Creation of Novel Sensitive Probe Substrate and Moderate Inhibitor Models for a Comprehensive Prediction of CYP2C8 Interactions for Tucatinib.

A physiologically-based pharmacokinetic (PBPK) model was developed to simulate plasma concentrations of tucatinib (TUKYSA®) after single-dose or multiple-dose administration of 300 mg b.i.d. orally. This PBPK model was subsequently applied to support evaluation of drug-drug interaction (DDI) risk as a perpetrator resulting from tucatinib inhibition of CYP3A4, CYP2C8, CYP2C9, P-gp, or MATE1/2-K. The PBPK model was also applied to support evaluation of DDI risk as a victim resulting from co-administration with CYP3A4 or CYP2C8 inhibitors, or a CYP3A4 inducer. After refinement with clinical DDI data, the final PBPK model was able to recover the clinically observed single and multiple-dose plasma concentrations for tucatinib when tucatinib was administered as a single agent in healthy subjects. In addition, the final model was able to recover clinically observed plasma concentrations of tucatinib when administered in combination with itraconazole, rifampin, or gemfibrozil as well as clinically observed plasma concentrations of probe substrates of CYP3A4, CYP2C8, CYP2C9, P-gp, or MATE1/2-K. The PBPK model was then applied to prospectively predict the potential perpetrator or victim DDIs with other substrates, inducers, or inhibitors. To simulate a potential interaction with a moderate CYP2C8 inhibitor, two novel PBPK models representing a moderate CYP2C8 inhibitor and a sensitive CYP2C8 substrate were developed based on the existing PBPK models for gemfibrozil and rosiglitazone, respectively. The simulated population geometric mean area under the curve ratio of tucatinib with a moderate CYP2C8 inhibitor ranged from 1.98- to 3.08-fold, and based on these results, no dose modifications were proposed for moderate CYP2C8 inhibitors for the tucatinib label.

The Pharmacokinetics and Safety of Tucatinib in Volunteers with Hepatic Impairment.

BACKGROUND AND OBJECTIVE: Tucatinib, a highly selective tyrosine kinase inhibitor of the human epidermal growth factor receptor 2 (HER2) approved for HER2-positive metastatic breast cancer, is cleared by hepatic metabolism and subsequent biliary excretion. Liver disease can alter drug disposition and pharmacokinetics (PK). The objective of this study is to characterize PK and safety of tucatinib in volunteers with hepatic impairment. METHODS: This Phase 1 study compared the PK and safety of a single 300-mg oral dose of tucatinib in volunteers with mild, moderate, and severe hepatic impairment (Child-Pugh A/B/C) to healthy volunteers matched for sex, age, and body mass index. Pharmacokinetic parameters were determined for tucatinib and its predominant metabolite ONT-993. RESULTS: Compared with healthy volunteers, tucatinib exposure was similar in volunteers with mild impairment and increased in those with moderate or severe impairment without reaching statistical significance. Respective fold increases in geometric mean ratios for AUC0-t and AUC0-∞ were 1.13 and 1.15 in moderate impairment, and 1.43 and 1.61 in severe impairment compared with healthy volunteers. Three treatment-emergent adverse events (nausea, dermatitis, and increased transaminases) were reported in three volunteers and showed no obvious association with hepatic impairment status. CONCLUSION: The 1.61-fold geometric mean ratio AUC0-∞ increase in volunteers with severe hepatic impairment supports the recommendation in the tucatinib prescribing information to reduce the dose from 300 mg twice daily to 200 mg twice daily in patients with severe impairment; no dose adjustment is recommended for patients with mild or moderate hepatic impairment. This trial (NCT03722823) was registered on October 29, 2018.

Mechanistic Modeling of Central Nervous System Pharmacokinetics and Target Engagement of HER2 Tyrosine Kinase Inhibitors to Inform Treatment of Breast Cancer Brain Metastases.

