Influence of Glutathione-S-Transferase A1*B Allele on the Metabolism of the Aromatase Inhibitor, Exemestane, in Human Liver Cytosols and in Patients Treated With Exemestane.

Exemestane (EXE) is used to treat postmenopausal women diagnosed with estrogen receptor positive (ER+) breast cancer. A major mode of metabolism of EXE and its active metabolite, 17ß-dihydroexemestane, is via glutathionylation by glutathione-S-transferase (GST) enzymes. The goal of the present study was to investigate the effects of genetic variation in EXE-metabolizing GST enzymes on overall EXE metabolism. Ex vivo assays examining human liver cytosols from 75 subjects revealed the GSTA1 *B*B genotype was associated with significant decreases in S-(androsta-1,4-diene-3,17-dion-6α-ylmethyl)-L-glutathione (P = 0.034) and S-(androsta-1,4-diene-17ß-ol-3-on-6α-ylmethyl)-L-gutathione (P = 0.014) formation. In the plasma of 68 ER+ breast cancer patients treated with EXE, the GSTA1 *B*B genotype was associated with significant decreases in both EXE-cysteine (cys) (29%, P = 0.0056) and 17ß-DHE-cys (34%, P = 0.032) as compared with patients with the GSTA1*A*A genotype, with significant decreases in EXE-cys (Ptrend = 0.0067) and 17ß-DHE-cys (Ptrend = 0.028) observed in patients with increasing numbers of the GSTA1*B allele. A near-significant (Ptrend = 0.060) trend was also observed for urinary EXE-cys levels from the same patients. In contrast, plasma and urinary 17ß-DHE-Gluc levels were significantly increased (36%, P = 0.00097 and 52%, P = 0.0089; respectively) in patients with the GSTA1 *B*B genotype. No significant correlations were observed between the GSTM1 null genotype and EXE metabolite levels. These data suggest that the GSTA1*B allele is associated with interindividual differences in EXE metabolism and may play a role in interindividual variability in overall response to EXE. SIGNIFICANCE STATEMENT: The present study is the first comprehensive pharmacogenomic investigation examining the role of genetic variability in GST enzymes on exemestane metabolism. The GSTA1 *B*B genotype was found to contribute to interindividual differences in the metabolism of EXE both ex vivo and in clinical samples from patients taking EXE for the treatment of ER+ breast cancer. Since GSTA1 is a major hepatic phase II metabolizing enzyme in EXE metabolism, the GSTA1*B allele may be an important biomarker for treatment outcomes and toxicities.

Variation in the UGT2B17 genotype, exemestane metabolism and menopause-related toxicities in the CCTG MAP.3 trial.

PURPOSE: To examine associations between the UGT2B17 gene deletion and exemestane metabolites, and commonly reported side effects (fatigue, hot flashes, and joint pain) among postmenopausal women participating in the MAP.3 chemoprevention trial. METHODS: The analytical samples for the UGT2B17 analysis comprised 1752 women on exemestane and 1721 women on placebo; the exemestane metabolite analysis included 1360 women on exemestane with one-year serum samples. Both the UGT2B17 gene deletion and metabolites were measured in blood. The metabolites were conceptualized as a ratio (17-DHE-Gluc:17-DHE). Symptoms were assessed using the CTCAE v4.0 at approximately 1-year intervals. Log-binomial regression was used to examine the associations between UGT2B17 deletion, exemestane metabolites and each side effect at 1 and up to 5-year follow-up, adjusting for potential confounders. RESULTS: Among individuals on exemestane with the UGT2B17 gene deletion (i.e., lower detoxification), a higher risk of severe fatigue (RR = 2.59 95% CI: 1.14-5.89) was observed at up to 5-year follow-up. Among individuals on placebo, those with the UGT2B17 gene deletion had a higher risk of any fatigue (RR = 1.39, 95% CI: 1.02-1.89) at year 1. A lower metabolite ratio (poor detoxification) was associated with a higher risk of any fatigue, hot flashes and joint pain at year 1 (fatigue: RR = 1.89, 95% CI: 1.16-3.09; hot flashes: RR = 1.77, 95% CI: 1.40-2.24; joint pain: RR = 2.05, 95% CI: 1.35-3.12); similar associations were observed at 5-year follow-up. CONCLUSION: Variation in the metabolism of exemestane through the UGT2B17-mediated pathway is associated with subsequent risk of commonly reported symptoms in MAP.3.

