Pharmacology Journal

The cytochrome P450 3A enzymes (CYP3A4 and CYP3A5) participate in the biotransformation of approximately half of the drugs that undergo metabolic clearance in human adults. CYP3A4 is highly expressed in the liver and the epithelium of the small intestine and represents on average 30% of total hepatic CYP protein and 33–87% of intestinal CYP. CYP3A5 exhibits a polymorphic expression pattern with high expression in 10–30% of Whites and is clinically important in the disposition of some CYP3A substrates such as tacrolimus and sirolimus. CYP3A enzymes metabolize a broad array of structurally diverse compounds, including macrolide antibiotics, benzodiazepines, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors, calcium channel blockers and human immunodeficiency virus protease inhibitors, and consequently are at the centre of many clinically significant drug interactions. Clarithromycin is a widely used macrolide antibiotic that is a potent inhibitor of CYP3A4 in vitro and in vivo and appears to exert inhibition by an irreversible mechanism. We have previously shown that clarithromycin irreversibly inhibits midazolam hydroxylation in intestinal biopsy tissue obtained from pretreated, young, healthy subjects. An important consequence of this irreversible mechanism is that the inhibition remains for up to 10 days after discontinuation of the inhibitor; this time delay is determined by the elimination half-life of CYP3A4. We have used the simultaneous intravenous (i.v.)/oral (p.o.) midazolam dosing paradigm to demonstrate that clarithromycin exerts a sex-dependent inhibition of intestinal and hepatic CYP3A in young, healthy subjects. Clinically important consequences of CYP3A inhibition by clarithromycin include excessive sedation following benzodiazepine administration, increased concentrations of ciclosporin A and increased risk of nifedipine-induced vasodilatory shock. However the extent of inhibition of CYP3A at hepatic and intestinal sites by potent inhibitors has not previously been examined in the elderly.

Elderly patients are more likely to be taking multiple medications and there is a corresponding increase in the likelihood of drug–drug interactions. It has been estimated that >75% of drug–drug interactions occur in persons >50 years old. Although expression of CYP3A proteins does not appear to change with age, elderly persons may exhibit altered clearance of CYP3A substrates for a variety of reasons, including disease and altered tissue perfusion and structure. The disposition of CYP3A inhibitors may also be altered in the elderly particularly when renal excretion is an important route of elimination. The renal clearance and total clearance of clarithromycin are significantly decreased in elderly persons with reduced creatinine clearance. However, the drug interaction consequences of the resulting increased exposure to inhibitor have not been previously addressed. Thus, this study was designed to examine the contribution of intestinal and hepatic CYP3A inhibition to the interaction between midazolam and clarithromycin in the elderly, and the effect of sex on the pharmacokinetics of midazolam and extent of interaction were evaluated.

Subjects

This study was approved by the Indiana University Purdue University Indianapolis and Clarian Health Partners institutional review board, and written informed consent was obtained from study volunteers prior to drug dosing. Healthy volunteers >65 years old with no significant medical history as assessed by physical examination and blood and urine chemistry screens were enrolled. Volunteers were excluded if they were taking medications known to influence CYP3A activity. Subjects with known allergies to either benzodiazepines or macrolide antibiotics were also excluded.

Study design

A fixed order study design was employed, because the duration of inhibition is unknown in this group and we wished to minimize interday variability in intestinal and hepatic CYP3A content which may occur during extended wash-out periods. Volunteers abstained from alcohol, grapefruit and grapefruit products, other citrus products and herbal supplements for at least 1 week prior to and during the study. However, participants were allowed to continue taking stable doses of medications which have not been previously identified as inducers, inhibitors or substrates of CYP3A4. Abstinence from alcohol and compliance with other dietary restrictions were assessed through volunteer questioning. All medications for the treatment of chronic conditions were withheld on the days of midazolam dosing. After an overnight fast, i.v. catheters were placed in one forearm for the withdrawal of blood samples and in the opposite forearm for the administration of drug. Prior to receiving the dose of midazolam, subjects emptied their bladder and a baseline blood sample was obtained. Subjects then simultaneously received midazolam (0.05 mg kg^−1) intravenously over 30 min and ¹⁵N₃-midazolam (3.0–4.0 mg) p.o. as a solution. Blood samples were obtained 5, 15, 30, 45 min and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, and 24 h following drug administration. Serum was obtained and frozen at −20°C until analysis. Urine was collected over the following time intervals: 0–12 and 12–24 h post dose and frozen at −20°C until analysis. After the 24-h blood draw, p.o. clarithromycin (500 mg) was initiated twice daily for 7 days. On day 8, 2 h after the morning clarithromycin dose, the midazolam portion of the study was repeated. Compliance with clarithromycin was confirmed by pill count and quantification of clarithromycin serum concentrations.

Sample analysis

Serum samples were processed using a liquid–liquid extraction technique and quantified following derivatization with N-methyl-N-t-butyldimethylsilyl trifluoroacetamide containing 1% t-butyldimethylchlorosilane using gas chromatography/mass specrometry (GC/MS) (Hewlett Packard 5971 mass selective detector and 5890A gas chromatograph) as described previously. The limit of quantification for midazolam and metabolite was 1 ng ml^−1. The interday coefficient of variation at 5 and 50 ng ml^−1 was 11% and 12%, and 4.6% and 5.2% for midazolam and ¹⁵N₃-midazolam, respectively. Urine samples were processed as described above following deconjugation with β-glucuronidase (Sigma Chemical Co., St Louis, MO, USA). The midazolam concentration in the i.v. infusion solution was determined by high-performance liquid chromatography (HPLC). Additionally, serum samples were processed through a liquid–liquid extraction method and clarithromycin serum concentrations were estimated using HPLC with electrochemical detection as previously described. The limit of quantification for clarithromycin was 2.5 ng ml^−1. The corresponding coefficients of variation and relative error for clarithromycin at 10 ng ml^−1 were <8% and 9%, respectively.

Filed under: Drug Interactions