Article

Evaluation of Mutual Drug-Drug Interaction within Geneva Cocktail for Cytochrome P450 Phenotyping using Innovative Dried Blood Sampling Method

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Abstract

Cytochrome P450 (CYP) activity can be assessed using a 'cocktail' phenotyping approach. Recently, we have developed a cocktail (Geneva cocktail) which combines the use of low-dose probes with a low-invasiveness dried blood spots (DBS) sampling technique and a single analytical method for the phenotyping of six major CYP isoforms. We have previously demonstrated that modulation of CYP activity after pre-treatment with CYP inhibitors/inducer could be reliably predicted using Geneva cocktail. In order to further validate this cocktail, in this study we have verified whether probe drugs contained in the latter cause mutual drug-drug interactions. In a randomized, 4-way, Latin-square cross-over study, 30 healthy volunteers received low-dose caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam (a previously validated combination with no mutual drug-drug interactions); fexofenadine alone; bupropion alone or all 7 drugs simultaneously (Geneva cocktail). Pharmacokinetic profiles of the probe drugs and their metabolites were determined in DBS samples using both conventional micropipette sampling and new microfluidic device allowing for self-sampling. The 90% confidence intervals for the geometric mean ratios of AUCmetabolite/AUCprobe for CYP probes administered alone or within Geneva cocktail fell within the 0.8-1.25 bioequivalence range indicating the absence of pharmacokinetic interaction. The same result was observed for the chosen phenotyping indices i.e. metabolic ratios at 2 hr (CYP1A2, CYP3A) or 3 hr (CYP2B6, CYP2C9, CYP2C19, CYP2D6) post-cocktail administration. DBS sampling could successfully be performed using a new microfluidic device. In conclusion, Geneva cocktail combined with an innovative DBS sampling device can be used routinely as a test for simultaneous CYP phenotyping. This article is protected by copyright. All rights reserved.

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... However, the default simulated concentration-time profile of omeprazole did not match the concentration-time profile of omeprazole in a population of Caucasian healthy volunteers (data not shown). 19 Based on a previously validated model, volume of distribution at steady state (Vss) of omeprazole was changed from 0.15 to 0.23 L/kg because the default value did not fit the Caucasian population. 20,21 The models used for midazolam and omeprazole are summarized in Table S1 and Table 1, respectively. ...
... (raw data not shown). 19 The esomeprazole model also needed to be validated as a previously validated model was modified. 25 We extracted the concentration-time profile of esomeprazole from one study with the same dose used in the cohort study to obtain observational data. ...
... The observed geometric mean AUC ratio of 5-OH-omeprazole/ omeprazole was 0.66. 19 Therefore, the AUC ratio between observation and simulation was 1.53, which is the accepted range of equivalence in PBPK modeling. PBPK models of omeprazole ( Figure 2a) and 5-OH-omeprazole (Figure 2b) accurately predict the observed data. ...
Article
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Xenobiotics can interact with cytochromes P450 (CYPs), resulting in drug-drug interactions, but CYPs can also contribute to drug-disease interactions, especially in the case of inflammation, which downregulates CYP activities through pretranscriptional and posttranscriptional mechanisms. Interleukin-6 (IL-6), a key proinflammatory cytokine, is mainly responsible for this effect. The aim of our study was to develop a physiologically based pharmacokinetic (PBPK) model to foresee the impact of elevated IL-6 levels in combination with drug interactions with esomeprazole on CYP3A and CYP2C19. Data from a cohort of elective hip surgery patients whose CYP3A and CYP2C19 activities were measured before and after surgery were used to validate the accurate prediction of the developed models. Successive steps were to fit models for IL-6, esomeprazole, and omeprazole and its metabolite from the literature and to validate them. The models for midazolam and its metabolite were obtained from the literature. When appropriate, a correction factor was applied to convert drug concentrations from whole blood to plasma. Mean ratios between simulated and observed areas under the curve for omeprazole/5-hydroxy omeprazole, esomeprazole, and IL-6 were 1.53, 1.06, and 0.69, respectively, indicating an accurate prediction of the developed models. The impact of IL-6 and esomeprazole on the exposure to CYP3A and CYP2C19 probe substrates and respective metabolites were correctly predicted. Indeed, the ratio between predicted and observed mean concentrations were <2 for all observations (ranging from 0.51 to 1.7). The impact of IL-6 and esomeprazole on CYP3A and CYP2C19 activities after a hip surgery were correctly predicted with the developed PBPK models.
