Bupropion sustained release for pregnant smokers: a randomized, placebo-controlled trial.

BACKGROUND: Bupropion is used to treat depression during pregnancy. However, its usefulness as a smoking cessation aid for pregnant women is not fully known. OBJECTIVE: The objective of the study was to evaluate the preliminary efficacy of bupropion sustained release for smoking cessation during pregnancy. STUDY DESIGN: We conducted a randomized, prospective, double-blind, placebo-controlled, pilot trial. Pregnant women who smoked daily received individualized behavior counseling and were randomly assigned to a 12 week, twice-a-day treatment with 150 mg bupropion sustained release or placebo. The primary study objectives were to determine whether bupropion sustained release reduces nicotine withdrawal symptoms on the quit date and during the treatment period compared with placebo and whether it increases 7 day point prevalence abstinence at the end of the treatment period and at the end of pregnancy. RESULTS: Subjects in the bupropion (n = 30) and placebo (n = 35) groups were comparable in age, smoking history, number of daily smoked cigarettes, and nicotine dependence. After controlling for maternal age and race, bupropion sustained release reduced cigarette cravings (1.5 ± 1.1 vs 2.1 ± 1.2, P = .02) and total nicotine withdrawal symptoms (3.8 ± 4.3 vs 5.4 ± 5.1, P = .028) during the treatment period. Administration of bupropion sustained release reduced tobacco exposure, as determined by levels of carbon monoxide in exhaled air (7.4 ± 6.4 vs 9.1 ± 5.8, P = .053) and concentrations of cotinine in urine (348 ± 384 ng/mL vs 831 ± 727 ng/mL, P = .007) and increased overall abstinence rates during treatment (19% vs 2%, P = .003). However, there was no significant difference in 7 day point prevalence abstinence rates between the 2 groups at the end of medication treatment (17% vs 3%, P = .087) and at the end of pregnancy (10% vs 3%, P = .328). CONCLUSION: Individual smoking cessation counseling along with the twice-daily use of 150 mg bupropion sustained release increased smoking cessation rates and reduced cravings and total nicotine withdrawal symptoms during the treatment period. However, there was no significant difference in abstinence rates between groups at the end of medication treatment and at the end of pregnancy, likely because of the small sample size. A larger study is needed to confirm these findings and to examine the potential benefit/ risk ratio of bupropion sustained release for smoking cessation during pregnancy.

Pharmacokinetics of Bupropion and Its Pharmacologically Active Metabolites in Pregnancy.

Bupropion sustained release is used to promote smoking cessation in males and nonpregnant females. However, its efficacy as a smoking cessation aid during pregnancy is not reported. The pregnancy-associated changes in maternal physiology may alter the pharmacokinetics and pharmacodynamics of bupropion and consequently its efficacy in pregnant smokers. Therefore, the aims of this study were to determine the steady-state pharmacokinetics of bupropion during pregnancy and the effect of functional genetic variants of CYP2B6 and CYP2C19 on bupropion pharmacokinetics in pregnant women. Plasma and urine concentrations of bupropion and its metabolites hydroxybupropion (OHBUP), threohydrobupropion, and erythrohydrobupropion were determined by liquid chromatography-mass spectrometry. Subjects were genotyped for five nonsynonymous single-nucleotide polymorphisms that result in seven CYP2B6 alleles, namely *2, *3, *4, *5, *6, *7, and *9, and for CYP2C19 variants *2, *3, and *17 The present study reports that the isoform-specific effect of pregnancy on bupropion-metabolizing enzymes along with the increase of renal elimination of the drug could collectively result in a slight decrease in exposure to bupropion in pregnancy. In contrast, pregnancy-induced increase in CYP2B6-catalyzed bupropion hydroxylation did not impact the plasma levels of OHBUP, probably due to a higher rate of OHBUP glucuronidation, and renal elimination associated with pregnancy. Therefore, exposure to OHBUP, a pharmacologically active metabolite of the bupropion, appears to be similar to that of the nonpregnant state. The predicted metabolic phenotypes of CYP2B6*6 and variant alleles of CYP2C19 in pregnancy are similar to those in the nonpregnant state.

Bupropion therapy during pregnancy: the drug and its major metabolites in umbilical cord plasma and amniotic fluid.