PURPOSE: This study evaluated the central nervous system (CNS) pharmacokinetics and target engagement of lapatinib, neratinib, and tucatinib in patients with cancer, using a physiologically based pharmacokinetic (PBPK) modeling approach. EXPERIMENTAL DESIGN: Drug-specific parameters for in vitro metabolism, binding to plasma proteins and brain tissues, transcellular passive permeability, and interactions with efflux transporters were determined. Whole-body PBPK models integrated with a 4-compartment permeability-limited brain model was developed and verified for predicting plasma and CNS pharmacokinetics. Target engagement ratio (TER), defined as the ratio of the average steady-state unbound drug brain concentration (Css,ave,br) to in vitro IC50 for HER2 inhibition, was used as a predictor of intracranial efficacy. RESULTS: PBPK models predicted that following 1 cycle of standard dosing, tucatinib and lapatinib achieved similar Css,ave,br (14.5 vs. 16.8 nmol/L), while neratinib Css,ave,br (0.68 nmol/L) was 20-fold lower. Tucatinib and neratinib were equally potent for HER2 inhibition (IC50, 6.9 vs. 5.6 nmol/L), while lapatinib was less potent (IC50, 109 nmol/L). The model-predicted population mean TER in the human normal brain was 2.1 for tucatinib, but < 0.20 for lapatinib and neratinib. CONCLUSIONS: The PBPK modeling suggests that tucatinib induces sufficient HER2 inhibition (TER > 2.0) in not only brain metastases with a disrupted blood-brain barrier (BBB), but also micrometastases where the BBB largely remains intact. These findings, in line with available clinical pharmacokinetics and efficacy data, support the therapeutic value of tucatinib for treatment of brain metastases and warrant further clinical investigation for the prevention of brain metastases in patients with HER2-positive breast cancer.

Elimination of tucatinib, a small molecule kinase inhibitor of HER2, is primarily governed by CYP2C8 enantioselective oxidation of gem-dimethyl.

PURPOSE: Tucatinib, a small molecule for the treatment of metastatic HER2-positive breast cancer, was extensively metabolized in humans to multiple oxidative metabolites. To fully understand the elimination and biotransformation pathways of tucatinib, we investigated the in vitro and in vivo metabolism of tucatinib, and also conducted a Phase I trial using [14C]tucatinib. METHODS: To identify the responsible enzymes for tucatinib clearance, we investigated the in vitro metabolism of tucatinib including enzyme phenotyping, which facilitated the discovery of several metabolites in human and monkey plasma and excreta, in particular M1 (ONT-993, an aliphatic hydroxylated metabolite). Stereoselective formation of M1 was further investigated in vitro, in vivo, and in silico. RESULTS: In humans, approximately 86% of the total radiolabeled dose was recovered in feces and 4% in urine; in plasma, approximately 76% of radioactivity circulated as parent drug, with 19% attributed to multiple metabolites. The primary isoforms responsible for the elimination of tucatinib were CYP2C8 and CYP3A4/5. CYP2C8 was shown to possess sole catalytic activity for the formation of M1, whereas CYP3A4/5 and aldehyde oxidase catalyzed the formation of the remaining metabolites. Subsequent investigation revealed that M1 was formed in a stereoselective manner. Examination of the enantiomeric ratio of M1 stereoisomers observed in humans relative to cynomolgus monkeys revealed comparable results, suggesting that the enantiomers that comprise M1 were not considered to be unique or disproportionately high in human. CONCLUSION: CYP2C8 and CYP3A4/5 are the primary drug-metabolizing enzymes involved in the in vitro metabolism of tucatinib, which provided the basis to describe human disposition of tucatinib and formation of the observed metabolites.

Risk-Based Pharmacokinetic and Drug-Drug Interaction Characterization of Antibody-Drug Conjugates in Oncology Clinical Development: An International Consortium for Innovation and Quality in Pharmaceutical Development Perspective.