Carbonyl reduction of NNK by recombinant human lung enzymes: identification of HSD17ß12 as the reductase important in (R)-NNAL formation in human lung.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant and carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke. The major metabolic pathway for NNK is carbonyl reduction to form the (R) and (S) enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung carcinogen. The goal of this study was to characterize NNAL enantiomer formation in human lung and identify the enzymes responsible for this activity. While (S)-NNAL was the major enantiomer of NNAL formed in incubations with NNK in lung cytosolic fractions, (R)-NNAL comprised ~60 and ~95% of the total NNAL formed in lung whole cell lysates and microsomes, respectively. In studies examining the role of individual recombinant cytosolic reductase enzymes in lung NNAL enantiomer formation, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and CBR1 all exhibited (S)-NNAL-formation activity. To identify the microsomal enzymes responsible for (R)-NNAL formation, 28 microsomal reductase enzymes were screened for expression by real-time PCR in normal human lung. HSD17ß6, HSD17ß12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5 were all expressed at levels ≥HSD11ß1, the only previously reported microsomal reductase enzyme with NNK-reducing activity, with HSD17ß12 the most highly expressed. Of these lung-expressing enzymes, only HSD17ß12 exhibited activity against NNK, forming primarily (>95%) (R)-NNAL, a pattern consistent with that observed in lung microsomes. siRNA knock-down of HSD17ß12 resulted in significant decreases in (R)-NNAL-formation activity in HEK293 cells. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD17ß12 is the major active microsomal reductase that contributes to (R)-NNAL formation in human lung.

Identification and Quantification of Novel Major Metabolites of the Steroidal Aromatase Inhibitor, Exemestane.

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of estrogen receptor-positive breast cancer. Although the known major metabolic pathway for EXE is reduction to form the active 17ß-dihydro-EXE (17ß-DHE) and subsequent glucuronidation to 17ß-hydroxy-EXE-17-O-ß-D-glucuronide (17ß-DHE-Gluc), previous studies have suggested that other major metabolites exist for exemestane. In the present study, a liquid chromatography-mass spectrometry (LC-MS) approach was used to acquire accurate mass data in MSE mode, in which precursor ion and fragment ion data were obtained simultaneously to screen novel phase II EXE metabolites in urine specimens from women taking EXE. Two major metabolites predicted to be cysteine conjugates of EXE and 17ß-DHE by elemental composition were identified. The structures of the two metabolites were confirmed to be 6-methylcysteinylandrosta-1,4-diene-3,17-dione (6-EXE-cys) and 6-methylcysteinylandrosta-1,4-diene-17ß-hydroxy-3-one (6-17ß-DHE-cys) after comparison with their chemically synthesized counterparts. Both underwent biosynthesis in vitro in three stepwise enzymatic reactions, with the first involving glutathione conjugation. The cysteine conjugates of EXE and 17ß-DHE were subsequently quantified by liquid chromatography-mass spectrometry in the urine and matched plasma samples of 132 subjects taking EXE. The combined 6-EXE-cys plus 6-17ß-DHE-cys made up 77% of total EXE metabolites in urine (vs. 1.7%, 0.14%, and 21% for EXE, 17ß-DHE, and 17ß-DHE-Gluc, respectively) and 35% in plasma (vs. 17%, 12%, and 36% for EXE, 17ß-DHE, and 17ß-DHE-Gluc, respectively). Therefore, cysteine conjugates of EXE and 17ß-DHE appear to be major metabolites of EXE in both urine and plasma.

Non-additivity of the functional properties of individual P450 species and its manifestation in the effects of alcohol consumption on the metabolism of ketamine and amitriptyline.

To explore functional interconnections between multiple P450 enzymes and their manifestation in alcohol-induced changes in drug metabolism, we implemented a high-throughput study of correlations between the composition of the P450 pool and the substrate saturation profiles (SSP) of amitriptyline and ketamine demethylation in a series of 23 individual human liver microsomes preparations from donors with a known history of alcohol consumption. The SSPs were approximated with linear combinations of three Michaelis-Menten equations with globally optimized KM (substrate affinity) values. This analysis revealed a strong correlation between the rate of ketamine metabolism and alcohol exposure. For both substrates, alcohol consumption caused a significant increase in the role of the low-affinity enzymes. The amplitudes of the kinetic components and the total rate were further analyzed for correlations with the abundance of 11 major P450 enzymes assessed by global proteomics. The maximal rate of metabolism of both substrates correlated with the abundance of CYP3A4, their predicted principal metabolizer. However, except for CYP2D6 and CYP2E1, responsible for the low-affinity metabolism of ketamine and amitriptyline, respectively, none of the other potent metabolizers of the drugs revealed a positive correlation. Instead, in the case of ketamine, we observed negative correlations with the abundances of CYP1A2, CYP2C9, and CYP3A5. For amitriptyline, the data suggest inhibitory effects of CYP1A2 and CYP2A6. Our results demonstrate the importance of functional interactions between multiple P450 species and their decisive role in the effects of alcohol exposure on drug metabolism.

