Investigation of discordant sibling pairs from hereditary breast cancer families and analysis of a rare PMS1 variant.

BACKGROUND: It is likely that additional genes for hereditary breast cancer can be identified using a discordant sib pair design. Using this design we identified individuals harboring a rare PMS1 c.605G>A variant previously predicted to result in loss of function. OBJECTIVES: A family-based design and predictive algorithms were used to prioritize candidate variants possibly associated with an increased risk of hereditary breast cancer. Functional analyses were performed for one of the candidate variants, PMS1 c.605G>A. METHODS: 1) 14 discordant sister-pairs from hereditary breast cancer families were identified. 2) Whole exome sequencing was performed and candidate risk variants identified. 3) A rare PMS variant was identified in 2 unrelated affected sisters but no unaffected siblings. 4) Functional analysis of this variant was carried out using targeted mRNA sequencing. RESULTS: Genotype-phenotype correlation did not demonstrate tracking of the variant with cancer in the family. Functional analysis revealed no difference in exon 6 incorporation, which was validated by analyzing PMS1 allele specific expression. CONCLUSIONS: The PMS1 c.605G>A variant did not segregate with disease, and there was no variant-dependent impact on PMS1 exon 6 splicing, supporting this variant is likely benign. Functional analyses are imperative to understanding the clinical significance of predictive algorithms.

Drug- and patient-related factors are the strongest predictors of endocrine therapy adherence in breast cancer patients.

PURPOSE: Medication non-adherence to treatment regimens can severely impact the mortality of patients afflicted with breast cancer.The purpose of this study was to identify factors that contribute to non-adherence to endocrine therapy in breast cancer treatment plans. METHODS: Thirty-two women with a breast cancer diagnosis were surveyed by pharmacists and pharmacy students to identify the patient- related factors (e.g. patient personal beliefs, education level), drug-related factors (e.g. patient drug allergies), socio-economic factors (e.g. patient ability to pay for the medication) and healthcare system factors (e.g. poor patient-healthcare provider relationship) that may impact non-adherence to endocrine therapy in breast cancer treatment plans. Medication adherence rates were measured using the Medication Adherence Rating Scale (MARS-8) system. Associations between adherence rate scores and clinical variables (e.g. age, tobacco use, alcohol consumption, cost of treatment, education level, personal beliefs, drug allergies, patient/provider relationship, adverse events) were carried out using Spearman Correlation, T-Test, Mann-Whitney U Test, and X2 tests. A p value of ≤ 0.05 was considered statistically significant. RESULTS: Our study found that 59% of survey respondents were non-adherent to their endocrine therapy in breast cancer treatment plans. Drug allergies (p = 0.000069), patient ability to pay (p = 0.005), poor personal beliefs about the prescribed therapy (p = 0.009), low education level (p = 0.025), adverse drug events (p = 0.026), and poor patient-provider relationship (p = 0.05) were found to play a role in patient non-adherence to treatment. CONCLUSIONS: Our study found that drug- (e.g. allergies), socio-economic (e.g. patient ability to pay), and patient-related factors (e.g. personal beliefs) are the strongest predictors of adherence among breast cancer patients undergoing endocrine therapy. These findings support the need for a better relationship between breast cancer patients and their healthcare providers, including drug experts such as pharmacists.

Relative Yield Loss From Stem Borers (Lepidoptera: Crambidae) and Rice Water Weevil (Coleoptera: Curculionidae) in Rice.

The rice water weevil, Lissorhoptrus oryzophilus Kuschel, is the most important economic pest of rice in the United States with the ability to substantially reduce rice yields. Stem borers, including the Mexican rice borer, Eoreuma loftini (Dyar), are emerging as more consistent pests in Louisiana and Texas, but the relationship between blanked panicles (whiteheads) caused by stem borer feeding and yield loss is not well understood. A series of field trials was conducted in Louisiana from 2019 to 2020 to determine the relative yield loss by manipulating infestations of both pests with selective insecticides. Results indicate losses from L. oryzophilus infestations are greater than those for E. loftini in early planted rice. In contrast, relative yield losses were approximately equal in later planted rice in 2019, in which E. loftini infestations were high. Additive impacts of the root-feeding L. oryzophilus and the stem-feeding E. loftini were not observed in our study. Linear regression revealed that each increase of one whitehead per square meter is associated with a 1.7% loss in yield. Results from ratoon rice suggest that protection from L. oryzophilus in the main crop also improves yield in the second crop. Collectively, results demonstrate the continued need for effective L. oryzophilus management programs to prevent economic losses. Conversely, findings suggest that benefits of E. loftini control may be minimal in many instances. Control of E. loftini is most likely to be economical under conditions where high populations are anticipated such as in late-planted fields or regions that previously had high infestations.