... They received a breakfast 30 min after taking the cocktail (but continued to avoid any caffeine-containing beverages or food and grapefruit juice for the whole trial duration) and left the sampling site. They returned to the sampling site three times during the day (at times + 2, + 3, and + 6 h after cocktail administration), each time informing the clinician about adverse events (AE) [see monitoring below], and standardized volume blood spots, as described previously [6], were taken from the volunteers for phenotype analysis at + 2, + 3, and + 6 h after having received the cocktail. Monitoring was thus performed through individual interviews of the volunteers by the clinician monitoring the trial each day. ...
... The principle behind this approach is to simultaneously administer a number of specific probes allowing the assessment of the activity of different CYPs and transporters at the same time. The Geneva cocktail is composed of a specific probe for six different CYPs and one for P-gp and has been validated through clinical studies that showed its usefulness with no interference of each drug with any of the others [6,7]. ...
... Another study that evaluated the usefulness and effectiveness of a dry blood spot sampling carried out on ten healthy volunteers showed no AEs after administration of the cocktail alone [7]. A third study that evaluated the incorporation of fexofenadine and bupropion in the cocktail with the dried blood spot sampling method in 30 healthy volunteers who completed four sessions showed no AEs [6]. A study aiming at investigating the association between CYP and P-gp activities and plasma antidepressant concentration in patients also showed the cocktail to be safe [10]. ...
Article
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Introduction and Objective Cytochrome P450 enzymes are the major drug-metabolizing enzymes in humans and the importance of drug transport proteins, in particular P-glycoprotein, in the variability of drug response has also been highlighted. Activity of cytochrome P450 enzymes and P-glycoprotein can vary widely between individuals and genotyping and/or phenotyping can help assess their activity. Several phenotyping cocktails have been developed. The Geneva cocktail is composed of a specific probe for six different cytochrome P450 enzymes and one for P-glycoprotein and was used in the context of a research aiming at exploring genotypes and phenotypes in distinct human populations (NCT02789527). The aim of the present study is to solely report the safety results of the Geneva cocktail in the healthy volunteers of these populations.Materials and Methods The Geneva cocktail is composed of caffeine, bupropion, flurbiprofen, omeprazole, dextromethorphan, midazolam, and fexofenadine. The volunteers fasted and avoided drinking caffeine-containing beverages or food and grapefruit juice overnight before receiving the cocktail orally. They provided blood spots for the probes’ concentrations at 2, 3, and 6 h after ingestion and were asked about adverse events.ResultsA total of 265 healthy adult volunteers were included from Ethiopia, Oman, and the Czech Republic. The mean plasma concentrations at the 2-h sampling time of each probe drug in the total sample were: 1663 ng/mL for caffeine, 8 ng/mL for bupropion, 789 ng/mL for flurbiprofen, 6 ng/mL for dextromethorphan, 2 ng/mL for midazolam, 35 ng/mL for fexofenadine, and 103 ng/mL for omeprazole. Four adverse events were observed representing an occurrence of 1.5%. All these events were categorized as mild to moderate, non-serious, and resolved spontaneously. A causal link with the cocktail cannot be excluded because of the temporal relationship but is at most evaluated as possible according to the World Health Organization-Uppsala Monitoring Centre causal assessment system.Conclusions In this research, healthy volunteers from three different human populations were phenotyped with the Geneva cocktail. Four adverse events were observed, confirming the safety of this cocktail that is given at lower than clinically relevant doses and therefore results in concentrations lower than those reported to cause adverse events.
