Assessment of Doxylamine Influence on Mixed Function Oxidase Activity upon Multiple Dose Oral Administration to Normal Volunteers GARY A. THOMPSON X ,JOHN V. ST.PETER*, MARK A. HEISE,ZEBULUN D. HOROWITZ,GEORGIANA C. SALYERS, TERRI T. CHARLES,CHRIS BREZOVIC,DARRELL A. RUSSELL,JULIE A. SKARE, AND JAMES H. POWELL Received October 16, 1995, from the Procter & Gamble Pharmaceuticals, Cincinnati, OH 45242, and *The Drug Evaluation Unit, Minneapolis, MN 55404. Accepted for publication July 24, 1996 X . Abstract 0 The primary purpose of this study was to assess the influence of doxylamine and phenobarbital on antipyrine/metabolites pharmaco- kinetics and 6-hydroxycortisol urinary excretion. This study was conducted in 48 healthy male human volunteers (16 per treatment group) using a parallel study design. Treatment groups consisted of 12.5 mg of doxylamine succinate, placebo, or 30 mg of phenobarbital administered orally every 6 h for 17 days. Results indicate that no statistically significant differences were observed between the doxylamine and placebo groups that are indicative of enzyme induction. For the phenobarbital group, a significant increase for antipyrine total (36 versus 45 mL/h/kg) and nonrenal (35 versus 44 mL/h/kg) clearances and 6-hydroxycortisol excretion (338 versus 529 µg) and a significant decrease in the terminal exponential half-life (11 versus 9 h) of antipyrine were observed. Introduction Doxylamine (2-[R-(2-(dimethylamino)ethoxy)-R-methyl- benzyl]pyridine) succinate is an ethanolamine antihistamine indicated for nonprescription use as a hypnotic and as an antihistamine. 1,2 In a 2 year rodent bioassay study conducted by the National Center for Toxicological Research, 3-5 dose- related increases in benign liver and thyroid tumors were observed in B6C3F1 mice orally administered approximately 70 and 140 mg/kg/day of doxylamine succinate. In addition, histologic changes that were consistent with microsomal enzyme induction were observed (i.e., centrilobular hepato- cellular hypertrophy), although no biochemical assessments of enzyme inducation were performed. In a subsequent study, doxylamine was shown to be a phenobarbital-type inducer of hepatic microsomal enzymes in B6C3F1 mice based on biochemical assays of enzyme activity and was shown to produce a 3-5-fold increase in thyroid stimulating hormone. 6 Additionally, in a study conducted in Fisher 344 rats, an increase in antipyrine total clearance was observed following oral administration of approximately 45 and 95 mg/kg/day of doxylamine for 8 days (personal communication, Robert C. Bookstaff, Procter and Gamble, Cincinnati, OH, January 25, 1995). Consistent with these findings are previous reports that have suggested a possible relationship between enzyme induction and alterations in thyroid and liver function. 7-9 Antipyrine is an analgesic which is widely used as a marker of mixed function oxidase activity. 10-14 Antipyrine is pre- dominately eliminated by oxidative metabolism to three major metabolites (3-(hydroxymethyl)antipyrine, norantipyrine, and 4-hydroxyantipyrine) which undergo further glucuronide conjugation. 11-14 As a result of the low hepatic extraction ratio and plasma protein binding (<10%) of antipyrine, total clearance for antipyrine is primarily a function of intrinsic metabolic clearance(s), making it an appropriate marker for oxidative metabolic induction/inhibition studies. The use of metabolic formation clearances for antipyrine metabolites also allows more specific identification of the cytochrome P-450 isozyme(s) involved (i.e., norantipyrine, CYP2C9/18; 4-hy- droxyantipyrine, CYP3A4; and 3-(hydroxymethyl)antipyrine, CYP1A2 and CYP2C9/18 families). 15 In addition, since an- tipyrine and metabolites undergo subsequent conjugation, changes in the ratio of urinary recovery of unconjugated to conjugated product may serve as an indicator of metabolic conjugation, as suggested for 3-(hydroxymethyl)antipyrine. 16 6-Hydroxycortisol, a metabolite of cortisol, is another commonly used marker of enzyme induction. 17-20 This en- dogenous substance has been shown to be a specific marker for CYP3A induction, 21,22 which is one of the most abundant and commonly induced forms of cytochrome P450 in man. 20,21,23-26 The purpose of this investigation was to determine whether doxylamine, administered at doses recommended in labeling, alters mixed function oxidase activity, as assessed using antipyrine/metabolites and 6-hydroxycortisol, and to char- acterize the pharmacokinetics of doxylamine upon multiple dose, oral administration of 12.5 mg doxylamine succinate every 6 h. Methods This was a randomized open-label, positive and placebo-controlled, parallel-designed study conducted in 48 healthy male human volun- teers (segregated into three treatment groups of 16 subjects each). Inclusion/exclusion criteria were established to avoid/minimize factors known to alter mixed function oxidase activity. 11,13,27-34 These criteria included inclusion of healthy male volunteers, on a normal diet and within a narrow age range (i.e., 18-40 years), and exclusion of smokers, subjects with positive drug screens, and subjects exposed to enzyme inducers or inhibitors within 30 days prior to the study. In addition, normal renal function (creatinine clearance) was also required in order to meet the underlying assumptions necessary to estimate metabolite formation clearances. 35 During the study, bal- anced diets were maintained and meals were prepared, excluding (e.g. cruciferous vegetables, charcoal broiled beef, etc.) or minimizing (e.g., protein) food stuffs known to alter mixed function oxidase activity. 36,37 This study was approved by the Institutional Review Board, prior to the start of the study. Either 12.5 mg doxylamine (Pfizer, lot number 92001), 30 mg phenobarbital (Lilly, lot number 5D239B), or placebo (Forest Phar- maceuticals, lot number 8911) was orally administered with 240 mL of water every 6 h for 17 days (69 doses). In order to minimize the potential influence of food on absorption, drugs were administered at least 1 h before or 2 h after a meal. The dose of doxylamine was based on the range recommended within the OTC monograph for antihistamines. 38 The dose of phenobarbital was based on previous studies demonstrating induction. 19,39 The duration of dosing, which is longer than recommended in the OTC monograph for doxylamine, was also based on phenobarbital results which indicated that induc- tion was consistently observed within 14 days. 19,39 Blood samples (lithium heparin) for doxylamine were obtained at 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 16, and 24 h on days 1, 8, and 15; immediately prior to the morning dose on days 11 and 13; and at 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 16, 24, 36, and 48 h on day 18. Urine samples for doxylamine were pooled prior to dosing and over 24 h on days 1, 8, and 15 and over 48 h on day 18. Safety assessments included clinical laboratory tests [chem- istry includingthyroid function (T4, T3 resin uptake, reverse T3, and TSH) and hematology]. Mixed function oxidase activity was assessed based on the phar- macokinetics of antipyrine and metabolites and the urinary recovery X Abstract published in Advance ACS Abstracts, September 1, 1996. S0022-3549(95)00443-6 CCC: $12.00 1242 / Journal of Pharmaceutical Sciences © 1996, American Chemical Society and Vol. 85, No. 11, November 1996 American Pharmaceutical Association + +