Metabolism of 17-alpha-hydroxyprogesterone caproate by human placental mitochondria.

Perfusion of 17-alpha-hydroxyprogesterone caproate (17HPC) via the maternal circuit of a dually perfused human placental lobule resulted in the extensive formation of 2 metabolites. On the other hand, human placental microsomes biotransformed 17HPC into 5 monohydroxylated metabolites, which did not correspond to those formed during perfusion. The goal of this investigation was to determine the subcellular localization of the enzymes responsible for the biotransformation of 17HPC during its perfusion in human placenta. Crude subcellular fractions of the human placental tissue were utilized. Six 17HPC metabolites were formed by the placental mitochondrial fraction, of which 4 were identical to those formed by the microsomes; whereas the other 2, namely MM and M19, were formed by the mitochondrial fraction only. The latter metabolites were identical to those formed during 17HPC perfusion, as determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Therefore, these data strongly suggest that the enzymes responsible for the biotransformation of 17HPC during its perfusion are predominantly localized in human placental mitochondria.

Transplacental transfer and metabolism of buprenorphine in preterm human placenta.

We sought to determine whether gestational age affects the transplacental transfer and metabolism of buprenorphine (BUP). Transfer of BUP (10 ng/mL) and its [ (3)H]-isotope was determined across placentas of 30 to 34 weeks of gestation utilizing the technique of dual perfusion of placental lobule. Concentration of the drug in trophoblast tissue and in maternal and fetal circuits was determined by liquid scintillation spectrometry. Microsomes prepared from placentas of 17 to 37 weeks of gestation were divided into three groups: late second, early third, and late third trimesters. Antibodies raised against human cytochrome P450 (CYP) isoforms were utilized to identify the enzyme(s) catalyzing BUP biotransformation by preterm placental microsomes. The amount of norbuprenorphine formed was determined by liquid chromatography-mass spectrometry (LC-MS). BUP transfer across the placentas of 30 to 34 weeks of gestation was similar to those at term. CYP19 antibodies caused 60% inhibition of BUP metabolism by microsomes of late second and early third trimesters and 85% by microsomes of late third trimester. The developmental changes occurring in human placenta between 30 weeks of gestation through term do not affect the transfer of BUP across human placenta. CYP19 is the major enzyme responsible for biotransformation of BUP beginning at 17 weeks of gestation until term.

Bupropion metabolism by human placenta.

Smoking during pregnancy is the largest modifiable risk factor for pregnancy-related morbidity and mortality. The success of bupropion for smoking cessation warrants its investigation for the treatment of pregnant patients. Nevertheless, the use of bupropion for the treatment of pregnant smokers requires additional data on its bio-disposition during pregnancy. Therefore, the aim of this investigation was to determine the metabolism of bupropion in placentas obtained from nonsmoking and smoking women, identify metabolites formed and the enzymes catalyzing their formation, as well as the kinetics of the reaction. Data obtained revealed that human placentas metabolized bupropion to hydroxybupropion, erythro- and threohydrobupropion. The rates for formation of erythro- and threohydrobupropion exceeded that for hydroxybupropion by several folds, were dependent on the concentration of bupropion and exhibited saturation kinetics with an apparent K(m) value of 40microM. Human placental 11beta-hydroxysteroid dehydrogenases were identified as the major carbonyl-reducing enzymes responsible for the reduction of bupropion to threo- and erythrohydrobupropion in microsomal fractions. On the other hand, CYP2B6 was responsible for the formation of OH-bupropion. These data suggest that both placental microsomal carbonyl-reducing and oxidizing enzymes are involved in the metabolism of bupropion.

Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide.

One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans-(M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.

Metabolism of 17alpha-hydroxyprogesterone caproate by hepatic and placental microsomes of human and baboons.

