than hyoscine methobromide but the removal of the proton is accompanied by a reduction in volume, such as is seen with other zwitterions. 4 These differences in volume indicate a reduction in entropy on solution which may allow a greater increase in entropy on binding to receptors and hence greater affinity. The higher activity of hyoscine itself could also be due to the presence of the N-methyl group in the axial position, rather than equatorial as in hyoscyamine or atropine.5 The different position of the N-methyl group may partly explain why the pKa of hyoscine is 2 units lower than that of hyoscyamine or atropine. It is also probable that the unionized form of hyoscine is stabilized by hydration. 6 Although hyoscine N-oxide is only weakly active at pH 7.6, it is present in a highly active form in the acid environment of the stomach and so might be expected to act selectively at this site.

THE EFFECTS OF pH ON THE AFFINITY OF PIRENZEPINE FOR MUSCARINIC RECEPTORS IN THE GUINEA‐PIG ILEUM AND RAT FUNDUS STRIP

“…The change in pH could itself alter the dose-ratio by affecting the receptors but the results obtained with hyoscine methobromide by Barlow & Winter (1981) indicate that for the guinea-pig ileum this effect is small: for the concentration of hyoscine methobromide used (50 nM), the graph of the doseratio against pH indicated a change from a dose-ratio of 355 at pH 7.6 to 350 at pH 8.6. Similar experiments with guinea-pig ileum and 10 Mm pirenzepine monomethiodide (Figure 3) suggest a bigger effect, from 107 at pH 7.6 to 88 at pH 8.6.…”

“…As has has been noted previously (Barlow & Winter, 1981) it is remarkable how alkaline solutions in contact with ileum can become and there must be considerable uncertainty about the exact pH at the receptor. There is the further uncertainty about the exact effect of pH on the receptor The material used was the dihydrochloride.…”

“…Experiments were done with the guinea-pig isolated ileum and the rat fundus preparations in which the dose-ratios produced by pirenzepine were measured over a range of pH as in previous work with the ileum with hyoscine and hyoscine-N-oxide (Barlow & Winter, 1981). In this work, however, experiments were carried out with more than one concentration of antagonist in order to observe the effect of concent- ration, B, on the slope of the graph of dose-ratio, DR, against the proportion of pirenzepine protonated.…”

“…The effects of pH were studied at 37°C: some experiments were done using modified Tyrode solutions as described by Barlow & Winter (1981). These all contained (mM): NaCl 137, KCl 2.7, MgSO4 1.0, CaCl2 1.8, glucose 5 and hexamethonium 0.28.…”

Influence of monovalent cations on the binding of a charged and an uncharged (‘carbo’‐)muscarinic antagonist to muscarinic receptors

“…(2) Measurement of the drugs' affinities at very high or very low pH, thereby neutralising the charge of the amine or of the aspartate residue (Barlow & Winter, 1981;Barlow & Chan, 1982;Asselin et al, 1983;Birdsall et al, 1989;Ehlert & Delen, 1990). An anionic site on the receptor indeed appears to be important for binding, since protons (acidic pH) behave as competitive antagonists and prevent drug binding to the receptor Ehlert & Delen, 1990).…”