BACKGROUND: Bupropion is used for treatment of depression during pregnancy. However, its use as a smoking cessation aid for pregnant women is currently under evaluation. OBJECTIVE: The aim of this opportunistic study was to investigate the transfer of bupropion and its major pharmacologically active metabolites, hydroxybupropion and threohydrobupropion, across the placenta in vivo. In addition, the concentrations of the drug and its metabolites were determined in the amniotic fluid. STUDY DESIGN: The following samples were collected at deliveries from 22 women taking bupropion: maternal blood (n = 22), umbilical cord venous blood (n = 22), and amniotic fluid (n = 9). The concentrations of the drug and its metabolites in blood plasma and amniotic fluid were determined by means of liquid chromatography-mass spectrometry. Placental passage was calculated as a ratio of umbilical cord venous plasma to maternal plasma concentrations. RESULTS: The levels of hydroxybupropion and threohydrobupropion in umbilical cord venous plasma were invariably lower than their corresponding concentrations in maternal plasma. The concentrations of bupropion in umbilical cord plasma were lower than in maternal plasma in the majority of the maternal-cord blood pairs. The median values of the umbilical cord venous plasma to maternal plasma ratios were: bupropion, 0.53 (interquartile range 0.35, n = 18), hydroxybupropion, 0.21 (interquartile range 0.12, n = 18), and threohydrobupropion, 0.61 (interquartile range 0.11, n = 21). In umbilical cord venous plasma, the median concentration of bupropion was 5.3 ng/mL; hydroxybupropion, 103.6 ng/mL; and threohydrobupropion, 59.6 ng/mL. Bupropion and its metabolites were detectable in the amniotic fluid but the concentrations of threohydrobupropion were higher than those in the corresponding umbilical cord venous plasma. CONCLUSION: Bupropion and its active metabolites cross the placenta to the fetal circulation. The concentrations of hydroxybupropion and threohydrobupropion in umbilical cord venous plasma were higher than bupropion concentrations suggesting a higher fetal exposure to the metabolites than the parent drug. The higher levels of threohydrobupropion in the amniotic fluid than those in umbilical cord venous plasma suggest that enzymes involved in the metabolism of bupropion to threohydrobupropion are most likely active in the fetus. The biological consequences of fetal exposure to maternally administered bupropion and/or its active metabolites via placental transfer and recirculation of the amniotic fluid are yet to be determined.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS.

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine.

Human adipose-derived mesenchymal stromal cell pigment epithelium-derived factor cytotherapy modifies genetic and epigenetic profiles of prostate cancer cells.

BACKGROUND AIMS: Adipose-derived mesenchymal stromal cells (ASCs) are promising tools for delivery of cytotherapy against cancer. However, ASCs can exert profound effects on biological behavior of tumor cells. Our study aimed to examine the influence of ASCs on gene expression and epigenetic methylation profiles of prostate cancer cells as well as the impact of expressing a therapeutic gene on modifying the interaction between ASCs and prostate cancer cells. METHODS: ASCs were modified by lentiviral transduction to express either green fluorescent protein as a control or pigment epithelium-derived factor (PEDF) as a therapeutic molecule. PC3 prostate cancer cells were cultured in the presence of ASC culture-conditioned media (CCM), and effects on PC3 or DU145. Ras cells were examined by means of real-time quantitative polymerase chain reaction, EpiTect methyl prostate cancer-focused real-time quantitative polymerase chain reaction arrays, and luciferase reporter assays. RESULTS: ASCs transduced with lentiviral vectors were able to mediate expression of several tumor-inhibitory genes, some of which correlated with epigenetic methylation changes on cocultured PC3 prostate cancer cells. When PC3 cells were cultured with ASC-PEDF CCM, we observed a shift in the balance of gene expression toward tumor inhibition, which suggests that PEDF reduces the potential tumor-promoting activity of unmodified ASCs. CONCLUSIONS: These results suggest that ASC-PEDF CCM can promote reprogramming of tumor cells in a paracrine manner. An improved understanding of genetic and epigenetic events in prostate cancer growth in response to PEDF paracrine therapy would enable a more effective use of ASC-PEDF, with the goal of achieving safer yet more potent anti-tumor effects.

Transplacental transfer of vancomycin and telavancin.

OBJECTIVE: We sought to determine the bidirectional transfer and distribution of vancomycin and telavancin across the dually perfused term human placental lobule. STUDY DESIGN: The technique of dually perfused placental lobule was used in its recirculating mode to determine the maternal to fetal (M→F) (n = 20) and fetal to maternal (n = 18) transfer of each antibiotic, which were coperfused with their radioactive isotopes. The concentrations of drugs were determined by liquid scintillation spectrometry. RESULTS: In the M→F direction, the transfer of vancomycin (9.6 ± 4%) and telavancin (6.5 ± 2%) were low; however, telavancin retention by the perfused lobule was greater than that of vancomycin (P < .01). The normalized transplacental transfer of telavancin across the placental lobule in the fetal to maternal direction was higher than in the M→F direction (P < .01), suggesting the involvement of placental efflux transporters. CONCLUSION: The ex vivo perfusion experiments revealed low transfer of vancomycin and telavancin to the fetal circuit.

