Hf Woods

Oxidation Phenotype — A Major Determinant of Metoprolol Metabolism and Response

GENETIC polymorphism in the oxidative metabolism of debrisoquine1 is largely responsible for individual differences in its plasma concentration and antihypertensive effect.2 Poor hydroxylators have...

Differential stereoselective metabolism of metoprolol in extensive and poor debrisoquin metabolizers

The hypothesis that variability in metoprolol metabolism stereoselectivity is related to debrisoquin oxidation phenotype was tested in six extensive (EM) and six poor (PM) debrisoquin metabolizers. In EM, plasma AUCs for (S)‐metoprolol were 35% higher than for (R)‐metoprolol, whereas in PM, AUCs for (S)‐metoprolol were lower than for (R)‐metoprolol. AUCs for total metoprolol correlated with the ratio of (S)‐ to (R)‐metoprolol AUC. The renal clearance of metoprolol was also stereoselective but to the same extent in both EM and PM. Findings suggest that the enzyme system responsible for polymorphic oxidation of the debrisoquin‐type is stereoselective. The relation between log total metoprolol plasma concentration and response (β‐blockade) was shifted to the right in PM relative to EM, which is compatible with a difference in pharmacologic activity of metoprolol enantiomers. Kinetic predictions based on total drug measurements will tend to overestimate dynamic differences between EM and PM, but the magnitude of the error is relatively small, and, in absolute terms, there is a large difference in pharmacologic activity between the phenotypes (β‐blockade at 24 hr: EM = 5.3 ± 5.6%; PM = 18.9 ± 3.8%).

The Polymorphic Oxidation of β-Adrenoceptor Antagonists

SummaryWide variability in response to some drugs such as debrisoquine can be attributed largely to genetic polymorphism of their oxidative metabolism. Mostβ-blockers undergo extensive oxidation. Anecdotal reports of high plasma concentrations of certain β-blockers in poor metabolisers (PMs) of debrisoquine have claimed that the oxidation of these drugs is under polymorphic control. Subsequently, controlled studies have shown that debrisoquine oxidation phenotype is a major determinant of the metabolism, pharmacokinetics and some of the pharmacological actions of metoprolol, bufuralol, timolol and bopindolol. The poor metaboliser phenotype is associated with increased plasma drug concentrations, a prolongation of elimination half-life and more intense and sustained β-blockade.Phenotypic differences have also been observed in the pharmacokinetics of the enantiomers of metoprolol and bufuralol. In vivo and in vitro studies have identified some of the metabolic pathways which are subject to the defect, viz. α-hydroxylation and O-de-methylation of metoprolol and 1′- and possibly 4- and 6-hydroxylation of bufuralol. In contrast, the overall pharmacokinetics and pharmacodynamics of propranolol, which is also extensively oxidised, are not related to debrisoquine polymorphism, although 4′-hydroxypropranolol formation is lower in poor metabolisers. As anticipated, the disposition of atenolol which is eliminated predominantly unchanged by the kidney and in the faeces, is unrelated to debrisoquine phenotype. The clinical significance of impaired elimination of β-blockers is not clear. If standard doses of β-blockers are used in poor metabolisers, these subjects may be susceptible to concentration-related adverse reactions and they may also require less frequent dosing for control of angina pectoris.

Altered plasma drug binding in cancer: Role of α1‐acid glycoprotein and albumin

Altered concentrations of serum proteins often accompany malignant disease. The effect of these changes on drug binding was studied with lidocaine, a basic drug, and tolbutamide, an acidic drug. Patients with cancer had increased serum concentrations of the acute‐phase protein α1‐acid glycoprotein (AAG) and lowered serum concentration of albumin. In association with these changes lidocaine binding was increased at all concentrations studied (predialysis concentrations 2, 6, and 10 μg · ml−1) and that of tolbutamide was decreased at the highest concentration (200 μg · ml−1). Not all of the increase in lidocaine binding was explicable on the basis of increased serum AAG concentration. Estimation of binding parameters with a model with two independent sites showed increased affinity at the high affinity site in cancer patients with no change in the calculated number of binding sites. Therefore, in cancer there is increased lidocaine binding in association with increased AAG concentrations. We also record the novel observation of a change in the intrinsic properties of the high affinity binding site.