Antibody-drug conjugates (ADCs) represent a rapidly evolving area of drug development and hold significant promise. To date, nine ADCs have been approved by the US Food and Drug Administration (FDA). These conjugates combine the target specificity of monoclonal antibodies with the anticancer activity of small-molecule therapeutics (also referred to as payload). Due to the complex structure, three analytes, namely ADC conjugate, total antibody, and unconjugated payload, are typically quantified during drug development; however, the benefits of measuring all three analytes at later stages of clinical development are not clear. The cytotoxic payloads, upon release from the ADC, are considered to behave like small molecules. Given the relatively high potency and low systemic exposure of cytotoxic payloads, drug-drug interaction (DDI) considerations for ADCs might be different from traditional small molecule therapeutics. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) convened an ADC working group to create an IQ ADC database that includes 26 ADCs with six unique payloads. The analysis of the ADC data in the IQ database, as well as nine approved ADCs, supports the strategy of pharmacokinetic characterization of all three analytes in early-phase development and progressively minimizing the number of analytes to be measured in the late-phase studies. The systemic concentrations of unconjugated payload are usually too low to serve as a DDI perpetrator; however, the potential for unconjugated payloads as a victim still exists. A data-driven and risk-based decision tree was developed to guide the assessment of a circulating payload as a victim of DDI.

Tucatinib Inhibits Renal Transporters OCT2 and MATE Without Impacting Renal Function in Healthy Subjects.

Tucatinib is a potent tyrosine kinase inhibitor selective for human epidermal growth factor receptor 2 (HER2) approved by the US Food and Drug Administration for the treatment of HER2-positive metastatic breast cancer and in development for other HER2-positive solid tumors. Modest, reversible serum creatinine (SCr) elevations have been observed in tucatinib clinical trials. SCr is conveyed by the renal drug transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) and 2-K (MATE2-K) and can increase in the presence of inhibitors of these transporters. In vitro, tucatinib inhibited OCT2-, MATE1-, and MATE2-K-mediated transport of metformin, with IC50 values of 14.7, 0.340, and 0.135 µM, respectively. Tucatinib also inhibited OCT2- and MATE1-mediated transport of creatinine, with IC50 values of 0.107 and 0.0855 µM, respectively. A phase 1 study with metformin administered orally in the absence and presence of tucatinib was conducted in 18 healthy subjects. Renal function was assessed by measuring glomerular filtration rate (GFR; based on iohexol plasma clearance) and endogenous markers (SCr, cystatin C-based estimated glomerular filtration rate [eGFR]) with and without tucatinib. Metformin exposure increased (1.4-fold) and renal clearance decreased (29.99-17.64 L/h) with tucatinib, with no effect on metformin maximum concentration. Creatinine clearance transiently decreased 23% with tucatinib. GFR and eGFR, which are unaffected by OCT2 and/or MATE1/2-K transport, were unchanged with tucatinib. These data demonstrate that tucatinib inhibits OCT2- and MATE1/2-K-mediated tubular secretion of creatinine, which may manifest as mild SCr elevations that are not indicative of renal impairment.

4-Hydroxyestrone, an Endogenous Estrogen Metabolite, Can Strongly Protect Neuronal Cells Against Oxidative Damage.

Earlier studies showed that endogenous estrogens have neuroprotective effect against oxidative damage. The present study seeks to investigate the protective effect of various endogenous estrogen metabolites against oxidative neurotoxicity in vitro and in vivo. Using immortalized mouse hippocampal neuronal cells as an in vitro model, 4-hydroxyestrone, an estrone metabolite with little estrogenic activity, is found to have the strongest neuroprotective effect against oxidative neurotoxicity among 25 endogenous estrogen metabolites tested, and its protective effect is stronger than 17ß-estradiol. Similarly, 4-Hydroxyestrone also exerts a stronger protective effect than 17ß-estradiol against kanic acid-induced hippocampal oxidative damage in rats. Neuroprotection by 4-hydroxyestrone involves increased cytoplasmic translocation of p53 resulting from SIRT1-mediated deacetylation of p53. Analysis of brain microsomal enzymes shows that estrogen 4-hydroxylation is the main metabolic pathway in the central nervous system. Together, these results show that 4-hydroxyestrone is an endogenous neuroestrogen that can strongly protect against oxidative neuronal damage.

No compelling evidence that more physically attractive young adult women have higher estradiol or progesterone.

