Abstract
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 theVmax values. TheVmax 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 6β-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 2β-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 2β-hydroxylase and nifedipine N-oxidase activities showed smaller differences between adult (or pediatric) and fetal liver microsomes than testosterone 6β-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), MK-0639, L-735,524), a potent HIV1protease 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 14C form (CRIXIVAN, MK-0639, L-735,524) [N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3-pyridylmethyl)-2(S)-N′-(t-butylcarboxamido)piperazinyl)) pentanamide] were synthesized at Merck Research Laboratories (West Point, PA). The labeled indinavir was prepared with14C in the 1-position of the pentanamide, with a specific activity of 33.08 μCi/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; HHM-124, 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 Research Laboratories (Rahway, NJ). They had original liver codes of 19007, 19008, and 19010. No significant difference in total P450 content was obtained between pediatric and adult liver microsomes (0.55 ± 0.12 nmol P450/mg protein for pediatricvs. 0.56 ± 0.04 nmol P450/mg protein for adult liver microsomes, data from Keystone Skin Bank). Because of limited availability, a similar quantitation of P450 was not conducted for fetal liver microsomes. However, the immunoquantitated content of CYP3A in the fetal liver microsomes was approximately one-third of that in the adult liver microsomes.2
The incubation mixture (final volume of 145 μl) consisted of an NADPH-generating system (10 mM G6P, 2 IU G6PDH and 10 mM MgCl2), various concentrations (0.17, 0.42, 0.83, 1.7, 4.2, 6.3, and 8.4 μM) of 14C-indinavir and 1.7 mg/ml human liver microsomes. After a 5-min preincubation at 37°C, the reaction was initiated by the addition of 5 μl of 50 mM NADPH. The incubated samples were analyzed by an HPLC method as previously described (1).
For characterization of P450 function by markers, the reaction mixture consisted of 500 μM substrate, 1 mg/ml human liver microsomes, and an NADPH-generating system in a final incubation volume of 250 μl (10 mM G6P, 2 IU G6PDH, and 10 mM MgCl2). Reactions were initiated by the addition of 1 mM NADPH and incubated for 20 min at 37°C after a 5 min preincubation. Testosterone and tolbutamide metabolites were assayed by HPLC methods as previously described (1). Nifedipine N-oxidized metabolite (2) and 4-hydroxylated debrisoquine (3) were assayed according to the referenced published procedures.
Kinetic parameters (Km andVmax) were obtained from the relation between metabolic velocity and indinavir concentration using the aid of a nonlinear curve-fitting in SCIENTIST (MicroMath, UT). All data were presented as mean ± SD, and the means were compared between adultvs. fetus or adult vs. pediatric subjects by using the Student’s t test with p<0.05 considered statistically significant.
Results and Discussion
The metabolic profiles for the formation of primary metabolites of indinavir were qualitatively similar among the different developmental stages in human, while quantitatively the ability of fetal liver to metabolize was less than that of pediatric and adult livers (fig.2). The lower metabolic activity of fetal liver was mainly a result of a decrease in theVmax values (table 1). TheVmax 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 (table 2). Debrisoquine 4-hydroxylase activity was comparable among the three age groups. In contrast, tolbutamide methyl hydroxylase activity, as well as the CYP3A marker, testosterone 6β-hydroxylase activity, in the fetal liver microsomes was much lower than the pediatric and adult by more than 40-fold. However, the difference in testosterone 2β-hydroxylase and nifedipineN-oxidase activities between fetus and adult was markedly smaller. The ratio of indinavir metabolism in adult or pediatric liver to fetus was 1.7 for pediatric and 3.6 for adult liver microsomes. Similarly, testosterone 2β-hydroxylase and nifedipineN-oxidase activities showed smaller differences between adult (or pediatric) and fetal liver microsomes than testosterone 6β-hydroxylase activity (i.e. 17.5 for pediatric and 45 for adult liver microsomes).
The present observations for P450 functional activities in the fetal liver microsomes suggest that CYP2C, CYP2D, and CYP3A are expressed in the fetal liver. These observations are consistent with previous reports (4-9). The reason for the observed marked differences in CYP3A marker activities between testosterone 6β-hydroxylase and 2β-hydroxylase/nifedipine oxidase/indinavir metabolism may lie in the differences of substrate specificities between CYP3A isoforms. It has been shown that the identity between CYP3A4 and CYP3A7 is 95% in nucleotides and 87% in amino acid sequences (4, 10). Studies on the expression of CYP3A4 and CYP3A7 revealed that CYP3A4 and CYP3A7 are expressed specifically in adult and fetal human livers, respectively (4).
In summary, our results suggest that indinavir is metabolized by the fetal liver. The formation of primary metabolites of indinavir are qualitatively similar among the different developmental stages in human. However, the formation of oxidative metabolites in fetal liver microsomes was about half and one-third of that in pediatric and adult liver, respectively.
Acknowledgments
The authors gratefully acknowledge the support and thoughtful discussion of Drs. Anthony Y. H. Lu and Thomas A. Baillie during the course of this study.
Footnotes
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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: Masato_Chiba{at}merck.com.
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Received March 6, 1997; accepted June 13, 1997
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↵2 Dr. J. L. Raucy, personal communication.
- Abbreviation used is::
- HIV
- human immunodeficiency virus
- The American Society for Pharmacology and Experimental Therapeutics