Abstract
The role of the gastrointestinal tract in drug metabolism was evaluated: (i) by measuring mixed-function oxidase and UDP-glucuronyltransferase activities in microsomal preparations of liver, stomach, duodenum, jejunum, ileum and caecum/colon from rat and ferret; (ii) by measuring the pharmacokinetics of labetalol in rat, ferret and dog; and (iii) by determining the rate of labetalol metabolism by in situ perfused intestine preparations from rat, ferret, and dog and in vitro perfused liver preparations from rat and ferret. The activities of four mixed-function oxidase enzymes, namely biphenyl 2-hydroxylase, biphenyl 4-hydroxylase, ethylmorphine N-demethylase and ethoxyresorufin O-deethylase, of cytochrome c reductase, and of UDP-glucuronyltransferase towards three group-1 substrates, namely 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone, were determined in each tissue preparation. Comparison of the specific enzyme activities in the various tissue preparations from both species used in this study showed that these enzymes were more active in rat than in ferret and that they were more active in the liver than in the gastrointestinal tract. Also, the specific activities of the enzyme systems studied indicated that detergent activated UDP-glucuronyltransferase were more active than the mixed-function oxidases. The greatest activities of the mixed-function oxidase enzymes along the gastrointestinal tract of rat and ferret occurred in the proximal section of the small intestine, and these progressively declined along the small intestine to the aboral end. The distribution of UDP-glucuronyltransferases along the small intestine of rat was similar to that of the mixed-function oxidases, but in the small intestine from ferret the greatest activities occurred in the ileum. The mixed-function oxidase activity in the stomach and caecum/colon from both species tended to be lower than that observed in the ileum, but glucuronylation of some substrates by these tissues may be similar to that occurring in the middle section of the small intestine. All substrates used in this study were metabolised by the microsomal preparations of livers from both rat and ferret. The various gastrointestinal preparations from rat and ferret did not metabolise ethylmorphine, nor did the various gastrointestinal preparations from the ferret metabolise ethoxyresorufin. Although treatment of the ferret with 3-methylcholanthrene induced ethoxyresorufin O-deethylase in the small intestine, phenobarbitone treatment of rat did not induce ethylmorphine N-demethylase in any section of the gastrointestinal tract. Phenobarbitone was a poor inducer of mixed-function oxidase, cytochrome c reductase and UDP-glucuronyltransferases in any of the various tissues of the rat. 3-Methylcholanthrene and Aroclor 1254 treatments of rat greatly increased the activities of all these enzymes, except ethylmorphine N-demethylase, in most of the tissue preparations. Glucuronylation was less sensitive than mixed-function oxidation to the inductive effects of these agents and the cytochrome P-448-related mixed-function oxidases in the gastrointestinal tract were particularly sensitive to induction by 3-methylcholanthrene and Arolor 1254. Treatment of rat and ferret with the inducing agents did not alter the relative distribution of enzyme activities in the various tissues. Three radioactive metabolites of labetalol, namely the N-, O-phenyl-, and secondary alcohol glucuronides, were isolated from the urine of dogs, collected after an oral dose of 14C-labetalol (20 mg/kg). The O-phenyl- and secondary alcohol glucuronides were purified sufficiently for use as standards (the N-glucuronide was lost during purification). A method of separating 14C-labetalol and its radioactive metabolites by HPLC in the ion-pair mode, followed by measuring the radioactivity in the HPLC eluate was investigated but the recovery of was poor (circa 50%), so this method of analysis could only be used on a semi-quantitative basis. The pharmacokinetics of labetalol in rat, ferret and dog was investigated. Oral doses of 14C-labetalol, to rat 25 mg/kg, to ferret 25 mg/kg, and to dog 30 mg/kg, and intravenous doses of the drug (1 mg/kg) were given to each species. The concentrations of labetalol and its metabolites measured in the plasma samples from rat and ferret were unsuitable for statistical analysis. In dog, the mean labetalol bioavailability oral was 0.82 which indicated that first-pass metabolism by the gastrointestinal tract had occurred. The in situ vascularly perfused intestines of rat and ferret both absorbed 10% of a 14C-labetalol dose (25 mg/kg and 22 mg/kg respectively) and metabolised approximately 2% of the dose within 3 h. The dog gut loop preparation, however, absorbed 10% of a 14C-labetalol dose (4.7 mg/kg) and metabolised 1.6% of the dose in 30 min. The in vitro perfused livers from rat and ferret were more effective at metabolising 14C-labetalol; 12.4% and 8.8%, respectively, of the dose (7 or 7.5 mg, respectively, circulating in the perfusion medium) were metabolised in 2 h. The pattern of radioactive labetalol metabolites detected, by HPLC in the ion-pair mode, in the urines and plasma samples from rat, ferret and dog differed. Also, the pattern of labetalol metabolites detected in the perfusate samples from the perfused organ preparations of rat, ferret and dog differed. Therefore, not only species differences in labetalol metabolism but also organ differences in labetalol metabolism occurred. Although five previously unidentified metabolites of labetalol were detected, insufficient concentrations of these metabolites occurred in the urine and plasma samples from the species used in this study for isolation and characterisation. On the basis of these observations and those from other workers it is concluded that the characteristics of the drug metabolising enzymes in the gastrointestinal tract are similar to those of the liver, but that the mechanisms of regulating these enzymes in the liver and various sections of the gastrointestinal tract differ. The enzyme capacity for drug metabolism in liver is much greater than the capacity of the mucosal epithelial cells of the gastrointestinal tract from both rat and ferret. However, the specific activity of UDP-glucuronyltransferase towards group-1 substrates in the small intestine of rats, and to a certain extent of ferrets, can account for significant first-pass metabolism of drugs by this tissue. Also, that significant first-pass metabolism of labetalol does occur in the intestine of rat, ferret, and dog. However, the first-pass metabolism and uptake of labetaiol by the livers of rat and ferret are more likely to be the reasons for the necessity to give high oral doses of labetalol in order that the drug may exert its therapeutic effect.