CLINICAL PHAKMACOLOGY & THERAPEUTICS VOLU'ME 73, NUMBER 2 American Society for Clinical Pharmacology and Therapeutics P 6 1

PDI-C-6 IRREVERSIBLE INHIBITION OF INTESTINAL CYTO-

CHROME 3A (CYP3A) BY CLARITHROMYCIN. A. G. Pinto. MD, N. Chalasani, MD, S. D. Hall, PhD, A. Aii, PhD, M. Hamman, MS, Division of Clinical Pharmacology, Wishard Memorial Hospital, Indiana University, Indianapolis, IN.

We have previously shown that clarithromycin (CLA) inhibits both hepatic and intestinal CYP3A in vivo using selective CYP3A substrate midazolam (increase in hepatic and intestinal bioavailability by 22% and 300% respectively). However, the exact mechanism of" intestinal CYP3A inhibition by CLR is not known. To differentiate between reversible and irreversible inhibition of CYP3A by CLA, 10 healthy volunteers were given CLR 500 mg twice a day for 7 days. Betbre and after receiving CLR small bowel lnucosal biopsies were obtained endoscopically, and intestinal CYP3A activity and CYP3A4 mRNA level was measured. Intestinal CYP3A activity was deter- mined by incubation of small bowel homogenate with midazolam and NADPH for 5 minutes and quantifying rate of formation of l-OH midazolam and 4- OH midazolam. All subjects had detectable serum CLA level on day 8 (3.71 _+ 2.43 IxM/L). While there was no significant difference in CYP3A4 mRNA levels between baseline and day 8 mucosal biopsy samples, the formation of 1-OH midazolam [1.36- + 0.46 pmol/min*mg (baseline) vs. 0.35 + - 0.16 pmol/min*mg (day 8)] and 4-OH midazolam [0.39_+ 0.12 pmol/min*mg (baseline) vs. 0.12 _+ .05 pmol/min*mg (day 8)] was significantly reduced on day 8 (p< 0.001). In conclusion, CLA reduced small bowel CYP3A activity by approximately 75% by irreversible inhibition of CYP3A with no corresponding change in CYP3A4 mRNA levels.

Supported by NIH grant RO1-GM67308-05A2 and FD-T- 001756-03

PDI-C-7 USE OF CPT-I 1 AS AN IN VITRO PHENOTYPIC PROBE FOR

CYP3A4/CYP3A5 ACTIVITY. J. Ramirez, MS, F. Innocenti, MD, L. P. Rivory, PhD, M. V. Relling, PharmD, E. G. Schuetz, PhD, M. J. Ratain, MD, University of Chicago, Sydney Cancer Centre, St. Jude Children's Research Hospital, Chicago, IL.

CYP3A4 and CYP3A5 have overlapping substrate specificities. Other than midazolam, a common substrate converted into CYP3A4- and CYP3A5-specific metabolites has not yet been found. Conse- quently, it is difficult to assess the individual contribution of these enzymes to drug metabolism. The major in vitro oxidation product of CPT-l l is NPC, a CYP3A4 metabolite. A new metabolite (M) of CPT-11, formed by CYP3A5, was recently discovered. We con- ducted a study to investigate if CPT-11 could be used as an in vitro phenotypic probe for determining CYP3A4/CYP3A5 activity. For- mation of NPC and M by human liver microsomes (HLM) and baculosomes expressing CYP3A4 and CYP3A5 was measured by HPLC. CYP3A4 and CYP3A5 content in HLM was measured by Western blot. Under our conditions, the contributions of CYP3A5 and CYP3A4 to M formation were 90% and 10%, respectively. NPC was formed only by CYP3A4 baculosomes. The Kms for the forma- tion of M by CYP3A5 and CYP3A4 baculosomes were 137.8_+25.63 IxM and 79.65_+4.03 p,M, respectively. However, the catalytic effi- ciency for M formation observed for CYP3A5 was five times higher than that observed with CYP3A4. The Km for NPC formation by CYP3A4 baculosomes was 26.42_+ 1.37 IxM. The metabolic ratio of M/NPC measured in HLM that express CYP3A5 correlated with CYP3A5 content (r=0.95, p=0.015). These experiments provide the basis for the development of a mathematical model for estimating in vitro CYP3A4/CYP3A5 activity using CPT-11 as probe substrate.