Varicocele complicating an anterior lumbar interbody fusion: a case report.

Anterior lumbar interbody fusion (ALIF) is performed for the surgical management of lumbar degenerative disc disease with excellent results, particularly for discogenic low back pain. Commonly reported complications associated with this approach include vessel injury, retrograde ejaculation, and ureteral and viscus organ injury. The development of a varicocele after ALIF has not been previously described in the literature. We report a case of varicocele in a 35-year-old patient who underwent ALIF via a left retroperitoneal approach. No intraoperative complications were identified. The postoperative course was uneventful. He was discharged from the hospital on the 5th postoperative day. Three months after surgery, he complained of discomfort and scrotal pain. Examination revealed a grade 3 varicocele according to the Dubin and Amelar classification. Scrotal Doppler US demonstrated dilatation of the veins of the pampiniform plexus. A lumbar CT scan revealed a bulky left spermatic vein closed to the ureter. The patient was treated with platelet anti-aggregation. He was seen at control intervals of 1, 3 and 5 months. Progress was seen as we had a regression of clinical signs. Varicocele appears as an uncommon complication of ALIF. After reviewing the literature, we describe the occurrence of a varicocele following ALIF, its pathophysiology, and its treatment options.

Vascular alterations among young adults with SARS-CoV-2.

While SARS-CoV-2 primarily affects the lungs, the virus may be inflicting detriments to the cardiovascular system, both directly through angiotensin-converting enzyme 2 receptor and initiating systemic inflammation. Persistent systemic inflammation may be provoking vascular dysfunction, an early indication of cardiovascular disease risk. To establish the potential effects of SARS-CoV-2 on the systemic vasculature in the arms and legs, we performed a cross-sectional analysis of young healthy adults (control: 5 M/15 F, 23.0 ± 1.3 y, 167 ± 9 cm, 63.0 ± 7.4 kg) and young adults who, 3-4 wk prior to testing, had tested positive for SARS-CoV-2 (SARS-CoV-2: 4 M/7 F, 20.2 ± 1.1 y, 172 ± 12 cm, 69.5 ± 12.4 kg) (means ± SD). Using Doppler ultrasound, brachial artery flow-mediated dilation (FMD) in the arm and single passive limb movement (sPLM) in the leg were assessed as markers of vascular function. Carotid-femoral pulse wave velocity (PWVcf) was asvsessed as a marker of arterial stiffness. FMD was lower in the SARS-CoV-2 group (2.71 ± 1.21%) compared with the control group (8.81 ± 2.96%) (P < 0.01) and when made relative to the shear stimulus (SARS-CoV-2: 0.04 ± 0.02 AU, control: 0.13 ± 0.06 AU, P < 0.01). The femoral artery blood flow response, as evidenced by the area under the curve, from the sPLM was lower in the SARS-CoV-2 group (-3 ± 91 mL) compared with the control group (118 ± 114 mL) (P < 0.01). PWVcf was higher in the SARS-CoV-2 group (5.83 ± 0.62 m/s) compared with the control group (5.17 ± 0.66 m/s) (P < 0.01). Significantly lower systemic vascular function and higher arterial stiffness are evident weeks after testing positive for SARS-CoV-2 among young adults compared with controls.NEW & NOTEWORTHY This study was the first to investigate the vascular implications of contracting SARS-CoV-2 among young, otherwise healthy adults. Using a cross-sectional design, this study assessed vascular function 3-4 wk after young adults tested positive for SARS-CoV-2. The main findings from this study were a strikingly lower vascular function and a higher arterial stiffness compared with healthy controls. Together, these results suggest rampant vascular effects seen weeks after contracting SARS-CoV-2 in young adults.

Effect of Aromatase Inhibition (Exemestane) on Urine Concentration of Osteoprotegerin in Healthy Postmenopausal Women.

