Stoichiometric Interaction of a β-Receptor Blocking Drug with Fraction of Liver Cytochrome P-450 as a Possible Cause for its Dose-dependent Availability

Abstract The liver's ability to bind and metabolize “first-pass” drugs is a major factor responsible for their blood concentrations on oral administration. Therefore we have characterized the binding of such a drug, the adrenergic β-receptor antagonist alprenolol, to rat liver. The microsomal fraction accounted for almost all the liver binding capacity. On titration of microsomes with alprenolol, the induced type I spectral change, reflecting alprenolol-cytochrome P-450 interaction, showed a high and a low affinity binding phase with identical “optical capacity”. The spectral dissociation constant for the high affinity binding was 0.17 μM. By use of spectral methods we found that 13–14 per cent of the total microsomal cytochrome P-450 participated in the alprenolol binding at the end of the first phase and 27 per cent at the end of the second phase. Spectral titrations showed that SKF 525-A and imipramine interact with the same sites of cytochrome P-450 as alprenolol. Binding studies with radioactive alprenolol showed the presence of a minimum of three classes of microsomal binding sites. The last had a very high capacity. The first phase caused a marked non-linearity in the overall microsomal binding of the drug, and it had almost identical capacity (0.12 mol alprenolol/mol P-450) and affinity (K_s = 0.21 μM) as the first type I spectral binding phase. This strongly indicates that this first “overall” binding phase reflects selective binding to P-450, which is further strengthened by the fact that SKF 525-A and imipramine markedly influenced this phase. Moreover, this phase was not present in plasma membranes, which do not contain cytochrome P-450. Hence we conclude that a stoichiometric (1:1) interaction of alprenolol with a 12–14 per cent fraction of rat liver microsomal cytochrome P-450 causes a non-linear overall microsomal binding. Such a phenomenon probably explains this drug's dose-dependent availability. We suggest that when this high affinity site is saturated the low affinity site of cytochrome P-450 oxidizes a large fraction of the alprenolol escaping ‘first pass’ liver extraction.