CYP2C19 Phenoconversion by Routinely Prescribed Proton Pump Inhibitors Omeprazole and Esomeprazole: Clinical Implications for Personalized Medicine.

The phenotype pantoprazole-(13)C breath test (Ptz-BT) was used to evaluate the extent of phenoconversion of CYP2C19 enzyme activity caused by commonly prescribed proton pump inhibitors (PPI) omeprazole and esomprazole. The Ptz-BT was administered to 26 healthy volunteers and 8 stable cardiovascular patients twice at baseline and after 28 days of PPI therapy to evaluate reproducibility of the Ptz-BT and changes in CYP2C19 enzyme activity (phenoconversion) after PPI therapy. The average intrapatient interday variability in CYP2C19 phenotype (n = 31) determined by Ptz-BT was considerably low (coefficient of variation, 17%). Phenotype conversion resulted in 25 of 26 (96%) nonpoor metabolizer (non-PM) volunteers/patients as measured by the Ptz-BT at baseline and after PPI therapy. The incidence of PM status by phenotype following administration of omeprazole/esomeprazole (known inhibitors of CYP2C19) was 10-fold higher than those who are genetically PMs in the general population, which could have critical clinical implications for personalizing medications primarily metabolized by CYP2C19, such as clopidogrel, PPI, cyclophosphamide, thalidomide, citalopram, clonazepam, diazepam, phenytoin, etc. The Ptz-BT can rapidly (30 minutes) evaluate CYP2C19 phenotype and, more importantly, can identify patients with phenoconversion in CYP2C19 enzyme activity caused by nongenetic factors such as concomitant drugs.

Does obesity interfere with anastrozole treatment? Positive association between body mass index and anastrozole plasma levels.

INTRODUCTION: The efficacy of adjuvant endocrine treatment with aromatase inhibitors (AIs), inhibiting the conversion of androgens to estrogen in adipose tissue, might depend on the overall volume of adipose tissue. However, little evidence is available regarding the pharmacokinetic behavior of AIs in women with obesity. The aim of this study was to investigate the interaction between body mass index (BMI) and anastrozole treatment as well as estrogenic activity. PATIENTS AND METHODS: A total of 216 postmenopausal patients with early-stage breast cancer who were receiving AI treatment with anastrozole constituted the final sample included in the analysis. During a regular 3-month after-care check-up, sociodemographic and clinical data and BMI were assessed. Blood samples were collected during routine blood testing. Measurement of AI plasma levels was performed by liquid chromatography-tandem mass spectrometry. Follicle stimulating hormone (FSH) and estradiol were measured within the routine blood examination. RESULTS: A median anastrozole plasma concentration of 34.7 ng/mL (mean, 37.4), with a large interindividual variability, was observed (SD, 15.1; range, 5.4-86.5). After age adjustment, it was found that anastrozole plasma concentrations significantly increased with BMI (r = 0.241; P = .001). Anastrozole serum concentrations in women with obesity (BMI ≥ 30) exceeded those of women with normal weight (BMI ≤ 25) by 25%. Women with excess weight had lower mean FSH levels, indicating higher estrogenic activity, compared with women with normal weight. CONCLUSION: This study indicates that BMI is a vital factor in anastrozole metabolism, as measured by anastrozole plasma concentration and FSH levels. Further research is mandatory to clarify results on the association of obesity and AI treatment efficacy to allow adapting AI treatment accordingly.

Association of polymorphisms in pharmacogenetic candidate genes (OPRD1, GAL, ABCB1, OPRM1) with opioid dependence in European population: a case-control study.