... Top-down estimation of CYP2D6-mediated intrinsic clearances of dextromethorphan, duloxetine, paroxetine, and fluoxetine Dextromethorphan and dextrorphan PK profiles in healthy volunteers with different CYP2D6 genotypes with ASs ranging from 1 to 3 were used to estimate the CYP2D6mediated intrinsic clearance (CLint CYP2D6 ) of dextromethorphan O-demethylation. 27 In this study, one subject had an AS of 0.5, 14 subjects were carriers of one fully functional with one nonfunctional allele (AS1), 9 subjects were carriers of two fully functional alleles (AS2), and one subject was a carrier of two functional alleles with one duplication (AS3). The CLint CYP2D6 value of dextromethorphan in Simcyp version 17 is 7.275 μL/minute per mg of microsomal proteins. ...
... The parameter was refined in each of the genotypes mentioned previously to obtain a best fit with the observed concentrations using 100 iterations around 10-fold the initial value mentioned previously, a maximum likelihood objective function, and expectation maximization as the minimization method. As clinical data were obtained in-house, 27 covariates such as age, sex, weight, height, and CYP2D6 genotype were taken into account for the parameter estimation. 28 Similarly, the genotype-dependent CYP2D6-mediated intrinsic clearance of duloxetine and paroxetine were estimated from available clinical data in AS1 and AS2 healthy volunteers 10 using the initial models' CLint CYP2D6 values as starting values for the parameter estimation. ...
... We used the full 0-8-hour PK profiles of dextromethorphan and its metabolite dextrorphan obtained from a previous clinical trial in our group 27 to estimate the CYP2D6-mediated O-demethylation CLint in AS1, AS2, and AS3 subjects using individual fitting. Similarly, we used the full PK profiles of duloxetine and paroxetine, both being CYP2D6 substrates as well, from another clinical trial in AS1 and AS2 subjects 10 to estimate their CYP2D6-mediated clearance. ...
Article
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The aim of this work was to predict the extent of Cytochrome P450 2D6 (CYP2D6)‐mediated drug–drug interactions (DDIs) in different CYP2D6 genotypes using physiologically‐based pharmacokinetic (PBPK) modeling. Following the development of a new duloxetine model and optimization of a paroxetine model, the effect of genetic polymorphisms on CYP2D6‐mediated intrinsic clearances of dextromethorphan, duloxetine, and paroxetine was estimated from rich pharmacokinetic profiles in activity score (AS)1 and AS2 subjects. We obtained good predictions for the dextromethorphan–duloxetine interaction (Ratio of predicted over observed area under the curve (AUC) ratio (Rpred/obs) 1.38–1.43). Similarly, the effect of genotype was well predicted, with an increase of area under the curve ratio of 28% in AS2 subjects when compared with AS1 (observed, 33%). Despite an approximately twofold underprediction of the dextromethorphan–paroxetine interaction, an Rpred/obs of 0.71 was obtained for the effect of genotype on the area under the curve ratio. Therefore, PBPK modeling can be successfully used to predict gene–drug–drug interactions (GDDIs). Based on these promising results, a workflow is suggested for the generic evaluation of GDDIs and DDIs that can be applied in other situations.
... Two of the more recent cocktails called the Basel and Geneva cocktails, including substrates for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (Geneva cocktail also including fexofenadine as an index for P-gp), have been specifically validated for DDI studies. [33][34][35] One of the biggest challenges in development of drug transporter cocktails is lack of selective index substrates. The first published cocktail approach for transporters included digoxin for P-gp, furosemide for OAT1 and OAT3, metformin for OCT2, MATE1, and MATE2-K, and rosuvastatin for OATP1B1, OATP1B3, and BCRP. ...
... 47 Such single time point metabolic ratios have been tested in index drug cocktails as well. 34,48 In cocktail approaches, however, a single sampling time represents a compromise between the different substrates and might not be optimal for each index substrate. ...
... One approach to limit blood sample volumes and to make blood sampling more feasible is to use capillary blood microsamples and dried blood as the sample matrix. 34 The main advantage of this approach is its low invasiveness and the flexibility it potentially offers, such as applicability in low resource environments or self-sampling by the study participants. However, the measurement of wholeblood concentrations in this matrix needs to be validated against samples collected by venipuncture, and whole blood concentrations of drugs often differ from those in plasma/serum. ...
