Development, validation and application of the Basel phenotyping cocktail

Personalized therapy is a matter of current discussion in the medical community. It consists of adapting the choice of a drug and its posology to the specific profile of a subject (genotype and phenotype) or to its disease (i.e. specific cancer biology). Activity of drug metabolizing enzymes depends on the genotype and extrinsic factors which together determine the phenotype of a subject. Therefore a precise characterization of a patient phenotype will guide the physicians efforts to personalize therapy and thus improve efficacy and reduce side effects. This approach requires specific and easy to use diagnostic tools that are not yet available in clinical routine. With our work, we explored possible ways to promote phenotyping of drug metabolizing enzymes as a valuable tool for personalized medicine. In this dissertation we describe the development of a new phenotyping cocktail (Basel cocktail) containing caffeine, efavirenz, losartan, omeprazole, metoprolol, and midazolam as probe drugs for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, respectively. We validated the new phenotyping procedure in two clinical studies in healthy volunteers: the Basel cocktail studies I and II. With the data acquired in study I, the absence of mutual interactions between the new combinations of probe drugs selected for the Basel phenotyping cocktail was established. Furthermore we showed that evaluation of the metabolic ratios in plasma at single time-points can replace conventional full AUC measurements. Additionally, the use of dried blood spots (DBS) and saliva as minimally or non-invasive sampling collection procedures were explored. For a comprehensive evaluation of the new phenotyping cocktail, the influence of genetic factors or co-administration of an enzyme inducer or inhibitor on the metabolic ratios of the Basel cocktail had to be assessed. The Basel cocktail study II was performed to characterize the range of the chosen phenotyping metrics under conditions of induction and inhibition. Additionally we measured the metabolic ratios in saliva and DBS. Bioanalysis and genotype determination were key tools for this project. We developed and validated bioanalytical methods for simultaneous detection of probe drugs and their main metabolites in different human matrices. To maintain clinical applicability, short return times were required. Therefore highly sensitive bioanalytical methods with simple sample work-up and short run-times were developed. Study subjects providing consent were genotyped for the most relevant single-nucleotide polymorphisms and when possible correlations between genotype and phenotype were assessed. In the Basel cocktail studies I and II, the lowest commercially available doses and formulations of the phenotyping drugs were used. To simplify probe drug administration, a prototype of a combi-pill containing all six cocktail probe drugs was developed and tested in a single subject in a clinical pilot study. Pharmacokinetic profiles of the new formulation were comparable with the ones obtained after intake of the individual probe drugs in the commercial formulations. This new cocktail combi-pill will be formally evaluated in a clinical study (Basel cocktail study III) in healthy volunteers. The use of phenotyping information to personalize cancer therapy with tyrosine kinase inhibitors is under investigation in an ongoing clinical trial. Individual CYP3A4 and CYP1A2 phenotype will be assessed using probe drugs of the Basel cocktail and the correlation of the phenotyping metrics with sunitinib, pazopanib or erlotinib exposure will be analyzed. Taken together the work performed during this dissertation was important to improve the clinical applicability of phenotyping, which is essential to move this important tool for personalized medicine closer to the clinical routine

In Vivo Evaluation of a Gastroretentive Drug Delivery System Based on Enteric-Coated Floating Tablets

Floating dosage forms are supposed to exhibit an enhanced gastric residence time. Their development is challenging as the prediction of the retention potential in humans based on in vitro studies and animal models is difficult. A strategy to determine the stomach residence time of a floating dosage form with an inherently low density in human without using imaging techniques was explored in a self-experiment. Floating tablets and non-floating controls were prepared containing caffeine as a model drug. The compacts had a pH-dependent entericcoating to assess their stomach residence time. Since caffeine is rapidly absorbed in the gastrointestinal tract, the prolonged gastric retention of tablets can be demonstrated by a delayed systemic exposure. Caffeine and paraxanthine were determined in capillary blood by liquid chromatography coupled to tandem mass spectrometry. An increase in caffeine and paraxanthine blood levels was observed in human volunteers after 90 to 180 min for the non-floating controls. For the floating tablets, no elevated blood concentrations were found in two out of three participants during 8 h of sample collection. The results demonstrate the technical feasibility of the proposed clinical study protocol. Follow-up clinical trials will be needed to confirm the preliminary data on stomach residence time of our floating dosage form