It is becoming increasingly evident that genetic variants contribute to the development of opioid addiction. An elucidation of these genetic factors is crucial for a better understanding of this chronic disease and may help to develop novel therapeutic strategies. In recent years, several candidate genes were implicated in opioid dependence. However, most study findings have not been replicated and additional studies are required before reported associations can be considered robust. Thus, the major objective of this study was to replicate earlier findings and to identify new genetic polymorphisms contributing to the individual susceptibility to opioid addiction, respectively. Therefore, a candidate gene association study was conducted including 142 well-phenotyped long-term opioid addicts undergoing opioid maintenance therapy and 142 well-matched healthy controls. In both study groups, 24 single nucleotide polymorphisms predominantly located in pharmacogenetic candidate genes have been genotyped using an accurate mass spectrometry based method. The most significant associations with opioid addiction (remaining significant after adjustment for multiple testing) were observed for the rs948854 SNP in the galanin gene (GAL, p = 0.001) and the rs2236861 SNP in the delta opioid receptor gene (OPRD1, p = 0.001). Moreover, an association of the ATP binding cassette transporter 1 (ABCB1) variant rs1045642 and the Mu Opioid receptor (OPRM1) variant rs9479757 with opioid addiction was observed. The present study provides further support for a contribution of GAL and OPRD1 variants to the development of opioid addiction. Furthermore, our results indicate a potential contribution of OPRM1 and ABCB1 SNPs to the development of this chronic relapsing disease. Therefore it seems important that these genes are addressed in further addiction related studies.

A novel amplification strategy for genotyping with liquid chromatography-electrospray ionization mass spectrometry.

Among numerous available genotyping techniques, mass spectrometry (MS) based methods play a major role in providing high quality genotype data at reasonable costs for research and diagnostics, e.g. for pharmacogenetic applications. Ion-pair reversed-phase liquid chromatography hyphenated to electrospray ionization time-of-flight MS (ICEMS) is, for example, a powerful instrument that allows a direct characterization of complex mixtures of polymerase chain reaction (PCR) amplified DNA fragments. Current limitations of PCR-ICEMS genotyping are mainly concerned with the multiplex PCR set-up. Assay development often requires time-consuming primer design and intensive optimization of PCR conditions. To overcome this restraint, a robust amplification strategy originally combined with arrayed primer extension genotyping was transferred and adapted to ICEMS genotyping. The modifications involved limitation of the primer length, application of two universal sequences and amplification with an appropriate DNA polymerase. To demonstrate the applicability of the novel amplification strategy for ICEMS, a 23-plex pharmacogenetic genotyping assay was developed. After slight optimization steps, an efficient and quantitatively balanced amplification of all targeted markers was achieved, resulting in a convenient characterization of the multiplexed PCR fragments with ICEMS. Expenditure of time, costs and hands-on work associated with assay design and optimization was dramatically lowered compared to previous multiplex PCR-ICEMS assays. The developed 23-plex assay was applied in a pharmacogenetic study including 284 individuals (genotype call rate 99.0%). A total of 399 SNPs were retyped by Sanger sequencing (concordance rate 99.8%). The PCR-ICEMS assay turned out to be an accurate, reliable, cost-effective and a ready-to-use tool for pharmacogenetic genotyping.

Adherence evaluation of endocrine treatment in breast cancer: methodological aspects.

BACKGROUND: Current studies on adherence to endocrine therapy in breast cancer patients suffer from methodological limitations due to a lack of well-validated methods for assessing adherence. There is no gold standard for measuring adherence. The aim of our study was to compare four different approaches for evaluating adherence to anastrozole therapy for breast cancer with regard to concordance between methods. METHODS: Outpatients with early breast cancer treated with anastrozole completed the multi-method assessment of adherence. We implemented a self-report scale (the Simplified Medication Adherence Questionnaire), physician- ratings, refill records and determination of anastrozole serum concentration. RESULTS: Comparison of the four approaches using Spearman rank correlation revealed poor concordance across all methods reflecting weak correlations of 0.2-0.4. Considering this data incomparability across methods, we still observed high adherence rates of 78%-98% across measures. CONCLUSION: Our findings contribute to the growing body of knowledge on the impact that methodological aspects exert on the results of adherence measurement in breast cancer patients receiving endocrine treatment. Our findings suggest that the development and validation of instruments specific to patients receiving endocrine agents is imperative in order to arrive at a more accurate assessment and to subsequently obtain more precise estimates of adherence rates in this patient population.

Is the toxicity of adjuvant aromatase inhibitor therapy underestimated? Complementary information from patient-reported outcomes (PROs).