This pilot study examined how exemestane (an aromatase inhibitor [AI]) affected osteoprotegerin (OPG) urine concentrations in postmenopausal women. Exemestane (25 mg, single dose) was given to 14 disease-free women past menopause in this nonrandomized, open-label study. Before dosing, urine specimens were gathered. Three days later, these women returned to provide urine specimens for pharmacokinetic (measurement of major parent drug and enzymatic product) and pharmacodynamic (profiling of OPG) analysis. Urine concentrations of the major parent drug (exemestane) and enzymatic product (17-hydroexemestane) were quantified using liquid chromatography-tandem mass spectrometry. An analyst software package was used for data processing. Following the manufacturer's guidelines, OPG urine concentrations were quantified using a human osteoprotegerin TNFRSF11b ELISA kit from Sigma-Aldrich. A microplate reader helped to carry out OPG data analysis and processing. Our results highlight that OPG urine concentrations were decreased 3 days after drug dosage (mean predosage OPG concentration, 61.4 ± 24.1 pg/mL; vs mean postdosage OPG concentration, 45.7 ± 22.1 pg/mL; P = .02, Wilcoxon rank test). Among the 14 volunteers enrolled in the study, 4 subjects had an increase of less than 1-fold, and the rest showed an average of a 2-fold decrease in OPG concentration (range, 1.1-5.4; standard deviation, 1.3) after exemestane administration. There was no association between fold decrease in OPG urine concentration and the pharmacokinetics of the major parent drug (exemestane) and its enzymatic product (17-hydroexemestane). We concluded that one of the off-target pharmacological effects of AIs (eg ,exemestane) may result in the reduction of osteoprotegerin.

Patients with laboratory evidence of West Nile virus disease without reported fever.

In 2013, the national surveillance case definition for West Nile virus (WNV) disease was revised to remove fever as a criterion for neuroinvasive disease and require at most subjective fever for non-neuroinvasive disease. The aims of this project were to determine how often afebrile WNV disease occurs and assess differences among patients with and without fever. We included cases with laboratory evidence of WNV disease reported from four states in 2014. We compared demographics, clinical symptoms and laboratory evidence for patients with and without fever and stratified the analysis by neuroinvasive and non-neuroinvasive presentations. Among 956 included patients, 39 (4%) had no fever; this proportion was similar among patients with and without neuroinvasive disease symptoms. For neuroinvasive and non-neuroinvasive patients, there were no differences in age, sex, or laboratory evidence between febrile and afebrile patients, but hospitalisations were more common among patients with fever (P < 0.01). The only significant difference in symptoms was for ataxia, which was more common in neuroinvasive patients without fever (P = 0.04). Only 5% of non-neuroinvasive patients did not meet the WNV case definition due to lack of fever. The evidence presented here supports the changes made to the national case definition in 2013.

Exemestane may be less detrimental than letrozole to bone health in women homozygous for the UGT2B17*2 gene deletion.

PURPOSE: UGT2B17 gene deletion (UGT2B17*2) has been reported to affect bone health as well as the pharmacokinetics of aromatase inhibitor (AI) drugs such as exemestane. The goal of this study was to assess associations between UGT2B17 gene deletion and bone health prior to and after 24 months of AI treatment in postmenopausal women with hormone receptor positive (HR+) breast cancer. METHODS: Bone health in women with HR+ breast cancer enrolled on the prospective randomized Exemestane and Letrozole Pharmacogenetics (ELPh) trial was determined by measuring bone turnover markers (BTM) and bone mineral density (BMD) pre-treatment and after 3 BTM and 24 BMD months of treatment with either the steroidal AI exemestane or the nonsteroidal AI letrozole. DNA samples were genotyped for UGT2B17*2. RESULTS: Of the 455 subjects included in the analyses, 244 (53.6%) carried at least one copy of UGT2B17*2. UGT2B17*2 was associated with lower pre-treatment BMD at the hip (P = 0.01) and spine (P = 0.0076). Letrozole treatment was associated with a greater decrease in BMD of the hip (P = 0.03) and spine (P = 0.03) than exemestane. UGT2B17 genotype was not associated with changes in BMD from 24 months of AI treatment, though in UGT2B17*2 homozygous patients, there was a trend toward greater decreases in BMD of the spine from treatment with letrozole compared with exemestane (P = 0.05). CONCLUSION: UGT2B17*2 may be associated with lower baseline BMD in women with HR+ breast cancer. Exemestane is less detrimental to bone health than letrozole in postmenopausal women treated with AI, and this effect may be confined to patients carrying UGT2B17*2, though this finding requires independent validation.