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8min. Recoveries of carnitine and acylcarnitines were between 98 and 105%. Both quantification methods were equally accurate (all values within 84 to 116% of target concentrations) and precise (day-to-day variation of less than 18%) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine. Figure A method is presented for the analysis of carnitine and acylcarnitines in urine which includes a precipitation step, on-column extraction and LC-MS/MS. The run time is 8 minutes and the method was validated for carnitine, acetylcarnitine, octanoylcarnitine and palmitoylcarnitine. Analysis of a patient sample with medium-chain acyl-CoA dehydrogenase deficiency is show

Movement Analysis Could Help in the Assessment of Chronic Low Back Pain Patients: Results from a Preliminary Explorative Study

Introduction: This study aimed to assess the reliability of a qualitative scoring system based on the movement analysis of the spine in different populations and after usual care rehabilitative intervention. If proven true, the results could further future research development in quantitative indexes, leading to a possible subclassification of chronic low back pain (cLBP). Methods: This was a preliminary exploratory observational study. Data of an optoelectronic spine movement analysis from a pathological population (cLBP population, 5 male, 5 female, age 58 ± 16 years) were compared to young healthy participants (5M, 5F, age 22 ± 1) and were analysed via a new qualitative score of the pattern of movement. Internal consistency was calculated. Two independent assessors (experienced and inexperienced) assessed the blinded data, and we calculated inter- and intrarater reliability. We performed an analysis for cLBP pre and post a ten session group rehabilitation program between and within groups. Results: Internal consistency was good for all movements (α = 0.84-0.88). Intra-rater reliability (Intraclass correlation coefficient-ICC) was excellent for overall scores of all movements (ICC(1,k) = 0.95-0.99), while inter-rater reliability was poor to moderate (ICC(1,k) = 0.39-0.78). We found a significant difference in the total movement scores between cLBP and healthy participants (p = 0.001). Within-group comparison (cLBP) showed no significant difference in the total movement score in pre and post-treatment. Conclusion: The perception of differences between normal and pathological movements has been confirmed through the proposed scoring system, which proved to be able to distinguish different populations. This study has many limitations, but these results show that movement analysis could be a useful tool and open the door to quantifying the identified parameters through future studies

Duloxetine Inhibits Effects of MDMA (“Ecstasy") In Vitro and in Humans in a Randomized Placebo-Controlled Laboratory Study

This study assessed the effects of the serotonin (5-HT) and norepinephrine (NE) transporter inhibitor duloxetine on the effects of 3,4–methylenedioxy­methamphetamine (MDMA, ecstasy) in vitro and in 16 healthy subjects. The clinical study used a double-blind, randomized, placebo-controlled, four-session, crossover design. In vitro, duloxetine blocked the release of both 5-HT and NE by MDMA or by its metabolite 3,4-methylenedioxyamphetamine from transmitter-loaded human cells expressing the 5-HT or NE transporter. In humans, duloxetine inhibited the effects of MDMA including elevations in circulating NE, increases in blood pressure and heart rate, and the subjective drug effects. Duloxetine inhibited the pharmacodynamic response to MDMA despite an increase in duloxetine-associated elevations in plasma MDMA levels. The findings confirm the important role of MDMA-induced 5-HT and NE release in the psychotropic effects of MDMA. Duloxetine may be useful in the treatment of psychostimulant dependence