Putative associations between sex hormones and attractive physical characteristics in women are central to many theories of human physical attractiveness and mate choice. Although such theories have become very influential, evidence that physically attractive and unattractive women have different hormonal profiles is equivocal. Consequently, we investigated hypothesized relationships between salivary estradiol and progesterone and two aspects of women's physical attractiveness that are commonly assumed to be correlated with levels of these hormones: facial attractiveness (N = 249) and waist-to-hip ratio (N = 247). Our analyses revealed no compelling evidence that women with more attractive faces or lower (i.e., more attractive) waist-to-hip ratios had higher levels of estradiol or progesterone. One analysis did suggest that women with more attractive waist-to-hip ratios had significantly higher progesterone, but the relationship was weak and the relationship not significant in other analyses. These results do not support the influential hypothesis that between-women differences in physical attractiveness are related to estradiol and/or progesterone.

Women's preferences for men's beards show no relation to their ovarian cycle phase and sex hormone levels.

According to the ovulatory shift hypothesis, women's mate preferences for male morphology indicative of competitive ability, social dominance, and/or underlying health are strongest at the peri-ovulatory phase of the menstrual cycle. However, recent meta-analyses are divided on the robustness of such effects and the validity of the often-used indirect estimates of fertility and ovulation has been called into question in methodological studies. In the current study, we test whether women's preferences for men's beardedness, a cue of male sexual maturity, androgenic development and social dominance, are stronger at the peri-ovulatory phase of the menstrual cycle compared to during the early follicular or the luteal phase. We also tested whether levels of estradiol, progesterone, and the estradiol to progesterone ratio at each phase were associated with facial hair preferences. Fifty-two heterosexual women completed a two-alternative forced choice preference test for clean-shaven and bearded male faces during the follicular, peri-ovulatory (validated by the surge in luteinizing hormone or the drop in estradiol levels) and luteal phases. Participants also provided for one entire menstrual cycle daily saliva samples for subsequent assaying of estradiol and progesterone. Results showed an overall preference for bearded over clean-shaven faces at each phase of the menstrual cycle. However, preferences for facial hair were not significantly different over the phases of menstrual cycle and were not significantly associated with levels of reproductive hormones. We conclude that women's preferences for men's beardedness may not be related to changes in their likelihood of conception.

Metabolism and Disposition of a Novel B-Cell Lymphoma-2 Inhibitor Venetoclax in Humans and Characterization of Its Unusual Metabolites.

Venetoclax (ABT-199), a B-cell lymphoma-2 (Bcl-2) protein inhibitor, is currently in clinical development for the treatment of hematologic malignancies. We characterized the absorption, metabolism, and excretion of venetoclax in humans. After a single oral dose of [14C]venetoclax to healthy volunteers, the recovery of total radioactive dose was 100%, with feces being the major route of elimination of the administered dose, whereas urinary excretion was minimal (<0.1%). The extent of absorption was estimated to be at least 65%. Venetoclax was primarily cleared by hepatic metabolism (∼66% of the administered dose). ∼33% of the administered dose was recovered as the parent drug and its nitro reduction metabolite M30 [2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-amino-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)benzamide] (13%) in feces. Biotransformation of venetoclax in humans primarily involves enzymatic oxidation on the dimethyl cyclohexenyl moiety, followed by sulfation and/or nitro reduction. Nitro reduction metabolites were likely formed by gut bacteria. Unchanged venetoclax was the major drug-related material in circulation, representing 72.8% of total plasma radioactivity. M27 (oxidation at the 6 position of cyclohexenyl ring followed by cyclization at the α-carbon of piperazine ring; 4-[(10aR,11aS)-7-(4-chlorophenyl)-9,9-dimethyl-1,3,4,6,8,10,10a,11a-octahydropyrazino[2,1-b][1,3]benzoxazin-2-yl]-N-[3-nitro-4-(tetrahydropyran-4-ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) was identified as a major metabolite, representing 12% of total drug-related material. M27 was primarily formed by cytochrome P450 isoform 3A4 (CYP3A4). Steady-state plasma concentrations of M27 in human and preclinical species used for safety testing suggested that M27 is a disproportionate human metabolite. M27 is not expected to have clinically relevant on- or off-target pharmacologic activities.