PDI-C-8 ALLELIC VARIATION OF THE PREGNANE X RECEPTOR

(PXR) AND CYP3A4 ACTIVITY. A. Gaedigk, MS, PhD~ J. Feld- meyer, BS, M. Czerwinski, PhD, A. Parkinson, PhD, J. S. Leeder, PharmD, PhD, Children's Mercy Hospital, Xenotech LLC, Kansas City, MO.

CYP3A4 activity ranges up to 60-fold in any given population and cannot solely be explained by genetic variability in the 3A4 gene. Modulation of CYP3A4 expression by endo- or xenobiotic com- pounds, such as rifampin (RIF), is mediated by the pregnane X receptor (PXR), and Zhang et al (Pharmacogenetics 11:555, 200l) have suggested that allelic variation of PXR may contribute to the range of CYP3A4 activities observed. To further investigate the role of PXR allelic variation on CYP3A4 activity, testosterone 6[3- hydroxylase (T6H) activity was measured in primary human hepato- cytes isolated from 48 donors following rifampin induction, and genotyping assays were established for 12 of the 42 known PXR SNPs. Basal T6H activity (-RIF) ranged from 63-9000 pmol/min/mg microsomal protein and induced activity (+RIF) ranged from 79- 17000 pmol/min/mg. Extent of induction ranged from 0.7-30 fold. No SNPs were observed (n=96 alleles) at positions 44826 (GenBank entry AF364606), 45342, 45671, 45928, 74710 [RI22Q] and 81582. The frequencies for SNPs at 45634, 45653, 46277, 81009, 81188 and 81545 were 0.03, 0.01, 0.25, 0.1t, 0.15 and 0.19, respectively. No correlation was observed between any SNP and -RIF or +RIF activ- ities, or fold induction. Conclusions: No single PXR variant signif- icantly affects basal or induced CYP3A4 activity in vitro. Haplotype analysis is necessary before definitive conclusions concerning PXR allelic variation and variability in CYP3A4 activity can be drawn.

PDI-C-9 DIFFERENTIAL MECHANISM-BASED INHIBITION OF

CYP3A4 AND CYP3A5 BY GRAPEFRUIT JUICE AND PROAD- WEN. A. Q. Adigun, MD, D. R. Jones, PhD, S. D. Hall, PhD, Indiana University School of Medicine, Indianapolis, IN.

The cytochrome P450 (CYP) 3A enzymes metabolize more than 50% of drugs that undergo metabolism. CYP3A5 expression is ge- netically determined but the significance of this variation is unclear. CYP3A4 appears to be more susceptible to reversible inhibition than CYP3A5. We examined mechanism-based inhibition of CYP3A su persomes CYP3A4 (b5) and CYP3A5 with grapefruit juice (GFJ) and proadifen (SKF-525A; SKF), a classical mechanism based inhibitor. GFJ (0.1%-8%) was preincubated with each enzyme for 5-20minutes while SKF (0.1-16lxM) was preincubated for 2-12minutes with each enzyme and NADPH. Enzyme activity was determined by a testos- terone 6-beta-hydroxylation assay. Both CYP3A4 and CYP3A5 dem- onstrated time and concentration dependent inhibition in the presence of GFJ and SKF. The Kinact for SKF was 0.46min-1 and 0.15rain-1 while the KI was 0.6 p~M and 3.6 IxM for CYP3A4 and CYP3A5, respectively. The Kinact for GFJ was 0.7min-1 vs. 0.42rain-1 while the KI was 8% and 12% for 3A4(b5) and 3A5, respectively. The clearance of inactivation (CLinact = Kinact/KI) differed 20-fold be- tween CYP3A4 and 3A5 for SKF (0.77 vs. 0.04 respectively), but only 3-fold for the GFJ (0.09 vs. 0.03). Thus CYP3A5 appears to be less susceptible to inhibition by GFJ and SKF but the magnitude of the difference varies with inhibitor. Individuals who express signif- icant amounts of 3A5 may have less pronounced drug interactions for some CYP3A inhibitors but not others.