A 3D QSAR study of betulinic acid derivatives as anti-tumor agents using topomer CoMFA: model building studies and experimental verification.

Betulinic acid (BA) is a natural product that exerts its cytotoxicity against various malignant carcinomas without side effects by triggering the mitochondrial pathway to apoptosis. Betulin (BE), the 28-hydroxyl analog of BA, is present in large amounts (up to 30% dry weight) in the outer bark of birch trees, and shares the same pentacyclic triterpenoid core as BA, yet exhibits no significant cytotoxicity. Topomer CoMFA studies were performed on 37 BA and BE derivatives and their in vitro anti-cancer activity results (reported as IC50 values) against HT29 human colon cancer cells in the present study. All derivatives share a common pentacyclic triterpenoid core and the molecules were split into three pieces by cutting at the C-3 and C-28 sites with a consideration toward structural diversity. The analysis gave a leave-one-out cross-validation q² value of 0.722 and a non-cross-validation r² value of 0.974, which suggested that the model has good predictive ability (q² > 0.2). The contour maps illustrated that bulky and electron-donating groups would be favorable for activity at the C-28 site, and a moderately bulky and electron-withdrawing group near the C-3 site would improve this activity. BE derivatives were designed and synthesized according to the modeling result, whereby bulky electronegative groups (maleyl, phthalyl, and hexahydrophthalyl groups) were directly introduced at the C-28 position of BE. The in vitro cytotoxicity values of the given analogs against HT29 cells were consistent with the predicted values, proving that the present topomer CoMFA model is successful and that it could potentially guide the synthesis of new betulinic acid derivatives with high anti-cancer activity. The IC50 values of these three new compounds were also assayed in five other tumor cell lines. 28-O-hexahydrophthalyl BE exhibited the greatest anti-cancer activities and its IC50 values were lower than those of BA in all cell lines, excluding DU145 cells.

Patient-derived microphysiological model identifies the therapeutic potential of metformin for thoracic aortic aneurysm.

BACKGROUND: Thoracic aortic aneurysm (TAA) is the permanent dilation of the thoracic aortic wall that predisposes patients to lethal events such as aortic dissection or rupture, for which effective medical therapy remains scarce. Human-relevant microphysiological models serve as a promising tool in drug screening and discovery. METHODS: We developed a dynamic, rhythmically stretching, three-dimensional microphysiological model. Using patient-derived human aortic smooth muscle cells (HAoSMCs), we tested the biological features of the model and compared them with native aortic tissues. Drug testing was performed on the individualized TAA models, and the potentially effective drug was further tested using ß-aminopropionitrile-treated mice and retrospective clinical data. FINDINGS: The HAoSMCs on the model recapitulated the expressions of many TAA-related genes in tissue. Phenotypic switching and mitochondrial dysfunction, two disease hallmarks of TAA, were highlighted on the microphysiological model: the TAA-derived HAoSMCs exhibited lower alpha-smooth muscle actin expression, lower mitochondrial membrane potential, lower oxygen consumption rate and higher superoxide accumulation than control cells, while these differences were not evidently reflected in two-dimensional culture flasks. Model-based drug testing demonstrated that metformin partially recovered contractile phenotype and mitochondrial function in TAA patients' cells. Mouse experiment and clinical investigations also demonstrated better preserved aortic microstructure, higher nicotinamide adenine dinucleotide level and lower aortic diameter with metformin treatment. INTERPRETATION: These findings support the application of this human-relevant microphysiological model in studying personalized disease characteristics and facilitating drug discovery for TAA. Metformin may regulate contractile phenotypes and metabolic dysfunctions in diseased HAoSMCs and limit aortic dilation. FUNDING: This work was supported by grants from National Key R&D Program of China (2018YFC1005002), National Natural Science Foundation of China (82070482, 81771971, 81772007, 51927805, and 21734003), the Science and Technology Commission of Shanghai Municipality (20ZR1411700, 18ZR1407000, 17JC1400200, and 20YF1406900), Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), and Shanghai Municipal Education Commission (Innovation Program 2017-01-07-00-07-E00027). Y.S.Z. was not supported by any of these funds; instead, the Brigham Research Institute is acknowledged.

Aorta smooth muscle-on-a-chip reveals impaired mitochondrial dynamics as a therapeutic target for aortic aneurysm in bicuspid aortic valve disease.