Metabolism and Disposition of Pan-Genotypic Inhibitor of Hepatitis C Virus NS5A Ombitasvir in Humans.

Ombitasvir (also known as ABT-267) is a potent inhibitor of hepatitis C virus (HCV) nonstructural protein 5A (NS5A), which has been developed in combination with paritaprevir/ritonavir and dasabuvir in a three direct-acting antiviral oral regimens for the treatment of patients infected with HCV genotype 1. This article describes the mass balance, metabolism, and disposition of ombitasvir in humans without coadministration of paritaprevir/ritonavir and dasabuvir. Following the administration of a single 25-mg oral dose of [(14)C]ombitasvir to four healthy male volunteers, the mean total percentage of the administered radioactive dose recovered was 92.1% over the 192-hour sample collection in the study. The recovery from the individual subjects ranged from 91.4 to 93.1%. Ombitasvir and corresponding metabolites were primarily eliminated in feces (90.2% of dose), mainly as unchanged parent drug (87.8% of dose), but minimally through renal excretion (1.9% of dose). Biotransformation of ombitasvir in human involves enzymatic amide hydrolysis to form M23 (dianiline), which is further metabolized through cytochrome P450-mediated oxidative metabolism (primarily by CYP2C8) at the tert-butyl group to generate oxidative and/or C-desmethyl metabolites. [(14)C]Ombitasvir, M23, M29, M36, and M37 are the main components in plasma, representing about 93% of total plasma radioactivity. The steady-state concentration measurement of ombitasvir metabolites by liquid chromatography-mass spectrometry analysis in human plasma following multiple doses of ombitasvir, in combination with paritaprevir/ritonavir and dasabuvir, confirmed that ombitasvir is the main component (51.9% of all measured drug-related components), whereas M29 (19.9%) and M36 (13.1%) are the major circulating metabolites. In summary, the study characterized ombitasvir metabolites in circulation, the metabolic pathways, and the elimination routes of the drug.

Naturally-occurring estradiol-17beta-fatty acid esters, but not estradiol-17beta, preferentially induce mammary tumorigenesis in female rats: implications for an important role in human breast cancer.

Because mammary glands are surrounded by adipose tissues, we hypothesize that the ultra-lipophilic endogenous estrogen-17beta-fatty acid esters may have preferential hormonal and carcinogenic effects in mammary tissues compared to other target organs (such as the uterus and pituitary). This hypothesis is tested in the present study. We found that all 46 rats implanted with an estradiol-17beta pellet developed large pituitary tumors (average weight=251+/-103 mg) and had to be terminated early, but only 48% of them developed mammary tumors. In addition, approximately one-fourth of them developed a huge uterus. In the 26 animals implanted with a mixture containing estradiol-17beta-stearate and estradiol-17beta-palmitate (two representative estradiol-17beta-fatty acid esters) or in the 29 animals implanted with estradiol-17beta-stearate alone (in the same molar dose as estradiol-17beta), 73% and 79%, respectively, of them developed mammary tumors, whereas only 3 or 2 animals, respectively, had to be terminated early due to the presence of a large pituitary tumor. Both tumorous and normal mammary tissues contained much higher levels of estrogen esterase than other tissues, which catalyzes the releases of bioactive estrogens from their fatty acid esters. In conclusion, while estradiol-17beta is much stronger in inducing pituitary tumor (100% incidence) than mammary tumor, estradiol-17beta-fatty acid esters have a higher efficacy than estradiol-17beta in inducing mammary tumor and yet it only has little ability to induce uterine out-growth and pituitary tumorigenesis. This study establishes the endogenous estrogen-17beta-fatty acid esters as preferential inducers of mammary tumorigenesis.

Cigarette smoking and erectile dysfunction: focus on NO bioavailability and ROS generation.

