CLINICAl, pHARMACOLOGY & THERAPEUTICS P 8 0 American Society for Clinical Pharmacology and Therapeutics FEBRUARY 2003

PIII-67 METABOLOMICS: STUDY OF SIMULTANEOUS ENZYME

KINETICS OF CYP450 BASED CATALYTIC REACTIONS. P. R. Jadhav, B. Rege, M. A. Sarkar, PhD, Virginia Commonwealth University, Richmond, VA.

Purpose: Traditionally, the study of metabolism has been limited to the characterization of single pathways, often ignoring the complex relationships between overlapping and competing metabolic paths. Most drugs and other xenobiotics are substrates for multiple en- zymes, particularly knowledge of relative contribution of CYP450 isoforms would provide valuable mechanistic insights. The objective of the study is to study simultaneous enzyme kinetics due to CYP1A1 and CYPIA2 on a single substrate.

Methods: In vitro incubation studies using CYP1A1 and CYP1A2 supersomes and mixture thereof were carried out using 7-Ethoxyresorufin (l-5000nM). Resorufin formation was monitored using a fluorometric assay.

Results: Michaelis-Menten parameters for pure enzymes were, Km (nM), CYP1A1- (90.5_+22.1) CYP1A2- (242.5±4.1), and Vmax (pmol of resorufin/pmol of enzyme/min) CYP1A1- 5.2(-+0.4), CYP1A2- 3.0 (±0.3). The apparent enzyme kinetic parameters for a mixture of the two enzymes were (Km=187.0_+3.6 nM and Vmax = 5.3 -+ 0.1 pmol res/pmol of enzyme/rain). The Eadie-Hofstee plot of v/S versus v yielded a linear relationship for CYP1A2 but exponential relationship for CYPIA1 indicating presence of high affinity low capacity as well as low affinity and high capacity site.

Conclusions: Substrate turnover is a complex process and a sim- ple additive enzyme kinetic model may not be sufficient to describe the data.

PIII-68 RELATIVE ORAL BIOAVAILABIL1TY OF METHOTREX-

ATE IN CROHN'S DISEASE. D. H. Ku_rnik, MD, Y. Chowers, MD, E. Fishbein, MD, S. Almog, PhD, R. Loebstein, MD, H. Halkin, MD, Chaim Sheba Medical Center, Ramat Gan, Israel.

Background: Methotrexate (Mtx) and folate are absorbed by a common intestinal transporter, possibly resulting in decreased ab- sorption in patients with small intestinal disease.

Purpose: To determine the relative bioavailability (BA) of oral Mtx administered with and without concomitant FA vs. subcutaneous (SC) administration in patients with stable Crohn's disease (CD).

Methods: 10 patients with active, stable CD on chronic Mtx therapy were randomized to receive their regular maintenance dose (15-25 mg) on 3 consecutive weeks: orally (O), orally with 5 mg FA (O+FA), or SC. Blood samples were drawn at specified intervals during 24 hours, and Mix levels determined by fluorescence immu- noassay (Abbott TDx). AUCs were extrapolated to infinity (AUC~) and compared among the three routes.

Results: Mean AUC~ (±SD) was 270.0-+72.0, 299.3-+ 117.3, and 370.7±99.9 mnol'h'L -1 per mg Mtx administered, for O, O+FA, and SC administration, respectively (p<0.05 for O vs. SC). Mean (±SD) relative BA, the ratio of oral to sc AUC~, was 0.74_+0.17 (signifi- cantly different from 1, p=0.01) and 0.80±0.27 (ns.) for O and O+FA, respectively. Cmax did not differ among the administration routes.

Conclusions: In patients with active CD, the systemic BA of oral Mtx is approximately 25% lower than that of SC administration. Concomitant FA has no effect on oral BA. Dose adjustment should be considered when switching patients fl'om parenteral to oral therapy.