Influence of Transporter Polymorphisms on Drug Disposition and Response: A Perspective From the International Transporter Consortium.

Advances in genomic technologies have led to a wealth of information identifying genetic polymorphisms in membrane transporters, specifically how these polymorphisms affect drug disposition and response. This review describes the current perspective of the International Transporter Consortium (ITC) on clinically important polymorphisms in membrane transporters. ITC suggests that, in addition to previously recommended polymorphisms in ABCG2 (BCRP) and SLCO1B1 (OATP1B1), polymorphisms in the emerging transporter, SLC22A1 (OCT1), be considered during drug development. Collectively, polymorphisms in these transporters are important determinants of interindividual differences in the levels, toxicities, and response to many drugs.

COX2 induction: a mechanism of endocrine breast cancer resistance?

PURPOSE: Urine prostaglandin E2 (PGE2) levels have shown to be a risk factor of breast cancer, and the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is known to be beneficial in preventing breast cancer risk and/or recurrence with or without aromatase inhibitors. We hypothesized that the use of an aromatase inhibitor triggers the activation of the inflammatory pathway via release of PGE2. METHODS: A single oral 25 mg dose of an aromatase inhibitor (exemestane) was given to 14 healthy postmenopausal female volunteers. Blood and urine samples were collected between 0 and 72 h post-dosing for pharmacokinetic and pharmacodynamic analysis. RESULTS: Our findings showed that urine PGE2 levels were markedly increased 72 h after exemestane administration (average pre-dosing PGE2 levels, 4061.1 pg/mL vs. post-dosing average PGE2 levels, 10732.5 pg/mL, P = 0.001, Wilcoxon Rank Test). Out of 14 subjects enrolled in the study, one subject showed no change in PGE2; another showed a 23-fold decreased in PGE2; and the remaining 12 showed an average of 8.4-fold increase in PGE2 levels (range 1.3-30.5, standard deviation 9.2) after exemestane administration. We found no statistically significant correlations between fold increase in urine PGE2 levels and the pharmacokinetics of either exemestane or 17-hydroexemestane (major in vivo metabolite of exemestane). CONCLUSION: Our results indicate that one of the pharmacological effects to aromatase inhibitors (e.g., exemestane) involves the activation of the inflammatory pathway via release of PGE2. Further in vitro mechanistic and in vivo translational studies designed to elucidate the role of this newly discovered effect are now warranted.

Association of stroke risk biomarkers with stroke symptoms: the Reasons for Geographic and Racial Differences in Stroke cohort.

Essentials Stroke symptom history predicts future stroke and may indicate prior unrecognized stroke. We studied associations of stroke symptoms with stroke risk biomarkers. Several stroke risk biomarkers were independently associated with stroke symptom history. Findings support a hypothesis that stroke symptoms may represent unrecognized stroke. SUMMARY: Background History of stroke symptoms in the absence of prior diagnosed stroke or transient ischemic attack (TIA) is associated with future stroke risk, as are biomarkers of inflammation, cardiac function and hemostasis. Objective To better elucidate the pathobiology of stroke symptoms, we studied associations of these biomarkers with history of stroke symptoms. Methods The Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort enrolled 30 239 black and white Americans age 45 years and older in 2003-7. In cross-sectional analyses in a random sample of 960 participants without prior stroke or TIA, levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), fibrinogen, factor VIII (FVIII), factor XI (FXI), C-reactive protein (CRP) and D-dimer were studied in relation to self-reported history of six sudden onset stroke symptoms. Results There were 190 participants with at least one stroke symptom and 770 without. Adjusting for age, race, sex and stroke risk factors, NT-proBNP, FXI, CRP and D-dimer in the top vs. bottom quartile were associated with prevalent stroke symptoms with odds ratios 2.69 (95% confidence interval [CI], 1.45-4.98), 1.65 (95% CI, 1.00-2.73), 2.21 (95% CI, 1.32-3.71) and 2.14 (95% CI, 1.22-3.75), respectively. Conclusions Strong associations of stroke risk biomarkers with stroke symptoms in persons without a clinical history of cerebrovascular disease support a hypothesis that some of these stroke symptoms represent unrecognized cerebrovascular disease. Future work is needed to determine whether these biomarkers identify persons with stroke symptoms who have a particularly high stroke risk.

Conditional Cash Transfers Improve Retention in PMTCT Services by Mitigating the Negative Effect of Not Having Money to Come to the Clinic.