INTRODUCTION: Thirty million men in the United States suffer from erectile dysfunction (ED) and this number is expected to double by 2025. Considered a major public health problem, which seriously affects the quality of life of patients and their partners, ED becomes increasingly prevalent with age and chronic smoking is a major risk factor in the development of ED. AIM: To review available evidence concerning the effects of cigarette smoking on vascular changes associated with decreased nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS) generation. METHODS: We examined epidemiological and clinical data linking cigarette smoking and ED, and the effects of smoking on vascular NO bioavailability and ROS generation. MAIN OUTCOME MEASURES: There are strong parallels between smoking and ED and considerable evidence supporting the concept that smoking-related ED is associated with reduced bioavailability of NO because of increased ROS. RESULTS: Cigarette smoking-induced ED in human and animal models is associated with impaired arterial flow to the penis or acute vasospasm of the penile arteries. Long-term smoking produces detrimental effects on the vascular endothelium and peripheral nerves and also causes ultrastructural damage to the corporal tissue, all considered to play a role in chronic smoking-induced ED. Clinical and basic science studies provide strong indirect evidence that smoking may affect penile erection by the impairment of endothelium-dependent smooth muscle relaxation or more specifically by affecting NO production via increased ROS generation. Whether nicotine or other products of cigarette smoke mediate all effects related to vascular damage is still unknown. CONCLUSIONS: Smoking prevention represents an important approach for reducing the risk of ED. The characterization of the components of cigarette smoke leading to ED and the mechanisms by which these components alter signaling pathways activated in erectile responses are necessary for a complete comprehension of cigarette smoking-associated ED.

Chemical synthesis of six novel 17beta-estradiol and estrone dimers and study of their formation catalyzed by human cytochrome P450 isoforms.

Our earlier studies have shown that over 20 nonpolar 17beta-estradiol metabolite peaks were detected following incubations of radioactive 17beta-estradiol with human liver microsomes or recombinant human cytochrome P450 isoforms in the presence of NADPH as a cofactor. The structures of two representative nonpolar metabolites were identified earlier as dimers of 17beta-estradiol linked through a diaryl ether bond between the C-3 phenolic oxygen of one molecule and the C-2 or C-4 aromatic carbon of another. Six additional putative dimers between estrone and 17beta-estradiol with structures similar to the two identified ones were synthesized in this study. Using these newly synthesized estrogen dimers as reference standards, we demonstrated that incubations of human liver microsomes or various human cytochrome P450 isoforms with estrone or 17beta-estradiol alone or two estrogens in combination in the presence of NADPH as a cofactor resulted in the formation of all eight estrogen dimers in varying quantities.

NADPH-dependent metabolism of 17beta-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms.

The endogenous estrogens, 17beta-estradiol (E(2)) and estrone (E(1)), undergo extensive metabolism in animals and humans, and a large number of their hydroxylated and keto metabolites have been identified in biological samples. The formation of most of the oxidative estrogen metabolites is catalyzed by cytochrome P450 (CYP) enzymes. Precise knowledge of the CYP-mediated formation of these metabolites, particularly those with unique biological activities (e.g., 4-hydroxy-E(2), 16alpha-hydroxy-E(1), 15alpha-hydroxy-E(2), 16-epiestriol, and 2-methoxyestradiol) in human liver and extrahepatic target tissues and cells, would add significantly to our understanding of the diverse biological functions that are associated with endogenous estrogens. In this article, we review recent results on the NADPH-dependent metabolism of endogenous estrogens to polar (hydroxylated and keto) metabolites as well as to nonpolar metabolites by human tissues and recombinant human CYP isoforms. The available data show that a large number of polar and nonpolar metabolites of E(2) and E(1) are formed by human tissues, and a variety of human CYP isoforms are involved in the NADPH-dependent formation of polar as well as nonpolar estrogen metabolites. These enzymes have varying degrees of catalytic activity and distinct regioselectivity.

Time-dependent inhibition and tetrahydrobiopterin depletion of endothelial nitric-oxide synthase caused by cigarettes.