Adjuvant endocrine treatment-related adverse effects have a strong impact on patients' quality of life and thereby limit therapy's risk benefit ratio resulting in morbidity and treatment discontinuation. Still, many AI adverse effects remain untreated given that they are unrecognized by conservative methods (e.g., proxy ratings). The ability of complementary patient-reported outcomes (PROs) to provide a more comprehensive assessment of side-effects is to be explored. A cross-sectional study sample of 280 postmenopausal, early stage breast cancer patients was subjected to a comprehensive PRO assessment (FACT-B/+ES) at their after-care appointment. Prevalence and severity of patient-reported physical side-effects and psychosocial burden related to adjuvant AI therapy were compared with prevalences derived from pivotal phase IV trials (ATAC 2005, BIG1-98 2005). Across all symptom categories, highest prevalence rates were found for joint pain (59.6%), hot flushes (52%), lost interest in sexual intercourse (51.4%), and lack of energy (40.3%). Overall, PROs resulted in significantly higher prevalence rates as compared to physician ratings for all symptoms published in pivotal clinical trials except vaginal bleeding and nausea. The treatment duration exerted no significant impact on symptom frequency (P > 0.05). Established prevalence rates of endocrine treatment-related toxicity seem to be underestimated. The incorporation of PRO data should be mandatory or at least highly recommended in clinical treatment planning to arrive at a more accurate assessment of a patient's actual symptom burden enabling improved individualized management of side-effects and mediating the preservation of treatment adherence.

CYP2D6 genotyping by liquid chromatography-electrospray ionization mass spectrometry.

Genetic polymorphisms can significantly affect the enzyme activity of the drug metabolizing enzyme Cytochrome P450 2D6 (CYP2D6; OMIM 124030). Accordingly, CYP2D6 genotyping is considered as a valid approach to predict the individual CYP2D6 metabolizing status. We introduce ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) as method for the characterization of single base variants, small deletions, and insertions in the CYP2D6 gene. A two-step polymerase chain reaction (PCR) was developed for the simultaneous amplification of nine polymorphic regions within the CYP2D6 gene. Cleanup, separation, and denaturation of PCR amplicons were achieved by high-performance liquid chromatography. High-performance molecular mass measurements provided nucleotide composition profiles that principally enable the resolution of 37 reported CYP2D6 alleles. The developed assay was applied to the genotyping of 93 unrelated Austrian individuals. For validation, a selected number of samples and polymorphic sites were retyped by alternative genotyping technologies. The PCR-ICEMS assay turned out to be an accurate, robust, and cost-effective CYP2D6 genotyping strategy.

CYP2D6 genotyping in breast cancer patients by liquid chromatography-electrospray ionization mass spectrometry.

The application of cytochrome P450 2D6 (CYP2D6) genotyping to allow a personalized treatment approach for breast cancer patients undergoing endocrine therapy has been repeatedly discussed. However, the actual clinical relevance of the CYP2D6 genotype in the endocrine treatment of breast cancer still remains to be elucidated. A major prerequisite for the successful and valid evaluation of the CYP2D6 genotype with regard to its pharmacokinetic and clinical relevance is the availability of a comprehensive, accurate and cost-effective CYP2D6 genotyping strategy. Herein we present a CYP2D6 genotyping assay employing polymerase chain reaction (PCR)-ion pair reversed-phase high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry (ICEMS). The genotyping strategy involves the simultaneous amplification of nine variable regions within the CYP2D6 gene by a two-step PCR protocol and the direct analysis of the generated PCR amplicons by ICEMS. The nucleotide composition profiles generated by ICEMS enable the differentiation of 37 of the 80 reported CYP2D6 alleles. The assay was applied to type the CYP2D6 gene in 199 Austrian individuals including 106 breast cancer patients undergoing tamoxifen treatment. The developed method turned out to be a highly applicable, robust and cost-effective approach, enabling an economical CYP2D6 testing for large patient cohorts.

3D versus 2D cell culture implications for electron microscopy.

Cell culture systems are indispensable tools for basic research and a wide range of clinical in vitro studies. However, conventional 2D cell cultures poorly mimic the conditions in the living organism. This limitation may seriously compromise the reliability and significance of data obtained from such approaches. Therefore, we present here a comparative study on selected 3D and 2D cell cultures of U87-MG human glioblastoma cells that were processed by means of high-pressure freezing and freeze-substitution as well as by conventional chemical fixation and Tokuyasu cryo-section immuno-labeling. Three-dimensional cultures comprised pseudo-vascularized cultures, fiber and bead scaffold cultures, and spheroid cultures. Cell cultures in dishes and on coverslips were the static 2D culture systems used as reference models. We will discuss morphological and immuno-cytochemical observations with respect to the feasibility of the cell culture systems investigated for the state-of-the-art electron microscopy.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of tamoxifen, anastrozole, and letrozole in human plasma and its application to a clinical study.