OBJECTIVE: To elucidate the mechanisms by which a cash incentive intervention increases retention in prevention of mother-to-child transmission services. METHODS: We used data from a randomized controlled trial in Kinshasa, Democratic Republic of Congo. Perceptual factors associated with loss to follow-up (LTFU) through 6 weeks postpartum were first identified. Then, binomial models were used to assess interactions between LTFU and identified factors, and the cash incentive intervention. RESULTS: Participants were less likely to be LTFU if they perceived HIV as a "very serious" health problem for their baby vs. not [risk difference (RD), -0.13; 95% confidence interval (CI): -0.30 to 0.04], if they believed it would be "very likely" to pass HIV to their baby if they did not take any HIV drug vs. not (RD, -0.15; 95% CI: -0.32 to 0.02), and if they anticipated that not having money would make it difficult for them to come to the clinic vs. not (RD, 0.12; 95% CI: -0.07 to 0.30). The effect of each of the 3 factors on LTFU was antagonistic to that of receiving the cash incentive intervention. The excess risk due to interaction between the cash incentive intervention and the anticipated difficulty of "not having money" to come to the clinic was exactly equal to the effect of removing this perceived barrier (excess risk due to interaction, -0.12; 95% CI: -0.35 to 0.10). CONCLUSIONS: Our analyses show that cash transfers improve retention in prevention of mother-to-child transmission services mainly by mitigating the negative effect of not having money to come to the clinic.

Conditional Cash Transfers to Increase Retention in PMTCT Care, Antiretroviral Adherence, and Postpartum Virological Suppression: A Randomized Controlled Trial.

BACKGROUND: Novel strategies are needed to increase retention in prevention of mother-to-child HIV transmission (PMTCT) services. We have recently shown that small, incremental cash transfers conditional on attending clinic resulted in increased retention along the PMTCT cascade. However, whether women who receive incentives to attend clinic visits are as adherent to antiretrovirals (ARV) as those who do not was unknown. OBJECTIVE: To determine whether HIV-infected women who received incentives to remain in care were as adherent to antiretroviral treatment and achieved the same level of viral suppression at 6 weeks postpartum as those who did not receive incentives but also remained in care. METHODS: Newly diagnosed HIV-infected women at ≤32 weeks gestational age were recruited at antenatal care clinics in Kinshasa, Democratic Republic of Congo. Women were randomized in a 1:1 ratio to an intervention or control group. The intervention group received compensation ($5, plus $1 increment at each subsequent visit) conditional on attending scheduled clinic visits and accepting offered PMTCT services, whereas the control group received usual care. The proportion of participants who remained in care, were fully adherent (took all their pills at each visit) or with undetectable viral load at 6 weeks postpartum were compared across group. RESULTS: Among 433 women randomized (216 in intervention group and 217 in control group), 332 (76.7%) remained in care at 6 weeks postpartum, including 174 (80.6%) in the intervention group and 158 (72.8%) in the control group, (P = 0.04). Data on pill count were available for 297 participants (89.5%), including 156 (89.7%) and 141 (89.2%) in the intervention and control groups, respectively; 69.9% (109/156) and 68.1% (96/141) in the intervention and control groups had perfect adherence [risk difference, 0.02; 95% CI: -0.06 to 0.09]. Viral load results were available for 171 (98.3%) and 155 (98.7%) women in the intervention and control groups, respectively; 66.1% (113/171) in the intervention group and 69.7% (108/155) in the control group had an undetectable viral load (risk difference, -0.04; 95% CI: -0.14 to 0.07). Results were similar after adjusting for marital status, age, education, baseline CD4 count, viral load, gestational age, and initial ARV regimen. CONCLUSIONS: Although the provision of cash incentives to HIV-infected pregnant women led to higher retention in care at 6 weeks postpartum, among those retained in care, adherence to ARVs and virologic suppression did not differ by study group.

Impact of UGT2B17 Gene Deletion on the Pharmacokinetics of 17-Hydroexemestane in Healthy Volunteers.