Smoking causes a dysfunction in endothelial nitric-oxide synthase (eNOS), which is ameliorated, in part, by administration of tetrahydrobiopterin (BH(4)). The exact mechanism by which the nitric oxide deficit occurs is unknown. We have previously shown that aqueous extracts of chemicals in cigarettes (CE) cause the suicide inactivation of neuronal NO synthase (nNOS) by interacting at the substrate-binding site. In the current study, we have found that CE directly inactivates eNOS by a process that is not affected by the natural substrate l-arginine and is distinct from the mechanism of inactivation of nNOS. We discovered that CE causes a time-, concentration-, and NADPH-dependent inactivation of eNOS in an in vitro system containing the purified enzyme, indicating a metabolic component to the inactivation. The CE-treated eNOS but not nNOS was nearly fully reactivated upon incubation with excess BH(4), suggesting that BH(4) depletion is a potential mechanism of inactivation. Moreover, in the presence of CE, eNOS catalyzed the oxidation of BH(4) to dihydrobiopterin and biopterin by a process attenuated by high concentrations of superoxide dismutase but not catalase. We speculate that a redox active component in CE, perhaps a quinone compound, causes oxidative uncoupling of eNOS to form superoxide, which in turn oxidizes BH(4). The discovery of a direct inactivation of eNOS by a compound(s) present in tobacco provides a basis not only for further study of the mechanisms responsible for the biological effects of tobacco but also a search for a potentially novel inactivator of eNOS.

NADPH-dependent formation of polar and nonpolar estrogen metabolites following incubations of 17 beta-estradiol with human liver microsomes.

By using a versatile high-pressure liquid chromatography method (total elution time approximately 135 min) developed in the present study, we detected the formation of some 20 nonpolar radioactive metabolite peaks (designated as M1 through M20), in addition to a large number of polar hydroxylated or keto metabolites, following incubations of [(3)H]17beta-estradiol with human liver microsomes or cytochrome P450 3A4 in the presence of NADPH as a cofactor. The formation of most of the nonpolar estrogen metabolite peaks (except M9) was dependent on the presence of human liver microsomal proteins, and could be selectively inhibited by the presence of carbon monoxide. Among the four cofactors (NAD, NADH, NADP, NADPH) tested, NADPH was the optimum cofactor for the metabolic formation of polar and nonpolar estrogen metabolites in vitro, although NADH also had a weak ability to support the reactions. These observations suggest that the formation of most of the nonpolar estrogen metabolite peaks requires the presence of liver microsomal enzymes and NADPH. Chromatographic analyses showed that these nonpolar estrogen metabolites were not the monomethyl ethers of catechol estrogens or the fatty acid esters of 17beta-estradiol. Analyses using liquid chromatography/mass spectrometry and NMR showed that M15 and M16, two representative major nonpolar estrogen metabolites, are diaryl ether dimers of 17beta-estradiol. The data of our present study suggest a new metabolic pathway for the NADPH-dependent, microsomal enzyme-mediated formation of estrogen diaryl ether dimers, along with other nonpolar estrogen metabolites.

Chemical synthesis of two novel diaryl ether dimers of estradiol-17beta.

We recently detected the formation of estradiol-17beta (estradiol) dimers, linked together through a diaryl ether bond between the C-3 phenolic oxygen of one estradiol molecule and the 2- or 4-position aromatic carbon of another estradiol, following incubations of [3H]estradiol with human liver microsomes or cytochrome p450 enzymes in the presence of NADPH. Using estradiol as the starting material, we designed a four-step method for the chemical synthesis of these two estrogen dimers with the Ullmann condensation reaction as a key step. Step 1: Synthesis of 2- or 4-bromoestradiol from estradiol. Step 2: Protection of the C-3 phenolic hydroxyl group of the 2- or 4-bromoestradiol. Step 3: The Ullmann condensation reaction between the phenol-protected bromoestradiol and the estradiol potassium salt under our modified reaction conditions (with a 41% product yield). Step 4: Removal of the C-3 benzyl group by catalytic hydrogenation. The chromatographic and various spectrometric properties of the two synthesized compounds were identical to those metabolically formed by human cytochrome p450 3A4.