Background: Bicuspid aortic valve (BAV) is the most common congenital cardiovascular disease in general population and is frequently associated with the development of thoracic aortic aneurysm (TAA). There is no effective strategy to intervene with TAA progression due to an incomplete understanding of the pathogenesis. Insufficiency of NOTCH1 expression is highly related to BAV-TAA, but the underlying mechanism remains to be clarified. Methods: A comparative proteomics analysis was used to explore the biological differences between non-diseased and BAV-TAA aortic tissues. A microfluidics-based aorta smooth muscle-on-a-chip model was constructed to evaluate the effect of NOTCH1 deficiency on contractile phenotype and mitochondrial dynamics of human aortic smooth muscle cells (HAoSMCs). Results: Protein analyses of human aortic tissues showed the insufficient expression of NOTCH1 and impaired mitochondrial dynamics in BAV-TAA. HAoSMCs with NOTCH1-knockdown exhibited reduced contractile phenotype and were accompanied by attenuated mitochondrial fusion. Furthermore, we identified that mitochondrial fusion activators (leflunomide and teriflunomide) or mitochondrial fission inhibitor (Mdivi-1) partially rescued the disorders of mitochondrial dynamics in HAoSMCs derived from BAV-TAA patients. Conclusions: The aorta smooth muscle-on-a-chip model simulates the human pathophysiological parameters of aorta biomechanics and provides a platform for molecular mechanism studies of aortic disease and related drug screening. This aorta smooth muscle-on-a-chip model and human tissue proteomic analysis revealed that impaired mitochondrial dynamics could be a potential therapeutic target for BAV-TAA. Funding: National Key R and D Program of China, National Natural Science Foundation of China, Shanghai Municipal Science and Technology Major Project, Shanghai Science and Technology Commission, and Shanghai Municipal Education Commission.

To function properly, the heart must remain a one-way system, pumping out oxygenated blood into the aorta ­ the largest artery in the body ­ so it can be distributed across the organism. The aortic valve, which sits at the entrance of the aorta, is a key component of this system. Its three flaps (or 'cusps') are pushed open when the blood exits the heart, and they shut tightly so it does not flow back in the incorrect direction. Nearly 1.4% of people around the world are born with 'bicuspid' aortic valves that only have two flaps. These valves may harden or become leaky, forcing the heart to work harder. This defect is also associated with bulges on the aorta which progressively weaken the artery, sometimes causing it to rupture. Open-heart surgery is currently the only way to treat these bulges (or 'aneurysms'), as no drug exists that could slow down disease progression. This is partly because the biological processes involved in the aneurysms worsening and bursting open is unclear. Recent studies have highlighted that many individuals with bicuspid aortic valves also have lower levels of a protein known as NOTCH1, which plays a key signalling role for cells. Problems in the mitochondria ­ the structures that power up a cell ­ are also observed. However, it is not known how these findings are connected or linked with the aneurysms developing. To answer this question, Abudupataer et al. analyzed the proteins present in diseased and healthy aortic muscle cells, confirming a lower production of NOTCH1 and impaired mitochondria in diseased tissues. They also created an 'aorta-on-a-chip' model where aortic muscle cells were grown in the laboratory under conditions resembling those found in the body ­ including the rhythmic strain that the aorta is under because of the heart beating. Abudupataer et al. then reduced NOTCH1 levels in healthy samples, which made the muscle tissue less able to contract and reduced the activity of the mitochondria. Applying drugs that tweak mitochondrial activity helped tissues from patients with bicuspid aortic valves to work better. These compounds could potentially benefit individuals with deficient aortic valves, but experiments in animals and clinical trials would be needed first to confirm the results and assess safety. The aorta-on-a-chip model developed by Abudupataer et al. also provides a platform to screen for drugs and examine the molecular mechanisms at play in aortic diseases.

Phase II metabolism of betulin by rat and human UDP-glucuronosyltransferases and sulfotransferases.