Exemestane is an aromatase inhibitor drug used for the treatment of hormone-dependent breast cancer. 17-Hydroexemestane, the major and biologically active metabolite of exemestane in humans, is eliminated via glucuronidation by the polymorphic UGT2B17 phase II drug-metabolizing enzyme. Previous microsomal studies have shown that UGT2B17 gene deletion affects the intrinsic hepatic clearances of 17-hydroexemestane in vitro. In this open-label study we set out to assess the effect of UGT2B17 gene deletion on the pharmacokinetics of 17-hydroexemestane in healthy female volunteers with and without UGT2B17. To achieve this goal, 14 healthy postmenopausal women (8 carriers of the homozygous UGT2B17 wild-type allele and 6 carriers of the homozygous UGT2B17 gene-deletion allele) were enrolled and invited to receive a single 25-mg oral dose of exemestane. Pharmacokinetics was assessed over 72 hours postdosing. Our results showed that there were statistically significant differences in plasma 17-hydroexemestane AUC0-∞ (P = .0007) and urine 17-hydroexemestane C24h (P = .001) between UGT2B17 genotype groups. Our data suggest that UGT2B17 gene deletion influences 17-hydroexemestane pharmacokinetics in humans.

In vitro cytochrome P450-mediated metabolism of exemestane.

Exemestane is a potent and irreversible steroidal aromatase inhibitor drug used for the treatment of estrogen receptor-positive breast cancer. Our aim was to identify and assess the contribution of the specific cytochromes P450 (P450s) responsible for exemestane primary in vitro metabolism. With the use of high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques, 17-hydroexemestane (MI) formation and 6-hydroxymethylexemestane (MII) formation were found to be the predominant exemestane metabolic pathways. In a bank of 15 well characterized human liver microsomes with known P450 isoform-specific activities, the MI formation rate correlated significantly with CYP1A2 (Spearman r = 0.60, p = 0.02) and CYP4A11 (Spearman r = 0.67, p = 0.01) isoform-specific activities, whereas the MII production rate significantly correlated with CYP2B6 (Spearman r = 0.57, p = 0.03) and CYP3A (Spearman r = 0.76, p = 0.005) isoform-specific activities. Recombinant CYP1A1 metabolized exemestane to MI with a catalytic efficiency (Cl(int)) of 150 nl/pmol P450 × min that was at least 3.5-fold higher than those of other P450s investigated. Recombinant CYP3A4 catalyzed MII formation from exemestane with a catalytic efficiency of 840 nl/pmol P450 × min that was at least 4-fold higher than those of other P450s investigated. Among a panel of 10 chemical inhibitors tested, only ketoconazole and troleandomycin (CYP3A-specific chemical inhibitors) significantly inhibited the formation of MII by 45 and 95%, respectively. None of them markedly inhibited the formation of MI. In summary, exemestane seems to be metabolized to MI by multiple P450s that include CYP4A11 and CYP1A1/2, whereas its oxidation to MII is primarily mediated by CYP3A.

In vitro and in vivo oxidative metabolism and glucuronidation of anastrozole.

AIMS: Little information is available regarding the metabolic routes of anastrozole and the specific enzymes involved. We characterized anastrozole oxidative and conjugation metabolism in vitro and in vivo. METHODS: A sensitive LC-MS/MS method was developed to measure anastrozole and its metabolites in vitro and in vivo. Anastrozole metabolism was characterized using human liver microsomes (HLMs), expressed cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs). RESULTS: Hydroxyanastrozole and anastrozole glucuronide were identified as the main oxidative and conjugated metabolites of anastrozole in vitro, respectively. Formation of hydroxyanastrozole from anastrozole was markedly inhibited by CYP3A selective chemical inhibitors (by >90%) and significantly correlated with CYP3A activity in a panel of HLMs (r= 0.96, P= 0.0005) and mainly catalyzed by expressed CYP3A4 and CYP3A5. The K(m) values obtained from HLMs were also close to those from CYP3A4 and CYP3A5. Formation of anastrozole glucuronide in a bank of HLMs was correlated strongly with imipramine N-glucuronide, a marker of UGT1A4 (r= 0.72, P < 0.0001), while expressed UGT1A4 catalyzed its formation at the highest rate. Hydroxyanastrozole (mainly as a glucuronide) and anastrozole were quantified in plasma of breast cancer patients taking anastrozole (1 mg day⁻¹); anastrozole glucuronide was less apparent. CONCLUSION: Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4 (and to some extent by CYP3A5 and CYP2C8). Once formed, this metabolite undergoes glucuronidation. Variable activity of CYP3A4 (and probably UGT1A4), possibly due to genetic polymorphisms and drug interactions, may alter anastrozole disposition and its effects in vivo.

Methadone: a substrate and mechanism-based inhibitor of CYP19 (aromatase).