Physicochemical and Biological Properties of Membrane Vesicles Derived from Human Term Placentas.

The purpose of this study was to conduct initial characterization of membrane vesicles isolated from human placenta by agitation of villous tissue (apical and basal) as well as vesicles obtained following dual perfusion of placental lobule. The morphology, physical and biological properties of the isolated vesicles were determined by electron microscopy, dynamic light scattering, and immunoblotting as well as nanoflow liquid chromatography-mass spectrometry proteomics analysis. CD-1 male mice were used to test the biocompatibility of the vesicles in vivo and assess the biodistribution of fluorescently labeled apical and perfusion vesicles. The vesicles obtained following placental perfusion and the apical vesicles had Z-average diameters of 199±23 nm and 246±24 nm, respectively, and demonstrated nanocarrier stability, low toxicity, and low immunogenicity. On the other hand, administration of basal vesicles resulted in animal demise with LD50 of 0.85 µgprotein/g. Both fluorescently labeled apical and perfusion vesicles were detected in the lungs, liver, kidneys, and spleen of CD-1 mice within 24 h of administration. However, there were differences in organ distribution of these vesicles over 24 hours time period. These data suggest that placental apical and perfusion vesicles have a potential for further development as biological vehicles for drug delivery.

Effect of deuteration on the single dose pharmacokinetic properties and postoperative analgesic activity of methadone.

Although methadone is effective in the management of acute pain, the complexity of its absorption-distribution-metabolism-excretion profile limits its use as an opioid of choice for perioperative analgesia. Because deuteration is known to improve the pharmacokinetic, pharmacodynamic and toxicological properties of some drugs, here we characterized the single dose pharmacokinetic properties and post-operative analgesic efficacy of d9-methadone. The pharmacokinetic profiles of d9-methadone and methadone administered intravenously to CD-1 male mice revealed that deuteration leads to a 5.7- and 4.4-fold increase in the area under the time-concentration curve and maximum concentration in plasma, respectively, as well as reduction in clearance (0.9 ± 0.3 L/h/kg vs 4.7 ± 0.8 L/h/kg). The lower brain-to-plasma ratio of d9-methadone compared to that of methadone (0.35 ± 0.12 vs 2.05 ± 0.62) suggested that deuteration decreases the transfer of the drug across the blood-brain barrier. The estimated LD50 value for a single intravenous dose of d9-methadone was 2.1-fold higher than that for methadone. Moreover, d9-methadone outperformed methadone in the efficacy against postoperative pain by primarily activating peripheral opioid receptors. Collectively, these data suggest that the replacement of three hydrogen atoms in three methyl groups of methadone altered its pharmacokinetic properties, improved safety, and enhanced its analgesic efficacy.

Role of Uptake Transporters OAT4, OATP2A1, and OATP1A2 in Human Placental Bio-disposition of Pravastatin.

Pravastatin is currently under evaluation for prevention of preeclampsia. Factors contributing to placental disposition of pravastatin are important in assessment of potential undesirable fetal effects. The purpose of this study was to identify the uptake transporters that contribute to the placental disposition of pravastatin. Our data revealed the expression of organic anion transporting polypeptide 1A2 (OATP1A2) and OATP2A1 in the apical, and OATP2B1 and OATP5A1 in the basolateral membranes of the placenta, while organic anion transporter 4 (OAT4) exhibited higher expression in basolateral membrane but was detected in both membranes. Preloading placental membrane vesicles with glutarate increased the uptake of pravastatin suggesting involvement of glutarate-dependent transporters such as OAT4. In the HEK293 cells overexpressing individual uptake transporters, OATP2A1, OATP1A2 and OAT4 were determined to accept pravastatin as a substrate at physiological pH, while the uptake of pravastatin by OATP2B1 (known to interact with pravastatin at acidic pH) and OATP5A1 was not detected at pH 7.4. These findings led us to propose that OATP1A2 and OATP2A1 are responsible for the placental uptake of pravastatin from the maternal circulation, while OAT4 mediates the passage of the drug across placental basolateral membrane in the fetal-to-maternal direction.

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.

The effect of esterases on 17alpha-hydroxyprogesterone caproate.