The natural product betulin is under investigation for several therapeutic indications, however little is known about its metabolism. In the present study, the glucuronidation and sulfation of betulin in human and rat liver microsomes and cytosol were tested. We further identified the main UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) involved in these two metabolism pathways. Results showed that one betulin glucuronide metabolite was observed after incubation with human and rat liver microsomes. The glucuronidation of betulin in human liver microsomes had a Km value of 21.1 ±â€¯5.93 µM and a Vmax value of 6.39 ±â€¯0.66 pmol/min/mg protein. The glucuronidation activity in rats was too low to get enzyme kinetic parameters. Among the 11 recombinant UGT enzymes investigated, UGT1A3 and UGT1A4 were identified as the major enzymes catalyzing the glucuronidation of betulin [Km values of 10.12 ±â€¯8.09 and 8.04 ±â€¯3.96 µM, Vmax values of 6.71 ±â€¯1.51 and 5.98 ±â€¯0.76 nmol/min/(mg protein)]. Two betulin sulfate metabolites were found in human and rat liver cytosols. Human and rat liver had similar affinity for the formation of these two metabolites, the apparent Vmax for betulin sulfate I was higher than that for betulin sulfate II in both species. Among the SULT isoforms studied, SULT2A1 was the major isoenzyme involved in the betulin sulfation metabolism in human liver cytosol. The results suggest that glucuronidation and sulfation are important metabolism pathways for betulin, and UGT1A3, UGT1A4 and SULT2A1 play the major roles in betulin glucuronidation and sulfation.

Association between Glucuronidation Genotypes and Urinary NNAL Metabolic Phenotypes in Smokers.

BACKGROUND: The most abundant and potent carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In vivo, NNK is rapidly metabolized to both the (R)- and (S)-enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which possesses similar carcinogenic properties as NNK. The major detoxification pathway for both NNAL enantiomers is glucuronidation by UDP-glucuronosyltransferase (UGT) enzymes including UGT2B10 and UGT2B17. The goal of the present study was to directly examine the role of UGT genotypes on urinary levels of NNAL glucuronides in smokers. METHODS: NNAL-N-Gluc, (R)-NNAL-O-Gluc, (S)-NNAL-O-Gluc, and free NNAL were simultaneously and directly quantified in the urine of smokers by LC/MS analysis. Genotypes were determined by TaqMan assay using genomic DNA. RESULTS: The functional knockout polymorphism in the UGT2B10 gene at codon 67 (Asp>Tyr) was significantly (P < 0.0001) associated with a 93% decrease in creatinine-adjusted NNAL-N-Gluc. The polymorphic whole-gene deletion of the UGT2B17 gene was associated with significant (P = 0.0048) decreases in the levels of creatinine-adjusted (R)-NNAL-O-Gluc, with a 32% decrease in the levels of urinary (R)-NNAL-O-Gluc/(S)-NNAL-O-Gluc among subjects with the UGT2B17 (*2/*2) genotype as compared to subjects with the UGT2B17 (*1/*1) genotype. CONCLUSIONS: These results suggest that functional polymorphisms in UGT2B10 and UGT2B17 are associated with a reduced detoxification capacity against NNAL and may therefore affect individual cancer risk upon exposure to tobacco. IMPACT: This is the first report to clearly demonstrate strong genotype-phenotype associations between both the UGT2B10 codon 67 Asp

Pharmacokinetic analysis and comparison of caffeine administered rapidly or slowly in coffee chilled or hot versus chilled energy drink in healthy young adults.

CONTEXT: There is a paucity of data describing the impact of type of beverage (coffee versus energy drink), different rates of consumption and different temperature of beverages on the pharmacokinetic disposition of caffeine. Additionally, there is concern that inordinately high levels of caffeine may result from the rapid consumption of cold energy drinks. OBJECTIVE: The objective of this study was to compare the pharmacokinetics of caffeine under various drink temperature, rate of consumption and vehicle (coffee versus energy drink) conditions. MATERIALS: Five caffeine (dose = 160 mg) conditions were evaluated in an open-label, group-randomized, crossover fashion. After the administration of each caffeine dose, 10 serial plasma samples were harvested. Caffeine concentration was measured via liquid chromatography-mass spectrometry (LC-MS), and those concentrations were assessed by non-compartmental pharmacokinetic analysis. The calculated mean pharmacokinetic parameters were analyzed statistically by one-way repeated measures analysis of variance (RM ANOVA). If differences were found, each group was compared to the other by all pair-wise multiple comparison. RESULTS: Twenty-four healthy subjects ranging in age from 18 to 30 completed the study. The mean caffeine concentration time profiles were similar with overlapping SDs at all measured time points. The ANOVA revealed significant differences in mean Cmax and Vd ss/F, but no pair-wise comparisons reached statistical significance. No other differences in pharmacokinetic parameters were found. DISCUSSION: The results of this study are consistent with previous caffeine pharmacokinetic studies and suggest that while rate of consumption, temperature of beverage and vehicle (coffee versus energy drink) may be associated with slightly different pharmacokinetic parameters, the overall impact of these variables is small. CONCLUSION: This study suggests that caffeine absorption and exposure from coffee and energy drink is similar irrespective of beverage temperature or rate of consumption.

Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3ß and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3ß and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.