Human cytochrome P450 3A7 has a distinct high catalytic activity for the 16alpha-hydroxylation of estrone but not 17beta-estradiol.

Like catechol estrogens, 16alpha-hydroxylated estrogens are hormonally active, chemically reactive, and potentially mutagenic. We report here our novel findings that human CYP3A7 has a distinct high catalytic activity for the NADPH-dependent 16alpha-hydroxylation of estrone (E(1); at 10 nM to 200 microM substrate concentrations) but not for the 16alpha-hydroxylation of 17beta-estradiol (E(2)). At a physiologically relevant low substrate concentration (10 nM), CYP3A7 had a strong catalytic activity for the 16alpha-hydroxylation of E(1), and the ratio of its 16alpha-hydroxylation to 2-hydroxylation was 107%. In addition to 16alpha-hydroxylation, CYP3A7 also had catalytic activity for the 2-, 4-, 6beta-, and 16beta-hydroxylation of E(1). However, when E(2) was the substrate, CYP3A7 had only very weak catalytic activity for its 16alpha-hydroxylation (<6% of E(1) 16alpha-hydroxylation), and the ratio of its 16alpha-hydroxylation to 2-hydroxylation was 10-33%. Enzyme kinetic analysis showed that the maximal velocity and substrate-binding affinity (1/K(m)) for CYP3A7-mediated 16alpha-hydroxylation of E(1) were both approximately 10 times higher than those for E(2), thereby giving the maximal velocity:K(m) ratio of >100 times higher for the 16alpha-hydroxylation of E(1) than for E(2). Given the recent findings that human CYP3A7 is a polymorphic isoform also expressed in adult liver and certain extrahepatic tissues (in addition to fetal tissues), our data raise the possibility that CYP3A7 may be an important catalyst for the local and/or systemic formation of the procarcinogenic 16alpha-hydroxyestrone in women.

Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms.

We systematically characterized the oxidative metabolites of 17beta-estradiol and estrone formed by 15 human cytochrome P450 (CYP) isoforms. CYP1A1 had high activity for 17beta-estradiol 2-hydroxylation, followed by 15alpha-, 6alpha-, 4-, and 7alpha-hydroxylation. However, when estrone was the substrate, CYP1A1 formed more 4-hydroxyestrone than 15alpha- or 6alpha-hydroxyestrone, with 2-hydroxyestrone as the major metabolite. CYP1A2 had the highest activity for the 2-hydroxylation of both 17beta-estradiol and estrone, although it also had considerable activity for their 4-hydroxylation (9-13% of 2-hydroxylation). CYP1B1 mainly catalyzed the formation of catechol estrogens, with 4-hydroxyestrogens predominant. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2D6 each showed a varying degree of low catalytic activity for estrogen 2-hydroxylation, whereas CYP2C18 and CYP2E1 did not show any detectable estrogen-hydroxylating activity. CYP3A4 had strong activity for the formation of 2-hydroxyestradiol, followed by 4-hydroxyestradiol and an unknown polar metabolite, and small amounts of 16alpha- and 16beta-hydroxyestrogens were also formed. The ratio of 4- to 2-hydroxylation of 17beta-estradiol or estrone with CYP3A4 was 0.22 or 0.51, respectively. CYP3A5 had similar catalytic activity for the formation of 2- and 4- hydroxyestrogens. Notably, CYP3A5 had an unusually high ratio of 4- to 2-hydroxylation of 17beta-estradiol or estrone (0.53 or 1.26, respectively). CYP3A4 and 3A5 also catalyzed the formation of nonpolar estrogen metabolite peaks (chromatographically less polar than estrone). CYP3A7 had a distinct catalytic activity for the 16alpha-hydroxylation of estrone, but not 17beta-estradiol. CYP4A11 had little catalytic activity for the metabolism of 17beta-estradiol and estrone. In conclusion, many human CYP isoforms are involved in the oxidative metabolism of 17beta-estradiol and estrone, with a varying degree of catalytic activity and distinct regioselectivity.