Short Communication In Vitro Metabolism of Indinavir in the Human Fetal Liver Microsomes

In vitro microsomal formation of primary metabolites of indinavir (CRIXIVAN, MK-0639, L-735,524), an HIV protease inhibitor, were qualitatively similar among the different developmental stages in humans, although the fetal liver had a lower capability to form the metabolites than the pediatric and adult liver. The lower activity of fetal liver was mainly owing to a decrease in the Vmax values. The Vmax value in the fetus was about one-third of that in the adult human, while no significant difference was found in Km values between groups. The liver microsomes were also characterized using P450 markers to examine the development-associated alteration in P450 functional activities. Debrisoquine 4-hydroxylase activity was comparable among the three age groups. In contrast, tolbutamide methyl hydroxylase activity, as well as the CYP3A marker, testosterone 6b-hydroxylase activity, in the fetal liver microsomes was much lower than in the pediatric and adult by more than 40-fold. However, the difference in testosterone 2b-hydroxylase and nifedipine N-oxidase activities between fetus and adult was markedly smaller. The ratio of indinavir metabolism in pediatric or adult liver to fetus was 1.7 for pediatric and 3.6 for adult liver microsomes. Similarly, testosterone 2b-hydroxylase and nifedipine N-oxidase activities showed smaller differences between adult (or pediatric) and fetal liver microsomes than testosterone 6b-hydroxylase activity. The reason for the observed marked differences in the development-associated alteration may lie in the differences of substrate specificities between CYP3A isoforms. Indinavir (CRIXIVAN (Merck & Co., Inc., West Point, PA), MK0639, L-735,524), a potent HIV protease inhibitor, has been approved recently for the treatment of AIDS. AIDS has become a global epidemic. The disease can infect every class and age, even the unborn. During pregnancy, women can pass the virus to their unborn children. Studies in pregnant rats and dogs revealed that a significant fraction of indinavir was subject to placental transfer to the fetus (Merck Research Laboratories, data on file). Therefore, it is important to study the fetal metabolism of indinavir. Our previous studies have shown that in humans, indinavir is metabolized exclusively by CYP3A4 to form all metabolites shown in fig. 1 (1) except for M7, which was not formed in humans. The present study was conducted to examine the in vitro metabolism of indinavir in human fetal liver microsomes and compare the metabolic profile and kinetics with those from adult and pediatric subjects. Materials and Methods Indinavir and its C form (CRIXIVAN, MK-0639, L-735,524) [N(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3pyridylmethyl)-2(S)-N9-(t-butylcarboxamido)piperazinyl)) pentanamide] were synthesized at Merck Research Laboratories (West Point, PA). The labeled indinavir was prepared with C in the 1-position of the pentanamide, with a specific activity of 33.08 mCi/mg. Testosterone, nifedipine, and tolbutamide were purchased from Sigma Chemical Co. (St. Louis, MO). Nifedipine N-oxidized metabolite was purchased from Ultrafine Chemicals (Manchester, UK). Debrisoquine and 4-hydroxydebrisoquine were obtained from Gentest Corporation (Woburn, MA). Methyl hydroxylated metabolite of tolbutamide was from Research Biochemical International (Natick, MA). Hydroxytestosterones were from Steraloids (Wilton, NH). All other reagents were of analytical grade. Microsomal fractions from adult and pediatric subjects (original liver sample code, age, sex, cause of death) (HHM-99, 10-year-old, male, anoxic encephalopathy; HHM-123, 11-year-old, female, closed head injury; HHM124, 6-year-old, male, anoxic brain injury; HHM-59, 50-year-old, female, gun shot wound; HHM-106, 45-year-old, female, sub arachnoid hemorrhage; HHM-127, 36-year-old, female, intracerebral hemorrhage) were obtained from Keystone Skin Bank (Exton, PA). Liver microsomes obtained from human fetus (20 weeks gestation) were generous gifts from Dr. A. Y. H. Lu at Merck Received March 6, 1997; accepted June 13, 1997 1 Abbreviation used is: HIV, human immunodeficiency virus. Send reprint requests to: Masato Chiba, Ph.D., WP 44B-100, Department of Drug Metabolism I, Merck Research Laboratories, West Point, PA 19486. E-mail: [email protected]. TABLE 1 Kinetics of indinavir metabolism in human liver microsomes obtained from fetal, pediatric and adult subjects Human Subjects Km (mM) Vmax (pmol/min/mg) Vmax/Km (ml/min/mg) Fetus HFM-1 2.87 18.2 6.35 HFM-2 1.70 8.57 5.03 HFM-3 2.06 12.0 5.82 Average 2.21 12.9** 5.73** SD 0.60 4.9 0.66 Pediatric HPM-1 2.57 32.0 12.5 HPM-2 1.33 15.7 11.8 HPM-3 1.10 13.6 12.4 Average 1.67 20.4* 12.2** SD 0.79 10.1 0.4 Adult HAM-1 2.18 38.8 17.7 HAM-2 2.76 51.2 18.5 HAM-3 1.98 35.4 17.9 Average 2.31 41.8 18.0 SD 0.41 8.3 0.4 a Fetal liver microsomes were obtained at 20 weeks gestation (N 5 3). Ages of pediatric and adult subjects were 9.0 6 2.6 (mean 6 SD, N 5 3) and 43.7 6 7.1 (mean 6 SD, N 5 3), respectively. Significant differences from the corresponding adult subject values are shown by asterisks (*p , 0.05; **p , 0.01). 0090-9556/97/2510-1219–1222$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 25, No. 10 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 1219 at A PE T Jornals on Jne 3, 2017 dm d.aspurnals.org D ow nladed from