PIII-69 POPULATION PHARMACOKINETIC MODEL OF CHLOR-

ZOXAZONE AND 6-HYDROXYCHLORZOXAZONE IN RENAL FAILURE: IMPACT OF PATIENT COVARIATES ON CYP2E1 ACTIVITY. T .D. Nolin~ PharmD, M. R. Gastonguay, PhD, R. R. Bies, PharmD, PhD, G. R. Matzke, PharmD, J. F. Bernardo, MD, N. Aslam, MD, R. F. Frye, PharmD, PhD, University of Pittsburgh School of Pharmacy, University of Connecticut School of Pharmacy, University of Pittsburgh Schools of Pharmacy and Medicine, Uni- versity of Pittsburgh School of Medicine, Pittsburgh, PA.

6-Hydroxychlorzoxazone (HCZ) is the primary metabolite of the CYP2E1 probe chlorzoxazone (CZ). Since HCZ is primarily elimi- nated renally, renal insufficiency may affect the HCZ to CZ plasma ratio. We assessed the impact of renal function, age, and weight (WT) on CYP2EI activity using nonlinear mixed-effects modeling (NON- MEM FOCE method). 310 plasma and urine samples were collected from 21 subjects with varying degrees of renal function (CLcr 10-126 ml/min) for up to 120 hours after a single oral dose of CZ 250 rag. A linear model with pt-order absorption for CZ and l~t-order formation for HCZ simultaneously described the dispositions of both CZ and HCZ. Inter-individual variability was described with a log-normal distribution, and residual error was characterized by separate addi- tive/proportional models for HCZ and CZ. Typical population pa- rameter estimates were [CZ: formation clearance (CLF) = 11.3 L/hr, biliary/other clearance (CLB) =5.83 L/hr, volume of distribution (Vd) =9.47 L, absorption rate constant = 0.564 l/hr; HCZ: total clearance (CLH) = 14.9 L/hr, Vd = 16.5 L]. Diagnostic scatter plots revealed no systematic bias. Significant covariates included WT on CLF and CLB, and CLcr on CLH (p<0.001). Therefore, WT and CLcr independently affected the HCZ:CZ plasma ratio. CYP2Et activity as estimated by HCZ formation clearance was not affected by renal failure. This model may serve as the basis for individual Bayesian estimation of CLF and subsequent CYP2E1 phenotyping.

PIII-70 SIGNIFICANT INTERACTIONS BETWEEN CYP PROBES IN

A 6-DRUG COCKTAIL. I. Q. Tran, P h a r m ~ X. Lin, PhD, M. Garrett, B. Hee, K. Wilner, PhD, Pfizer Global R&D, San Diego, CA.

The previous "Cooperstown cocktail" showed no interaction be- tween probes for CYPs IA2 (caffeine, CFN), 2Cl9 (omeprazole, OMP), 2D6 (dextromethorphan, DM), and 3A (IV midazolam, MDZ). To assess a 6-drug cocktail containing the previously vali- dated probes (CFN + OMP + DM) and probes for CYPs 2C9 (losartan, L), 2E1 (chlorzoxazone, CZX), and 3A (oral MDZ), a drug interaction study was conducted. These 6 probes were selected for ease of use and are available across countries. Fifteen healthy sub- jects were randomized to receive each of the following regimens separated by a 7-day washout: A) CZX (250 mg); B) L (50 rag); C) oral MDZ (2 rag); D) CFN (t00 rag) + OMP (20 rag) + DM (30 rag); and E) CZX + L + oral MDZ + CFN + OMP + DM. Blood and/or urine samples were collected for PK assessments. Statistical analyses were performed using ANOVA with e~ = 0.05. Results are listed below:

Ratio (90% CI)* P-values

CYP Probe AUC~ C ..... for AUC

IA2 CFN 0.99 (0.88-1.10) 0.95 (0.85-1.06) NS 2C19 OMP 1.22 (1.12-1.34) 1.18 (0.99 1.40) 0.002 2C9 L 0.93 (0.85-1.03) 0.60 (0.51-0.69) NS 2El CZX 1.05 (0.89-1.24) 0.87 (0.71 1.07) NS 3A MDZ 1.92 (1.67-2.21) 1.27 (1.08-1.48) <0.000!

* Regimen E vs A, B, C, o1" D

No significant differences between regimens were detected for 12-h urine ratios of DM/dextrorphan (P=0.84) and L/E-3174 (P=0.61). L T ~ x was delayed by 1 hr when administered as part of regimen E. Results from this study do not support the concomitant use of these probes for the assessment of CYP activities.