Polymorphic metabolism of metoprolol: Clinical studies

SummaryAfter a single 200 mg oral dose of metoprolol tartrate the mean metoprolol AUC was found to be six-fold higher in poor metabolizers (PMs) of debrisoquine than in extensive metabolizers (EMs). This was associated with impaired metabolic clearance via α-hydroxylation and O-dealkylation. A population study (n=143) has shown a bimodal distribution in the ratio of metoprolol: α-hydroxymetoprolol recovered in urine which was correlated highly with the debrisoquine metabolic ratio. Nine per cent of the population were PMs. Plasma metoprolol concentrations three hours after a 100 mg oral dose of metoprolol were greater than 200 ng/ml in PMs but were lower than this in most EMs. This dose of metoprolol given once daily provided a clinically significant reduction (16%) in exercise heart rate in PMs after 24 hours. EMs require conventional doses (100 mg b.d.) to achieve the same degree of β-blockade. Preliminary data from family studies support the view that the defect in metoprolol oxidation is inherited. In 12 hypertensive patients who were EMs we compared the β-blocking activity and antihypertensive effect of chronic treatment with metoprolol 200 mg once daily (conventional and long-acting formulations), with those of atenolol 100 mg once daily and placebo. The effects of all active preparations were similar at 3.5 hours but atenolol was superior to all metoprolol formulations at 24 hours after dosing. It is concluded that for the majority of patients metoprolol should be prescribed twice daily when using currently available dosage forms. Relationships between oxidation phenotype and side-effects should be examined.

The expression of human cytochrome P450IA1 in the yeast Saccharomyces cerevisiae

Data from animal studies suggest that cytochrome P450IA1 catalyses the metabolic activation of several procarcinogenic compounds. In the present study, we have expressed human cytochrome P450IA1 in yeast cells. A 1.70 kb BclI/BamHI fragment containing a full-length human cytochrome P450IA1 cDNA was inserted into the BglII expression site of the yeast expression plasmid pMA91 thereby allowing the ATG initiation codon to be located adjacent to the PGK (phosphoglycerate kinase) promoter. The resulting recombinant plasmid, pCK-1, was introduced into Saccharomyces cerevisiae strains ATCC 44773 and AH22. Microsomes prepared from yeast transformatants of strain ATCC 44773 contained undetectable levels of cytochrome P450. In contrast, microsomes from strain AH22 contained cytochrome P450 with a specific content of 33.3 +/- 10.8 pmol/mg of microsomal protein and showed a reduced carbon monoxide difference spectrum with a peak at 448 nm. Control yeast cells transformed with pMA91 showed no cytochrome P450. Western blots were carried out using an antibody that reacts against rat cytochrome P450IA1 and an antibody that reacts against a synthetic peptide representing a short sequence of human cytochrome P450IA1. A band with a molecular weight of 54 kD was observed in microsomes of yeast transformed with pCK-1, but not with pMA91. When microsomes from yeast transformed with pCK-1 were incubated with benzo(a)pyrene (10 min, 10-160 microM), an estimated Km value of 7 microM was obtained. The availability of yeast cells with functionally active human cytochrome P450IA1 will facilitate molecular structure-activity studies of procarcinogen and drug metabolism by this enzyme in man.

Time-dependent kinetics of lignocaine in the isolated perfused rat liver

The kinetics of lignocaine have been investigated in the isolated perfused rat liver preparation. After a low dose (0.3 mg) the drug was eliminated according to first-order kinetics, but after higher doses (7.5, 15.0 mg) and multiple doses (3×1.5 mg at 15 min intervals), nonlinear kinetics were observed, which appeared to show time dependence. This was not due to deterioration of the preparation nor was there any evidence of a hepatotoxic effect of lignocaine. The kinetics of lignocaine were also found to be sex-dependent since it was eliminated at a faster rate by livers from male rats compared to those from female rats. Exogenous MEGX (7.5 mg), the mono-N-deethylated metabolite of lignocaine, inhibited the elimination of parent drug (1.5 mg dose). However, evidence was obtained suggesting that a direct effect of this and other end-product metabolites may not be responsible for the observed changes in lignocaine kinetics with time when the compounds are produced endogenously. Studies of the hepatic binding of lignocaine in the preparation showed the presence of high affinity-low capacity and low affinity-high capacity binding sites, which may be the enzymes responsible for aromatic hydroxylation and N-deethylation of the drug, respectively. Further experiments supported the view that an intermediate product of lignocaine, related to the N-deethylation pathway, might be inhibiting its further metabolism.

