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.

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.

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.

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.

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.

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.

Dominant contribution of P450 3A4 to the hepatic carcinogenic activation of aflatoxin B1.

The hepatic carcinogen aflatoxin B1 (AFB1) is metabolized in the liver by at least four different P450s, all of which exhibit large interindividual differences in the expression levels. These differences could affect the individual risk of hepatocellular carcinoma (HCC). We investigated the metabolism of AFB1 in a panel of 13 human liver microsomal preparations using a hepatic abundance model, which takes into account the specific kinetic parameters and the expression levels of these P450s. We found a 12-fold variability in the production rate of the carcinogenic metabolite AFB1-8,9-epoxide (AFBO) and a 22-fold variability in the production of the detoxification product AFQ1. The ratio between the AFBO and the AFQ1 production rates varied between 1:19 and 1:1.7. P450 3A4 contributed a majority of AFBO and AFQ1, and its expression level was the most important determinant of the AFB1 disposition toward these primary metabolites. P450 3A5, which exclusively produced AFBO, was the second-most important enzyme activating AFB1 to AFBO, followed by P450 3A7 and P450 1A2. The relative contribution of AFBO by P450 3A5 strongly depended on the concomitant expression of P450 3A4, and it was as high as 15% in a P450 3A5 high expressor with the lowest P450 3A4 expression of all livers. The P450 1A2-specific AFB1 detoxification product AFM1 was not detected. In conclusion, the variable expression of P450s has a major effect on the carcinogenic activation of AFB1, which may affect the individual predisposition to HCC. P450 3A4 expression is the most important determinant of AFB1 activation to AFBO. The contribution of P450 1A2 to AFB1 metabolism appears to be negligible and may have been overestimated. Targeted chemoprevention of AFB1-associated HCC should consider P450 3A4 inhibitors and avoidance of P450 3A4 inducers.

Genetic signature consistent with selection against the CYP3A4*1B allele in non-African populations.

Cytochrome P450 3A enzymes (CYP3A) play a major role in the metabolism of steroid hormones, drugs and other chemicals, including many carcinogens. The individually variable CYP3A expression, which remains mostly unexplained, has been suggested to affect clinical phenotypes. We investigated the CYP3A locus in five ethnic groups. The degree of linkage disequilibrium (LD) differed among ethnic groups, but the most common alleles of the conserved LD regions were remarkably similar. Non-African haplotypes are few; for example, only four haplotypes account for 80% of common European Caucasian alleles. Large LD blocks of high frequencies were suggestive of selection. Accordingly, European Caucasian and Asian cohorts each contained a block of single nucleotide polymorphism (SNPs) with very high P excess values. The overlap between these blocks in these two groups contained only two of the investigated 26 SNPs and one of them was the CYP3A4*1B allele. The region centromeric of CYP3A4*1B exhibited high haplotype homozygosity in European Caucasians as opposed to African-Americans. CYP3A4*1B showed a moderate effect on CYP3A4 mRNA and protein expression, as well as on CYP3A activity assessed as Vmax of testosterone 6beta-hydroxylation in a liver bank. Our data are consistent with a functional relevance of CYP3A4*1B and with selection against this allele in non-African populations. The elimination of CYP3A4*1B involved different parts of the CYP3A locus in European Caucasians and Asians. Because CYP3A4 is involved in the vitamin D metabolism, rickets may have been the underlying selecting factor.

Contribution of CYP3A5 to the in vitro hepatic clearance of tacrolimus.

BACKGROUND: Tacrolimus is metabolized predominantly to 13-O-demethyltacrolimus in the liver and intestine by cytochrome P450 3A (CYP3A). Patients with high concentrations of CYP3A5, a CYP3A isoenzyme polymorphically produced in these organs, require higher doses of tacrolimus, but the exact mechanism of this association is unknown. METHODS: cDNA-expressed CYP3A enzymes and a bank of human liver microsomes with known CYP3A4 and CYP3A5 content were used to investigate the contribution of CYP3A5 to the metabolism of tacrolimus to 13-O-demethyltacrolimus as quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Demethylation of tacrolimus to 13-O-demethyltacrolimus was the predominant clearance reaction. Calculated K(m) and V(max) values for CYP3A4, CYP3A5, and CYP3A7 cDNA-expressed microsomes were 1.5 micromol/L and 0.72 pmol x (pmol P450)(-1) x min(-1), 1.4 micromol/L and 1.1 pmol x (pmol P450)(-1) x min(-1), and 6 micromol/L and 0.084 pmol x (pmol P450)(-1) x min(-1), respectively. Recombinant CYP3A5 metabolized tacrolimus with a catalytic efficiency (V(max)/K(m)) that was 64% higher than that of CYP3A4. The contribution of CYP3A5 to 13-O-demethylation of tacrolimus in human liver microsomes varied from 1.5% to 40% (median, 18.8%). There was an inverse association between the contribution of CYP3A5 to 13-O-demethylation and the amount of 3A4 protein (r = 0.90; P <0.0001). Mean 13-O-demethylation clearances in CYP3A5 high and low expressers, estimated by the parallel-tube liver model, were 8.6 and 3.57 mL x min(-1) x (kg of body weight)(-1), respectively (P = 0.0088). CONCLUSIONS: CYP3A5 affects metabolism of tacrolimus, thus explaining the association between CYP3A5 genotype and tacrolimus dosage. The importance of CYP3A5 status for tacrolimus clearance is also dependent on the concomitant CYP3A4 activity.

Limited contribution of CYP3A5 to the hepatic 6beta-hydroxylation of testosterone.

The polymorphic expression of CYP3A5 in human livers is well established, but its significance for the entire hepatic CYP3A activity is disputed. We investigated the contribution of CYP3A5 to the CYP3A activity assessed as 6beta-hydroxylation of testosterone using baculovirus-expressed CYP3A4 and CYP3A5 and microsomes isolated from 47 Caucasian human livers. Under comparable conditions, the specific activities of baculovirus-expressed CYP3A4 and CYP3A5 were nearly identical. Among human livers tested, the Vmax of 6beta-testosterone hydroxylation varied 28-fold. Of the enzymes that are capable of catalyzing 6beta-hydroxylation of testosterone (CYP3A and CYP1A1), only CYP3A4 mRNA and protein expression correlated significantly with the Vmax values (r=0.51, p<0.001 and r=0.66, p<0.001, respectively). Neither consideration of the CYP3A5 polymorphism nor combining CYP3A4 mRNA expression with the expression of other CYP3A mRNA species increased the correlation. The five livers heterozygous for the CYP3A5*1 allele had a mean 6beta-testosterone hydroxylation Vmax value of 2,976 pmol/mg/min, compared with 3,798 pmol/mg/min in the homozygous CYP3A5*3 livers. Together, these data suggest that the specific activities of CYP3A4 and CYP3A5 towards testosterone are comparable. However, the contribution of CYP3A5 to 6beta-hydroxylation of testosterone in Caucasian livers is limited, due to the much lower expression levels of CYP3A5.