CLINICAL PHARMACOLOGY & THERAPE UTICS P 1 8 American Society /br Clinical Pharmacology and Therapeutics FEBRUARY2003

PI-61 THE EFFECT OF AGE ON TRIAZOLAM HYDROXYLATION

IN HUMAN LIVER MICROSOMES FROM MALES. K.C. Patki, L. L. yon Moltke, MD, J. S. Harmatz, BA, D. J. Greenblatt, MD,

Tufts University School of Medicine, Boston, MA. We studied age related changes in enzyme kinetic parameters in

human liver microsomes (HLMs) in vitro. We evaluated the biotrans- formation of the benzodiazepine triazolam (TRZ), an index of CYP3A activity, using HLMs made from male livers from four groups: A (1-20 years, n =7), B (21-40 years n =5), C (41-60 years n =5), and D (61-80 years n =5). TRZ is metabolized to two major products, 1-OH and 4-OH-TRZ, which were quantified by high performance liquid chromatography. Mean Vmax values in B and C for both 1-OH and 4-OH-TRZ were significantly greater as compared to A and D. The mean intrinsic clearance (Vmax/Km) for both pathways was significantly greater in B and C than A and D, indi- vidually, as well as the net intrinsic clearance (sum of the two pathways). The mean net intrinsic clearance values were 19.4, 65, 59 and 15.6 microt/lnin/mg protein in A, B, C and D respectively. We also evaluated effect of testosterone (TST) on TRZ metabolism in HLMs in the four groups. In all groups, TST inhibited 1-OH-TRZ formation to 31-47% of control and activated 4-OH-TRZ tbrmation to 173-230% of control. There were no significant differences among any of the groups with respect to the effect of TST on TRZ metabolite formation. Reduced Vmax and intrinsic clearance for TRZ hydroxy- lation in liver samples from elderly men suggest reduced CYP3A expression in this group. These findings are consistent with many clinical studies showing reduced clearance of CYP3A substrates in elderly men.

PI-62 EFFECTS OF INCREASING TESTOSTERONE ON ZOLPI-

DEM CLEARANCE 1N VITRO. J.O. Olubodun, MD, MRCP, L. L. von Moltke, MD, D. J. Greenblatt, MD, Tufts University, Tufts University School of Medicine, Boston, MA.

Our recent in vivo study demonstrated reduction of zolpidem (Z) clearance associated with decreasing free sermn testosterone levels in elderly men. Testosterone effects on cytochrome P (CYP) 450 me- diated metabolite formation rate and intrinsic clearance of Z were studied using human liver microsomes (HLM). Z (0 to 1500~LM) was incubated with testosterone (0 to 501xM) and the formation of the principal metabolite (M3) was quantitated by HPLC. At Z concen- trations of 10txM and 251xM, M3 formation consistently increased with testosterone, being maximum at 5 to 25~M testosterone. The maximum % increase in M3 formation ranged from 127 to 239 % (mean 179_+48 {SD}) at 10txM, and 116 to 149 % (131_+16) at 25jxM of Z. M3 formation rates were maximum at testosterone concentrations of 25~M for Z concentrations of 10 and 25~M. The overall mean Z intrinsic clearance increased from 15-+ 10 to 18-+ 16 Ixl/min/mg protein at testosterone concentrations of 10 to 25~M. For the 3 HLMs with the highest CYP3A4 activity, the increase was from 16- + 12 to 2t .7_ + 18 ~l/min/mg protein.

Thus increasing testosterone levels activate hydroxylation of Z to its major metabolite in HLMs. The increase occurred mainly at lower testosterone concentrations. This is consistent with the significant contribution (60%) of CYP3A isoforms to Z clearance, and the known property of testosterone as an allosteric activator of CYP3A function.

PI-63 RITONAVIR IMPAIRS CLEARANCE AND ENHANCES TOX-

ICITY OF TRAZODONE. D.J. Greenblatk MD, L. L. yon Moltke, MD, J. S. Harmatz, S. Fogetman, MD, G. Chen, R. I. Shader, MD, Tufts University, Boston, MA.

In a randomized 4-way crossover study, healthy volunteers re- ceived: A. Trazodone (TRAZ) 50 mg, along with ritonavir (RIT) 200 mg every 12 hours for 48 hours (800 mg total dose); B. TRAZ, plus placebo to match RIT; C. Placebo to match TRAZ, plus RIT; D. Placebo plus placebo. Compared to TRAZ plus placebo (Trial B), TRAZ plus RIT (Trial A) caused elevation of TRAZ C ..... (1.13 vs. 0.84 ng/ml p<0.05), increased total AUC (13.9 vs. 5.9 ixg/ml x hr, p<0.01) prolonged half-life (14.9 vs. 6.7 hours, p<0.05) and reduced clearance (75 vs. 155 ml/min, p <0.001). TRAZ plus RIT also increased impairment on the DSST (p<0.05) compared to TRAZ plus placebo. During Trial A, three subjects experienced nausea and hypotension; one also experienced syncope. Transformation of TRAZ to its principal metabolite by human liver microsomes in vitro was inhibited by RIT (K~ = 0.14 IxM). Measured total plasma RIT levels (pre-dose minimum: 0.73 b~g/ml = 1.0 txM) and the in vitro K i overpredicted the in vivo impairment of TRAZ clearance (observed: 47% of control; predicted: 12%). However use of unbound plasma RIT levels predicted essentially no interaction (TRAZ clearance: 96% of control). Thus short-term exposure to low-dose RIT impairs clear- ances and enhances adverse effects of single doses of TRAZ.

PI-64 INDUCTION OF THE MRP3 TRANSPORTER BY ACTIVA-

TORS OF THE PREGNANE X RECEPTOR. S. Teng, M. Piquette- Miller, PhD, University of Toronto, Toronto, Canada.

Purpose. The pregnane X receptor (PXR) has been shown to be involved in mediating the induction of the MDRI, MRP2 and OATP2 transporters by xenobiotics. Furthermore, PXR activators induce MRP1 and MRP2 expression in human cell lines. Our studies were aimed at determining the involvement of PXR in the regulation of MRP3 expression. Methods. The human hepatoma cell lines HepG2 and HuH7 were treated for 24h with the PXR activators clotrimazole, rifampicin, RU486 (10-30 b~M) or phenobarbital (0.5 - 2 mM). Levels of mRNA of MRP3, MRP2 and MRP6 were measured by RT-PCR. Reactive oxygen species (ROS) levels were measured by dichlo- rofluorescein production. Results. MRP3 mRNA levels were induced 2- to 8-fold in a dose-dependent manner in both cell lines by all of the compounds tested. MRP6 expression was not affected whereas MRP2 expression was induced by clotrimazole and rifampicin as in agreement with published reports. Clotrimazole, but not rifampicin, caused oxidative stress as indicated by dose-dependent ROS forma- tion, however the antioxidant N-acetylcysteine did not prevent induc- tion of MRP3 by clotrimazole. Conclusions. These results suggest that PXR may play a role in the regulation of MRP3 and MRP2 expression but not that of MRP6. Furthermore, MDR1 and MRP2 have previously been shown to be induced by oxidative stress how- ever ROS does not appear to play a role in clotrimazole- or rifampicin-mediated induction of MRP3.