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine

Toxicity of thienopyridines on human neutrophil granulocytes and lymphocytes

Thienopyridines can cause neutropenia and agranulocytosis. The aim of the current investigations was to compare cytotoxicity of ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel for human neutrophil granulocytes with the toxicity for lymphocytes and to investigate underlying mechanisms. For granulocytes, clopidogrel, ticlopidine, clopidogrel carboxylate and prasugrel were concentration-dependently toxic starting at 10μM. Cytotoxicity could be prevented by the myeloperoxidase inhibitor rutin, but not by the cytochrome P450 inhibitor ketoconazole. All compounds were also toxic for lymphocytes, but cytotoxicity started at 100μM and could not be prevented by rutin or ketoconazole. Granulocytes metabolized ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel, and metabolization was inhibited by rutin, but not by ketoconazole. Metabolism of these compounds by lymphocytes was much slower and could not be inhibited by ketoconazole or rutin. In neutrophils, all compounds investigated decreased the electrical potential across the inner mitochondrial membrane, were associated with cellular accumulation of ROS, mitochondrial loss of cytochrome c and induction of apoptosis starting at 10μM. All of these effects could be inhibited by rutin, but not by ketoconazole. Similar findings were obtained in lymphocytes; but compared to neutrophils, the effects were detectable only at higher concentrations and were not inhibited by rutin. In conclusion, ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel are toxic for both granulocytes and lymphocytes. In granulocytes, cytotoxicity is more accentuated than in lymphocytes and depends on metabolization by myeloperoxidase. These findings suggest a mitochondrial mechanism for cytotoxicity for both myeloperoxidase-associated metabolites and, at higher concentrations, also for the parent compounds

Comparison Of Liver Cell Models Using The Basel Phenotyping Cocktail

Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH) cultures. We investigated the suit-ability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4]) in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture) regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6 and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least 4-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20µM rifampicin, mRNA increased 3 to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6) and 17-fold (CYP3A4) for 2D-PHH and 4-fold (CYP3A4) for HepG2. In 3D-PHH at least a 2-fold in-crease in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model.Our studies show that 3D-PHH and (with some limitations) HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to as-sess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro

Toxicity of clopidogrel and ticlopidine on human myeloid progenitor cells: importance of metabolites

Ticlopidine and clopidogrel are thienopyridine derivatives used for inhibition of platelet aggregation. Not only hepatotoxicity, but also bone marrow toxicity may limit their use. Aims of the study were to find out whether non-metabolized drug and/or metabolites are responsible for myelotoxicity and whether the inactive clopidogrel metabolite clopidogrel carboxylate contributes to myelotoxicity. We used myeloid progenitor cells isolated from human umbilical cord blood in a colony-forming unit assay to assess cytotoxicity. Degradation of clopidogrel, clopidogrel carboxylate or ticlopidine (studied at 10 and 100 μM) was monitored using LC/MS. Clopidogrel and ticlopidine were both dose-dependently cytotoxic starting at 10 μM. This was not the case for the major clopidogrel metabolite clopidogrel carboxylate. Pre-incubation with recombinant human CYP3A4 not only caused degradation of clopidogrel and ticlopidine, but also increased cytotoxicity. In contrast, clopidogrel carboxylate was not metabolized by recombinant human CYP3A4. Pre-incubation with freshly isolated human granulocytes was not only associated with a myeloperoxidase-dependent degradation of clopidogrel, clopidogrel carboxylate and ticlopidine, but also with dose-dependent cytotoxicity of these compounds starting at 10 μM. In conclusion, both non-metabolized clopidogrel and ticlopidine as well as metabolites of these compounds are toxic towards myeloid progenitor cells. Taking exposure data in humans into account, the myelotoxic element of clopidogrel therapy is likely to be secondary to the formation of metabolites from clopidogrel carboxylate by myeloperoxidase. Concerning ticlopidine, both the parent compound and metabolites formed by myeloperoxidase may be myelotoxic in vivo. The molecular mechanisms of cytotoxicity have to be investigated in further studies