Metoprolol oxidation by rat liver microsomes: Inhibition by debrisoquine and other drugs

The oxidative metabolism of metoprolol has been shown to display genetic polymorphism of the debrisoquine-type. The use of in vitro inhibition studies has been proposed as a means of defining whether one or more forms of cytochrome P-450 are involved in the monogenically-controlled metabolism of two substrates. We have, therefore, tested the ability of debrisoquine and other substrates to inhibit the oxidation of metoprolol by rat liver microsomes. Debrisoquine and guanoxan were potent competitive inhibitors of the alpha-hydroxylation and O-desmethylation of metoprolol as well as its metabolism by all routes (measured by substrate disappearance). Cimetidine and ranitidine, drugs which are known to impair the clearance of metoprolol in man, showed an inhibitory action comparable to that of debrisoquine in rat liver microsomes. Antipyrine, a compound whose metabolism is not impaired in poor metabolisers of debrisoquine, was found to be only a weak inhibitor of the metabolism of metoprolol. These findings suggest that the oxidation of metoprolol is linked closely to that of debrisoquine, cimetidine and ranitidine but not to that of antipyrine in the rat.

Histamine inhibition of mixed function oxidase activity in rat and human liver microsomes and in the isolated perfused rat liver

The imidazole ring is a common structural feature of some xenobiotics that inhibit cytochrome P-450-catalysed reactions. Histamine is a 4-substituted imidazole and a preliminary study has shown it to be an inhibitor of rat liver microsomal drug oxidation. This work has now been extended. Histamine appears to be a competitive inhibitor of the alpha-hydroxylation (HM) (Ki = 164 microM; IC50 at 20 microM = 308 microM) and O-demethylation (ODM) (Ki = 243 microns; IC50 at 20 microM = 400 microM) of metoprolol in rat liver microsomes. Of the metabolites of histamine only N-acetylhistamine showed comparable inhibitory potency to that of the parent compound. Histamine impaired the disappearance of lignocaine when incubated with rat liver microsomes. This was accompanied by a corresponding inhibition of 3-hydroxy-lignocaine appearance. Histamine produced a type II spectral interaction with rat liver microsomes (lambda max = 432 nm, lambda min = 408 nm; Ks = 0.11 mM). When histamine was incubated alone with rat liver microsomes no loss of substrate was observed. The oxidation of metoprolol by human liver microsomes was impaired by histamine (IC50 values for ODM appearance at 25 microM: liver HL1 greater than 10, HL3 = 3.8 and HL4 = 3.7 mM). In comparison, cimetidine had an IC50 value of 1.5 mM using microsomes from liver HL3. Addition of histamine impaired the elimination of metoprolol by the isolated perfused rat liver in a dose-dependent manner (P less than 0.001, one-way analysis of variance). These data demonstrate that histamine can enter hepatocytes, interact with cytochrome P-450 and inhibit some drug oxidation reactions. The physiological relevance of inhibition of drug metabolism by histamine remains to be determined.

Disopyramide elimination in the isolated perfused rat liver

The elimination kinetics of disopyramide, [14C]disopyramide and [2H]disopyramide have been studied in the isolated perfused rat liver. Disappearance of disopyramide from perfusate was dose- and time-dependent over the dose range 0.3-7.5 mg. Although the mechanism underlying these observations is unclear, the data are consistent with the presence of enzyme saturation and product inhibition. Biliary secretion of conjugated metabolites appeared to be the rate-limiting step in the perfusate clearance of total radioactivity. At doses of 0.3 and 7.5 mg the kinetics of [2H]disopyramide showed a small isotope effect probably of negligible importance.