OBJECTIVE: The aim of this investigation is to determine whether 17alpha-hydroxyprogesterone caproate is hydrolyzed, in vitro, to 17alpha-hydroxyprogesterone and caproate. STUDY DESIGN: The in vitro hydrolysis of dual radioactively labeled 17alpha-hydroxy-[3H] progesterone [14C] caproate by human plasma, hepatic and placental S9 fractions as well as recombinant esterases was investigated. The formation of [3H]-17alpha-hydroxyprogesterone and [14C]-caproate were determined with the use of high-performance liquid chromatography equipped with an online radioactivity detector. The presence and activity of carboxylesterase and butyrylcholinesterase in the human-derived preparations was confirmed by the hydrolysis of their prototypic substrates p-nitrophenyl acetate, p-nitrophenyl butyrate, and butyrylthiocholine, respectively. RESULTS: The aforementioned human-derived preparations hydrolyzed p-nitrophenyl acetate, p-nitrophenyl butyrate, and butyrylthiocholine. However, when 17alpha-hydroxyprogesterone caproate was incubated with the human-derived preparations under identical experimental conditions neither [3H]-17alpha-hydroxyprogesterone nor [14C]-caproate was detected. CONCLUSION: 17Alpha-hydroxyprogesterone caproate is not hydrolyzed in vitro by the esterase enzymes present in human plasma, liver, preterm, or term placenta.

Effect of albumin on transplacental transfer and distribution of rosiglitazone and glyburide.

OBJECTIVE: The aims of this investigation were (i) to determine the rate and extent of rosiglitazone transfer across term human placenta, and (ii) to determine the effect of human serum albumin (HSA) on rosiglitazone and glyburide transfer and distribution. METHODS: These aims were achieved by utilizing the technique of dual perfusion of placental lobule (DPPL). Each hypoglycemic drug was coperfused with the marker compound antipyrine (AP). In each experiment, the [3H]-isotope of the hypoglycemic drug and the [14C]-isotope of AP were added to enhance the detection limits of each drug. Human serum albumin (HSA) was added to both the maternal and fetal circuits in the experiments in which it was investigated. RESULTS: Transplacental transfer of rosiglitazone and glyburide from the maternal to fetal circuits in media devoid of HSA was similar. However, the addition of HSA to the maternal and fetal circuits had different effects on the transfer and distribution of the two drugs, though their binding to HSA (99.8%) was almost identical. HSA increased the maternal (M) to fetal (F) transfer of rosiglitazone, as revealed by an increase in its F/M concentration ratio from 0.17 +/- 0.01 (in the absence of albumin) to 0.33 +/- 0.07 (p < 0.001). Moreover, the addition of albumin decreased the amount of rosiglitazone retained by placental tissue from 539 +/- 148 to 60 +/- 8 ng/g (p < 0.001). Conversely, the addition of HSA to the perfusion media resulted in a decrease in glyburide transfer, as revealed by the change of its F/M concentration ratio from 0.09 +/- 0.02 (in the absence of albumin) to 0.03 +/- 0.01 (p < 0.01). However, similar to rosiglitazone, glyburide retention by the tissue decreased from 103 +/- 26 to 19 +/- 6 ng/g (p < 0.001). CONCLUSIONS: These data indicate that the binding of the two drugs to albumin, though similar, is only one of the factors that could affect their placental transfer and distribution.

Role of the efflux transporters BCRP and MRP1 in human placental bio-disposition of pravastatin.

The expression and activity of human placental transporters during pregnancy could be altered by several factors including pathological changes associated with preeclampsia. The aims of this study were to identify the placental efflux transporters involved in the bio-disposition of pravastatin, determine the protein expression of these transporters and their encoding genes as well as the activity of pravastatin uptake in placentas obtained from patients with preeclampsia. ATP-dependent uptake of [3H]-pravastatin by trophoblast tissue apical and basal membrane vesicles exhibited sigmoidal kinetics. The curved shapes of Eadie-Hofstee plots indicate that more than one placental transporter are involved in the uptake of pravastatin. ATP-dependent uptake of [3H]-pravastatin into vesicles expressing MRP1-5, BCRP, and P-gp, as well as the results of inhibition studies suggest that BCRP and MRP1 are the major placental efflux transporters responsible for the in vitro uptake of pravastatin. Compared to placentas from healthy pregnancies, preeclamptic placentas had increased number of syncytial knots with increased expression of BCRP in their apical membrane and increased expression of MRP1 in the cytoplasm of the syncytiotrophoblast and in cytoplasm of syncytial knots. There was a concomitant increase in ABCC1 but not in ABCG2 gene expressions in preeclamptic placentas. ATP-dependent uptake of [3H]-pravastatin by vesicles prepared from apical membranes of preeclamptic placentas was similar to the uptake by vesicles prepared from placentas obtained after uncomplicated pregnancies (13.9 ±â€¯6.5 vs 14.1 ±â€¯5.8 pmol·mg protein-1 min-1). The transporter-specific changes in the expression of BCRP and MRP1 in preeclamptic placentas did not affect the efflux activity of transporters localized on the apical membrane of the syncytiotrophoblast.