There is substantial evidence that circulating estrogens promote the proliferation of breast cancer. Consequently, adjuvant hormonal treatment strategies targeting estrogen action have been established. Such hormonal therapies include selective estrogen receptor modulators, such as tamoxifen, which interfere at the estrogen receptors directly, or non-steroidal aromatase inhibitors, such as anastrozole and letrozole, which inhibit estrogen synthesis through blocking the aromatase, a key enzyme of estrogen production. Despite considerable therapeutic success, in several cases, the use of these drugs is limited by side effects that have been described to significantly impair the adherence of patients to endocrine treatment. However, objective data concerning patient adherence and its clinical relevance are limited. One promising approach to check patient-reported adherence is drug monitoring in human plasma. Therefore, a liquid chromatography-tandem mass spectrometry method to determine the plasma concentrations of tamoxifen, anastrozole, and letrozole has been developed and fully validated according to guidelines for clinical and forensic toxicology. The validation criteria evaluated were selectivity, linearity, accuracy and precision, limit of quantification, recovery and matrix effects, sample stability, and carryover. The six-point calibration curves showed linearity over the range of concentrations from 25 to 500 ng/ml for tamoxifen, 5 to 200 ng/ml for anastrozole, and 10 to 300 ng/ml for letrozole. The intra- and inter-day precision and accuracies were always better than 15%. The validated procedure was successfully applied to a clinical study (Patient-Reported Outcomes in Breast Cancer Patients undergoing Endocrine Therapy, PRO-BETh). A major aim of PRO-BETh study is the comprehensive evaluation of adherence to treatment in pre- and post-menopausal women with breast cancer. Plasma samples of 310 breast cancer patients undergoing anti-estrogen therapy were analyzed. Eight samples did not contain a quantifiable amount of drug, strongly indicating non-adherence of the corresponding patients to adjuvant breast cancer treatment. Furthermore, plasma concentrations at the lower end of the observed plasma level distribution might represent a hint but not a confirmation for non-adherence in terms of non-daily and irregular intake of the prescribed drug.

[Impact of slow-release oral morphine on drug abusing habits in Austria]. / Der Einfluss von retardiertem Morphin auf die Drogensituation in Osterreich.

A well-established possibility to treat opiate addiction is the participation in opiate maintenance treatment programmes. For this purpose the opioids methadone and buprenorphine have been evaluated and are used nowadays in many countries. However, since 1998 also the use of slow-release oral morphine (SROM) has been legally permitted in Austria. Our data show that these morphine preparations are frequently abused and are dominating the black market in the meantime. Especially the intravenous consumption of SROM goes along with highly dangerous side effects that exceed the risks of needle sharing alone. Special galenics are supposed to ensure a 24 h effect of the otherwise quickly metabolised morphine. If dissolved and injected, insoluble contents such as talcum cause microembolisms, leading to severe damages of the inner organs. Furthermore, SROM, i.e. a drug prescribed by physicians, has been proved to be the main responsible substance in most drug related deaths since its permission and has nearly replaced heroin. Forensic physicians play a major role in the profound examination of these cases, including extensive toxicological analyses and interpretation of results. For instance, a differentiation between a recent morphine and heroin consumption is certainly possible, provided appropriate methods are used. A reliable estimation of the current situation of drug abusing habits is a premise for adequate therapeutic offers and preventive measures. Thus, well-founded and comparable data have to be collected. To facilitate data report a standardized report form has been developed that includes an obligatory statement regarding morphine or heroin consumption. This should help to enlighten the ongoing discussion on the role of SRM in drug abuse cases. Our results indicate that the prescription of SROM in opiate maintenance therapy has to be handled very strictly and should be reserved for special patients only. A slackening of the Austrian law concerning SROM is therefore objected.

A fatal intoxication case involving topiramate.