Recent data from our laboratory revealed the formation of an unknown metabolite of 17 hydroxyprogesterone caproate (17-HPC), used for treatment of preterm deliveries, during its perfusion across the dually perfused human placental lobule. Previously, we demonstrated that the drug is not hydrolyzed, neither in vivo nor in vitro, to progesterone and caproate. Therefore, the hypothesis for this investigation is that 17-HPC is actively metabolized by human and baboon (Papio cynocephalus) hepatic and placental microsomes. Baboon hepatic and placental microsomes were investigated to validate the nonhuman primate as an animal model for drug use during pregnancy. Data presented here indicate that human and baboon hepatic microsomes formed several mono-, di-, and tri-hydroxylated derivatives of 17-HPC. However, microsomes of human and baboon placentas metabolized 17-HPC to its mono-hydroxylated derivatives only in quantities that were a fraction of those formed by their respective livers, except for two metabolites (M16' and M17') that are unique for placenta and contributed to 25% and 75% of the total metabolites formed by human and baboon, respectively. The amounts of metabolites formed, relative to each other, by human and baboon microsomes were different suggesting that the affinity of 17-HPC to CYP enzymes and their activity could be species-dependent.

Glyburide metabolism by placentas of healthy and gestational diabetics.

The aim of this investigation was to determine the metabolism of glyburide (GL) by microsomes prepared from placentas obtained from uncomplicated pregnancies (UP), women with gestational diabetics (GD) on a diabetic diet, and those on a diet and GL. Term placentas were obtained from UP and GD. Crude microsomal fractions were prepared by differential centrifugation and stored at -80 degrees C. The activity of the microsomes in metabolizing glyburide to the trans-4-hydroxycyclohexyl glyburide (THCGL) and cis-3-hydroxycyclohexyl glyburide (CHCGL) was determined and quantified using high-performance liquid chromatography-mass spectrometer (HPLC-MS). The activity of the placental microsomes varied widely between individual placentas in each group. The median values (pmol.mg (-1) P.min (-1)) for the rates of THCGL formation were 0.34, 0.3, and 0.23 for placentas of UP, GD on diet, and GD on GL and a diet, respectively. The median values for CHCGL formation were 0.13 for UP, 0.11 for GD on a diet, and 0.10 (pmol.mg (-1) P.min (-1)) for GD on GL and a diet. A pool of individual microsomal fractions from each group was prepared and its activity revealed the following: greater formation of THCGL in the UP (0.36 +/- 0.10) than GD (0.22 +/- 0.03) ( P = 0.058 for GD on a diet, 0.04 for GD on GL). There was greater formation of CHCGL in UP (0.26 +/- 0.04) than GD (0.12 +/- 0.003) ( P < 0.006). There was no difference in GD on a diet and GD on GL plus diet. We concluded that the apparent differences in the formation of metabolites may be statistically significant, but it is unlikely to be of physiological importance, given the sample size and other experimental factors. Therefore, a more comprehensive investigation is underway.

Kinetics of glyburide metabolism by hepatic and placental microsomes of human and baboon.

Glyburide (glibenclamide) is under investigation for treatment of gestational diabetes. Two metabolites of glyburide have been previously identified in patients, namely, 4-trans-(M1) and 3-cis-(M2) hydroxycyclohexyl glyburide. Recently, the metabolism of glyburide by microsomes of liver and placenta from humans and baboons revealed the formation of four additional metabolites: 4-cis-(M2a), 3-trans-(M3), and 2-trans-(M4) hydroxycyclohexyl glyburide, and ethyl-hydroxy glyburide (M5). The aim of this investigation was to determine the kinetics for the metabolism of glyburide by cytochrome P450 (CYP) isozymes of human and baboon placental and hepatic microsomes. The metabolism of glyburide by microsomes from the four organs revealed saturation kinetics and apparent K(m) values between 4 and 12 microM. However, the rates for formation of the metabolites varied between organs and species. M1 was the major metabolite (36% of total), formed by human hepatic microsomes with V(max) of 80+/-13 pmol mg protein(-1)min(-1), and together with M2, accounted for only 51% of the total. M5 was the major metabolite (87%) formed by human placental microsomes with V(max) of 11 pmol mg protein(-1)min(-1). In baboon liver, M5 had the highest rate of formation (V(max) 135+/-32 pmol mg protein(-1)min(-1), 39% of total), and in its placenta, was M4 (V(max) 0.7+/-0.1 pmol mg protein(-1)min(-1), 65%). The activity of human and baboon hepatic microsomes in metabolizing glyburide was similar, but the activity of human and baboon placental microsomes was 7% and 0.3% of their respective hepatic microsomes. The data obtained suggest that more than 1 CYP isozyme is responsible for catalyzing the hydroxylation of glyburide.