The Role of Placental Carbonyl Reducing Enzymes in Biotransformation of Bupropion and 4-methylnitrosamino-1-(3-pyridyl)-1-butanone.

BACKGROUND: Bupropion (BUP) has a potential to be an effective pharmacotherapy for smoking cessation during pregnancy. Smoking during pregnancy stimulates placental carbonyl reductases that catalyze the biotransformation of BUP. 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen of cigarette smoke. Carbonyl reduction of NNK into 4- methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) constitutes a major step in NNK detoxification. Thus, placentas of pregnant smokers on BUP therapy can become a site of drug-drug interaction. Therefore, we investigated the effect of continuous exposure to BUP and cigarette smoke on the activity of placental carbonyl reductases in the formation of NNAL from NNK. METHODS: The reductive metabolism of NNK was determined using microsomal and cytosolic subcellular fractions of placentas obtained from non-smoking women treated with BUP for depression, and women not exposed to BUP: non-smokers (control) and smokers. The effect of BUP and its metabolites on the reductive metabolism of NNK was investigated using subcellular fractions of control placentas. RESULTS: The formation of NNAL from NNK by placental cytosolic fractions of heavy smokers (≥20 cigarettes per day) was lower than that of control (12.1±3.5 nmol.mgP-1 vs 16.5±6.0 nmol.mgP-1, P<0.05). While being exposed to BUP, the activity of placental carbonyl reductases remained unaffected, the formation of NNAL in the placental cytosolic fraction decreased only in the presence of high concentrations of BUP metabolites. CONCLUSION: Smoking during pregnancy decreases the detoxifying capacity of soluble carbonyl reductases towards NNK. Given the experimental conditions, exposure to BUP and its metabolites should not impede the reductive metabolism of NNK by placenta in vivo.

The expression pattern of opticin during chicken embryogenesis.

We isolated the chicken homologue of opticin (cOptc), a member of the small leucine-rich repeat protein (SLRP) family. cOptc is expressed in the brain and the neural tube from stage 9 onward. At later stages, cOptc is expressed in the ciliary epithelium of the eye, optic stalk, Rathke's pouch, pharyngeal pouches, nasal pit and otic vesicle.

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.

Metabolism of bupropion by baboon hepatic and placental microsomes.

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (Km 36±6 µM, Vmax 258±32 pmol mg protein(-1) min(-1)), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent Km values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11ß-hydroxysteroid dehydrogenases (18ß-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.

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.

Expression patterns of Wnt genes during development of an anterior part of the chicken eye.

To address the roles of Wnts in the development of the anterior eye, we used a chicken model to perform comprehensive expression analysis of all Wnt genes during anterior eye development. In analyzing the available genomic sequences, we found that the chicken genome encodes 18 Wnt proteins that are homologous to corresponding human and mouse proteins. The mRNA sequences for 12 chicken Wnt genes are available in GenBank, and mRNAs for six other Wnt genes (Wnt2, Wnt5b, Wnt7b, Wnt8b, Wnt9b, and Wnt16) were identified and cloned based on the homology to the genes from other species. In addition, we found that chicken Wnt3a and Wnt7b genes encode two alternative mRNA isoforms containing different first exons. Following in situ hybridization, we found that out of 18 Wnt genes, 11 genes were expressed in the anterior eye, exhibiting distinct temporal-spatial patterns. Several Wnts were expressed in the lens, including Wnt2 and Wnt2b in the anterior epithelium and Wnt5a, Wnt5b, Wnt7a, and Wnt7b in the differentiating lens fiber cells. In the cornea, we detected Wnt3a, Wnt6, and Wnt9b in the ocular surface ectoderm, including the corneal epithelium, and Wnt9a in the corneal endothelium from the onset of its differentiation. In the optic cup, Wnt2, Wnt2b, and Wnt9a were localized in the rim of the optic cup (presumptive iris), while Wnt5a and Wnt16 were detected in the ciliary epithelium/iris zone of the differentiated optic cup, and Wnt6 was expressed in the iridial mesenchyme. These data suggest that Wnt signaling might play important roles in anterior eye development.