Topiramate belongs to a new group of anticonvulsive drugs primarily applied in treatment of epilepsy and in preventive therapy of migraines. Topiramate is structurally unrelated to other antiepileptic drugs and acts by multiple neurostabilizing mechanisms. However, the pharmacology of topiramate appears to be complex and some of its pharmacodynamic actions still remain to be elucidated. This case report documents a fatal intoxication involving topiramate. A 41-year old woman with a known history of psychiatric disorder was found unresponsive by her husband. Resuscitation efforts did not succeed and the woman was pronounced dead at the intensive care unit four hours later. At the scene, drug packages of topiramate, citalopram and flunitrazepam were found. Autopsy including histological examination revealed morphological signs of an acute intoxication and shock. A comprehensive toxicological analysis with GC-MS was performed on the deceased's autopsy samples (femoral blood, bile, kidney, gastric content). The results revealed the presence of topiramate at a concentration of 49mg/L in the femoral blood sample, thus clearly exceeding the therapeutic range. Additionally, citalopram (0.85mg/L) and flunitrazepam in traces (<2µg/L) were detected in peripheral blood. Based on the autopsy findings and toxicological results, the cause of death was primarily attributed to an intoxication with topiramate in combination with citalopram.

Challenging small human hepatocytes with opiates: further characterization of a novel prototype bioartificial liver.

Bioartificial liver (BAL) systems can take over liver functions in patients undergoing liver failure until transplantation. Recently, a novel prototype rotary BAL has been developed using small human hepatocytes (SH). This study investigated the metabolism of opiates morphine and methadone in the BAL and their influence on the basic cell culture parameters, viability, and growth of SH. Opiates may be present in patients due to pain therapy, anticancer treatment, or drug abuse. Cells were cultivated in the BAL for a total of 12 days and exposed twice to 100 microg/L of morphine or methadone. Morphine and methadone concentrations were analyzed using gas chromatography with a mass spectrometry detector. Further, the production of albumin, lactate dehydrogenase release, lactate release, urea production, and glucose consumption were measured. Cell viability and growth were determined by confocal microscopy. Cytochrome P 3A4 and uridindiphosphat (UDP) glucuronosyl transferase 2B7 in SH were analyzed by western blot. The mean cell density during treatment was 5.5 +/- 0.7 x 10(6) cells/mL (n = 6) and was not altered significantly by the opiates. Cell viability stayed above 90%. Morphine was not reduced by SH and was a stress factor as determined by decreased metabolic activity. On the other hand, SH metabolized methadone showing first-order kinetics: the first-order rate constant k = 0,019, half-life t(1/2) = 36 h. Methadone metabolism led to decreased urea and albumin production. The expression of cytochrome P 3A4, mainly responsible for methadone metabolism, was proved in SH. The prototype BAL is basically suited to support liver functions, provided patients receive therapy with methadone.

Optimized conformal paraaortic lymph node irradiation is not associated with enhanced renal toxicity.

BACKGROUND AND PURPOSE: For patients with gynecologic carcinomas, irradiation of paraaortic lymph nodes (PLNs) is a routine treatment concept. Planning target volumes (PTVs) individually optimized by radiation field delineations along the big vessels permit the inclusion of at least 97% of potentially involved PLNs. However, this novel treatment technique might increase radiation-induced nephrotoxicity. Therefore, the actual incidence of kidney damage after PLN irradiation has to be assessed in order to validate the safety of this treatment concept. PATIENTS AND METHODS: 19 patients were treated with irradiation alone (50.4 Gy; 5 x 1.8 Gy/week) and monitored for up to 90 months. Functional renal parameters, namely renal plasma flow (RPF) and glomerular filtration rate (GFR), were assessed by dynamic renal scintigraphy. Additionally, patients were clinically observed (i.e., hypertension, proteinuria) and calculations of normal-tissue complication probability (NTCP) values for nonuniform kidney irradiation were performed using the Lyman-Wolbarst algorithm. RESULTS: Two patients with anticipated moderate NTCP values (12.6% and 8.7%) showed slightly impaired RPF rates at 12, 24, and after 48 months of follow-up. Only one patient in the subgroup showing NTCP values > 50% (n = 9) developed a notable impairment of renal RPF. However, all patients including those with elevated complication probabilities exhibited neither impaired GFR nor clinically apparent symptoms related to a loss of functioning renal tissue from 12 to > 48 months post irradiation. CONCLUSION: Conformal irradiation of retroperitoneal lymph nodes with individual PTV delineation appears not to be associated with clinically relevant functional impairment of the kidneys.