The effect of opiates on the activity of human placental aromatase/CYP19.

Aromatase, cytochrome P450 19, is a key enzyme in the biosynthesis of estrogens by the human placenta. It is also the major placental enzyme that metabolizes the opiates L-acetylmethadol (LAAM), methadone, and buprenorphine (BUP). Methadone and BUP are used in treatment of the opiate addict and are competitive inhibitors of testosterone conversion to estradiol (E(2)) and 16alpha-hydroxytestosterone (16-OHT) to estriol (E(3)) by aromatase. The aim of this investigation is to determine the effect of 20 opiates, which can be administered to pregnant patients for therapeutic indications or abused, on E(2) and E(3) formation by placental aromatase. Data obtained indicated that the opiates increased, inhibited, or had no effect on aromatase activity. Their effect on E(3) formation was more pronounced than that on E(2) due to the lower affinity of 16-OHT than testosterone to aromatase. The K(i) values for the opiates that inhibited E(3) formation were sufentanil, 7 +/- 1 microM; LAAM, 13 +/- 8 microM; fentanyl, 25 +/- 5 microM; oxycodone, 92 +/- 22 microM; codeine, 218 +/- 69 microM; (+)-pentazocine, 225 +/- 73 microM. The agonists morphine, heroin, hydromorphone, oxymorphone, hydrocodone, propoxyphene, meperidine, levorphanol, dextrorphan, and (-)-pentazocine and the antagonists naloxone and naltrexone caused an increase in E(3) formation by 124-160% of control but had no effect on E(2) formation. Moreover, oxycodone and codeine did not inhibit E(2) formation and the IC(50) values for fentanyl, sufentanil, and (+)-pentazocine were >1000 microM. It is unlikely that the acute administration of the opiates that inhibit estrogen formation would affect maternal and/or neonatal outcome. However, the effects of abusing any of them during the entire pregnancy are unclear at this time.

Identification of glyburide metabolites formed by hepatic and placental microsomes of humans and baboons.

Glyburide (glibenclamide) is a second-generation sulfonylurea used for treatment of type-2 and gestational diabetes mellitus. To date, two glyburide metabolites have been identified in maternal urine: namely, 4-trans-hydroxycyclohexyl glyburide and 3-cis-hydroxycyclohexyl glyburide. The use of glyburide to treat gestational diabetes prompted us to investigate its metabolism by the placenta. The metabolism of glyburide by microsomal preparations from human and baboon placenta was compared with metabolism by their livers. The metabolites formed by the microsomes of the four tissues were identified by high-performance liquid chromatography-mass spectrometry using retention times, ion current (extracted at m/z 510), and selected-ion monitoring. The data obtained revealed the formation of six distinct hydroxylated derivatives of glyburide by each of the four microsomal preparations. However, the amounts of the six metabolites formed by the placentas were a fraction of that formed by the livers. Moreover, the relative quantities of each metabolite formed differed between species as well as between the two tissues. Also, the structure of the unidentified metabolites was determined by comparison with synthesized standards. These metabolites were identified as the 4-cis-hydroxycyclohexyl glyburide, 3-trans-hydroxycyclohexyl glyburide, and 2-trans-hydroxycyclohexyl glyburide. Therefore, one glyburide metabolite remains to be identified, but the data we obtained allowed us to suggest its structure.

The effect of methadone and buprenorphine on human placental aromatase.

