SOI-A-1 IDENTIFICATION OF KEY AMINO ACID RESIDUES REQUIRED FOR RIFAMPIN-MEDIATED ACTIVATION OF HUMAN PREGNANE X RECEPTOR. R.G . T ir ona, PhD , R.B. Kim, MD, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, TN. The pregnane X receptor (PXR) is a nuclear receptor importantly involved in xenobiotic sensing and the transcriptional regulation of drug metabolizing enzymes and transporters. Activation of human PXR by drugs such as rifampin, is thought to be the basis for their inductive effects in vivo. Interestingly, rifampin can readily induce human and rabbit but not rodent CYP3A isoforms. Recent studies suggest that this phenomenon is related to species-dependent struc- tural differences among PXR proteins. However, the key domain(s) or amino acids responsible for species-dependent, rifampin-mediated PXR activation are unknown. To identify such a domain(s), we cre- ated an array of human-rat and rat-human chimeric PXR cDNAs in a tandem head-to-tail configuration, using a random chimeragenesis method. Upon expression of chimeric PXRs in HepG2 cells and analysis of CYP3A4 promoter-based luciferase reporter activities, we were able to localize the rifampin-sensitive region to a 31 base pair stretch within the ligand-binding domain of human PXR. Based on the recent structural determination of human PXR, it appears that the rifampin-sensitive region lies within the flexible loop adjacent to the ligand-binding pocket. Studies designed to identify the particular amino acid(s) responsible for rifampin-sensitive PXR activation are ongoing. SOI-A-2 ALFENTANIL MIOSIS—A POTENTIAL NONINVASIVE PROBE FOR CYP3A ACTIVITY. E.D . Khar asc h, MD , PhD , S. Phimmasone, BS, University of Washington, Seattle, WA. The search continues for an ideal CYP3A4 probe. The opioid alfentanil (ALF) is a CYP3A4 substrate. ALF clearance is dependent on hepatic CYP3A4 activity, and has been used as an in vivo probe for human hepatic CYP3A4 activity and drug interactions. PK stud- ies, however, are invasive, time-consuming & expensive. We tested the hypothesis that ALF-induced miosis can be used as a surrogate measure for ALF plasma concentration & pharmacokinetic clear- ance, and hence serve as a non-invasive probe for CYP3A4 activity. Midazolam (MDZ) was used as a CYP3A probe. Normal volunteers (n=6, 18-40 yr, IRB approved consent) were studied in a 3-way ran- domized crossover. They received MDZ (1 mg IV) followed later by ALF (15 ug/kg IV) under 3 conditions: 1) no pretreatment (control); 2) hepatic CYP3A induction by rifampin (600 mg po for 5 d), 3) hepatic CYP3A inhibition by troleandomycin (TAO, 500 mg po q12hr). Plasma MDZ was determined by GC-MS. ALF effects were measured by infrared pupilometry, evaluating dark-adapted pupil diameter change vs baseline. MDZ concentrations and miosis vs time data were analyzed by noncompartmental modeling; pupil diameter change was treated analogously to plasma concentration data. ALF miosis was substantially affected by CYP3A activity. ALF effect AUC, effect clearance, and half-life after CYP3A induction & inhi- bition were different (p<0.05) vs control. ALF effect AUC and clear- ance were significantly correlated with MDZ plasma AUC and clear- ance, respectively. ALF effect (miosis) is a sensitive and reliable sur- rogate for ALF plasma concentrations. ALF effect kinetics may be a noninvasive in vivo probe for CYP3A4 activity. American Society for Clinical Pharmacology and Therapeutics Abstracts of papers, * 2002 Annual Meeting Marriott Marquis Hotel Atlanta, Georgia, March 24-27, 2002 *Abstracts appear in presenting order. The first author is the presenter. MPI, TPII, and WPIII designate Poster Sessions I (Monday), II (Tuesday), and III (Wednesday); SOI A-1 through C-4, MOII A-1 through C-4, and TOIII A-1 through C-4 designate the first, second, and third oral presenta- tion sessions. FEBRUARY 2002 P1 CLINICAL PHARMACOLOGY & THERAPEUTICS 1-104February CPT 1/31/02 3:18 PM Page 1