The peripheral conversion of testosterone to estradiol by aromatase is the primary source of endogenous estrogen in postmenopausal women. Studies indicating that placental aromatase is able to metabolize methadone to its primary metabolite, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidin (EDDP), led us to test the hypothesis that methadone is able to act as an inhibitor of aromatase. Using recombinant human CYP19, we examined the ability of methadone to bring about either reversible or mechanism-based inhibition of the conversion of testosterone to estradiol. To test for reversible inhibition, racemic methadone or its metabolite EDDP or 2-ethyl-5-methyl-3, 3-diphenylpyrroline (EMDP) was incubated for 30 min with testosterone at the K(m) (4 microM). To test for mechanism-based inhibition, microsomal preincubations were performed for up to 30 min using racemic methadone (1-1000 microM), R- or S-methadone (0.5-500 microM), or EDDP or EMDP (10 and 100 microM) followed by incubation with testosterone at a V(max) concentration (50 microM). Racemic methadone, EDDP, and EMDP did not act as competitive inhibitors of CYP19. Preincubation of methadone, EDDP, or EMDP with CYP19 resulted in time- and concentration-dependent inhibition, indicating a mechanism-based reaction that destroys CYP19 activity. The K(I) and k(inact) values for racemic methadone were calculated to be 40.6 +/- 2.8 microM and 0.061 +/- 0.001 min(-1), respectively. No stereoselectivity was observed. Methadone is metabolized by CYP19 and may act as a potent inhibitor of CYP19 in vivo. These findings may contribute to variability in methadone clearance, to drug-drug interactions, and to side effects observed in individual patients.

Genetic predictors of glucocorticoid-induced hypertension in children with acute lymphoblastic leukemia.

OBJECTIVE: Glucocorticoids are used universally in the remission induction therapy for acute lymphoblastic leukemia (ALL). One of the adverse effects of glucocorticoids is hypertension. Our aim was to define the frequency of and clinical and genetic risk factors for steroid-induced hypertension. METHODS: We determined the genotypes for 203 candidate polymorphisms in genes previously linked to hypertension or to the pharmacokinetics or pharmacodynamics of antileukemic agents. Hypertension was defined according to the guidelines of the American Academy of Pediatrics; patients were evaluated during the 28-day period of prednisone at 40 mg/m2/day during remission induction of childhood ALL. RESULTS: Of the 602 children with newly diagnosed ALL who were normotensive pretherapy, 270 (45%) developed hypertension during remission induction. None of the putative risk factors (age, sex, race, white blood cell count, risk group, body mass index, or serum creatinine) was associated with hypertension. Among the polymorphisms genotyped, we identified eight genes (CNTNAP2, LEPR, CRHR1, NTAN1, SLC12A3, ALPL, BGLAP, and APOB) containing variants that were associated with hypertension (chi2 P values 0.002-0.048), several of which interact with the hypothalamus-pituitary-adrenal axis. Polymorphisms in CYP3A4 and CYP3A5 were not associated with hypertension. CONCLUSION: Hypertension is common during ALL remission induction and is related to germline genetic variation.

Altered gene expression in cells from patients with lysosomal storage disorders suggests impairment of the ubiquitin pathway.

By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.

Clinical implications of CYP3A polymorphisms.

Due to their enormous substrate spectrum CYP3A4, -3A5 and -3A7 constitute the most important drug-metabolising enzyme subfamily in humans. CYP3As are expressed predominantly, but not exclusively, in the liver and intestine, where they participate in the metabolism of 45 - 60% of currently used drugs and many other compounds such as steroids and carcinogens. CYP3A expression and activity vary interindividually due to a combination of genetic and nongenetic factors such as hormone and health status, and the impact of environmental stimuli. Over the past several years, genetic determinants have been identified for much of the variable expression of CYP3A5 and -3A7, but not for CYP3A4. Using these markers, an effect of CYP3A5 expression status has been demonstrated beyond doubt for therapies with the immunosuppressive drug tacrolimus. Further associations are likely to emerge for drugs metabolised predominantly by CYP3A5 or -3A7, especially for individuals or tissues with concomitant low expression of CYP3A4. However, as exemplified by the controversial association between CYP3A4*1B and prostate cancer, the detection of clinical effects of CYP3A gene variants will be difficult. The most important underlying problems are the continuing absence of activity markers specific for CYP3A4 and the strong contribution of nongenetic factors to CYP3A variability.