Methadone and buprenorphine (BUP) are used for treatment of the pregnant opiate addict. CYP19/aromatase is the major placental enzyme responsible for the metabolism of methadone to 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and BUP to norbuprenorphine (norBUP). The aim of this investigation was to determine the effects of methadone and BUP on the activity of placental microsomal aromatase in the conversion of its endogenous substrates testosterone to 17beta-estradiol (E(2)) and 16alpha-hydroxytestosterone (16-OHT) to estriol (E(3)). The conversion of testosterone and 16-OHT by human placental microsomes exhibited saturation kinetics, and the apparent K(m) values were 0.2 +/- 1 and 6 +/- 3 microM, respectively. V(max) values for E(2) and E(3) formation were 70 +/- 16 and 28 +/- 10 pmol/mg proteinmin, respectively. Also, data obtained revealed that methadone and BUP are competitive inhibitors of testosterone conversion to E(2) and 16-OHT to E(3). The K(i) for methadone inhibition of E(2) and E(3) formation were 393 +/-144 and 53 +/- 28 microM, respectively, and for BUP the K(i) was 36 +/- 9 and 6 +/- 1 microM. The higher potency of the two opiates and their metabolites in inhibiting E(3) formation is in agreement with the lower affinity of 16-OHT than testosterone to aromatase. Moreover, the metabolites EDDP and norBUP were weaker inhibitors of aromatase than their parent compounds. The determined inhibition constants of methadone and BUP for E(3) formation by a cDNA-expressed CYP19 preparation were similar to those for placental microsomes. Therefore, data reported here suggest that methadone, BUP, and their metabolites are inhibitors of androgen aromatization in the placental biosynthesis of estrogens.

Bidirectional transfer of methadone across human placenta.

Methadone maintenance programs are considered the standard of care for the pregnant opiate addict. However, data on changes in methadone pharmacokinetics (PK) during pregnancy are limited and do not include its disposition by the placenta due to obvious ethical and safety considerations. Accordingly, investigations in our laboratory are focusing on human placental disposition of opiates including methadone. Recently, we reported on methadone metabolism by placental aromatase and provide here data on its bidirectional transfer across the tissue utilizing the technique of dual perfusion of placental lobule. The concentrations of the opiate transfused into the term placental tissue were those reported for its in vivo levels in the maternal serum of women under treatment with the drug. Data obtained indicated that the opiate has no adverse effects on placental viability and functional parameters and that it is retained by the tissue. Also, methadone transfer and its clearance index in the fetal to maternal direction (0.97+/-0.05) was significantly higher (P<0.05) than in the maternal to fetal (0.83+/-0.09). The observed asymmetry in methadone transfer could be explained by the unidirectional activity of the efflux transporter P glycoprotein (P-gp) that is highly expressed in variable amounts in trophoblast tissue. Therefore, placental disposition of methadone might be an important contributor to the regulation of its concentration in the fetal circulation and consequently may affect the incidence and intensity of neonatal abstinence syndrome for women treated with the drug during pregnancy.

Methadone metabolism by human placenta.

Methadone pharmacotherapy is considered the standard for treatment of the pregnant heroin/opioid addict. One of the factors affecting the transfer kinetics of opioids across human placenta and their levels in the fetal circulation is their metabolism by the tissue. The aim of this investigation is to identify the enzyme(s) responsible for the metabolism of methadone, determine the kinetics of the reaction and the metabolites formed utilizing placental tissue obtained from term healthy pregnancies. Microsomal fractions of trophoblast tissue homogenates had the highest activity in catalyzing the metabolism of methadone. The product formed was identified by HPLC-UV as 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Inhibitors selective for cytochrome P450 (CYP) isozymes were used to identify the enzyme catalyzing the biotransformation of methadone. Aminoglutethimide and 4-hydroxyandrostenedione inhibited EDDP formation by 88 and 70%, respectively, suggesting that CYP19/aromatase is the enzyme catalyzing the reaction. This was confirmed by the effect of monoclonal antibodies raised against CYP19 that caused an 80% inhibition of the reaction. The apparent K(m) and V(max) values for the CYP19 catalyzed metabolism of methadone to EDDP were 424 +/- 92 microM and 420 +/- 89 pmol(mgprotein)(-1)min(-1), respectively. Kinetic analysis of a cDNA-expressed CYP19 for the metabolism of methadone to EDDP was identical to that by placental microsomes. Taken together, these data indicate that CYP19/aromatase is the major enzyme responsible for the metabolism of methadone to EDDP in term human placentas obtained from healthy pregnancies.