Article
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Many drug‐drug interactions (DDIs) are based on alterations of the plasma concentrations of a victim drug due to another drug causing inhibition and/or induction of the metabolism or transporter‐mediated disposition of the victim drug. In the worst case, such interactions cause more than tenfold increases or decreases in victim drug exposure, with potentially life‐threatening consequences. There has been tremendous progress in the predictability and modeling of DDIs. Accordingly, the combination of modeling approaches and clinical studies is the current mainstay in evaluation of the pharmacokinetic DDI risks of drugs. In this article, we focus on the methodology of clinical studies on DDIs involving drug metabolism or transport. We specifically present considerations related to general DDI study designs, recommended enzyme and transporter index substrates and inhibitors, pharmacogenetic perspectives, index drug cocktails, endogenous substrates, limited sampling strategies, physiologically‐based pharmacokinetic modeling, complex DDIs, methodological pitfalls, and interpretation of DDI information. This article is protected by copyright. All rights reserved.
... This further verifies the cocktail's ability to predict normal and modified CYP activity accurately. Due to the combination of a straightforward, user-friendly capillary sample device [19], this low-dose, high-throughput concoction can be utilized a a phenotyping instrument in various settings, including hospitals, private clinical practice and research in countries with limited resources. A persistent, low-grade form of inflammation is obesity. ...
... This further verifies the cocktail's ability to predict normal and modified CYP activity accurately. Due to the combination of a straightforward, user-friendly capillary sample device [19], this low-dose, high-throughput concoction can be utilized as a phenotyping instrument in various settings, including hospitals, private clinical practice, and research in countries with limited resources. ...
Article
Full-text available
The inter-individual variability of CYP450s enzyme activity may be reduced by comparing the effects of bariatric surgery on CYP-mediated drug elimination in comparable patients before and after surgery. The current research will use a low-dose phenotyping cocktail to simultaneously evaluate the activities of six CYP isoforms and P-gp. The results showed that following weight reduction after surgery, the activity of all enzymes increased compared to the obese period, which was statistically significant in the case of CYP3A, CYP2B6, CYP2C9, and CYP1A2. Furthermore, the activity of P-gp after surgery decreased without reaching a statistical significance (p-value > 0.05). Obese individuals had decreased CYP3A and CYP2D6 activity compared with the control group, although only CYP3A was statistically important. In addition, there was a trend toward increased activity for CYP1A2, CYP2B6, CYP2C9, and CYP2C19 in obese patients compared to the control group, without reaching statistical insignificance (p-value ≥ 0.05). After six months (at least), all enzymes and the P-gp pump activity were significantly higher than the control group except for CYP2D6. Ultimately, a greater comprehension of phenoconversion can aid in altering the patient’s treatment. Further studies are required to confirm the changes in the metabolic ratios of probes after bariatric surgery to demonstrate the findings’ clinical application. As a result, the effects of inflammation-induced phenoconversion on medication metabolism may differ greatly across persons and drug CYP pathways. It is essential to apply these results to the clinic to recommend dose adjustments.
... The major CYP enzymes assessed in these cocktails included CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4. [52][53][54][55][56][57][58][59][60][61][62][63] Caffeine was selected as the CYP1A2 probe substrate in all these cocktails. Four CYP2C9 probe substrate drugs were used in these cocktails, including flurbiprofen, losartan, tolbutamide, and warfarin. ...
... ,63 In addition, the Pittsburgh T A B L E 3 Clinically validated cocktails involving CYP mediated DDIs. ...
Article
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It is well recognized that therapeutic proteins (TPs) with proinflammatory activities elevate the proinflammatory cytokines and result in cytokine‐drug interactions. In the current review, several proinflammatory cytokines including IL‐2, IL‐6, IFN‐ γ and TNF‐α as well as an anti‐inflammatory cytokine IL‐10 were summarized for their respective effect on major cytochrome P450 enzymes and efflux transporter Pgp. Proinflammatory cytokines are generally associated with suppression of CYP enzymes across assay systems but have varied effect on Pgp expression levels and activities depending on the individual cytokines and assay systems, whereas IL‐10 had no significant impact on CYP enzymes and Pgp. A cocktail DDI study design could be an ideal approach for simultaneously assess the impact of TPs with proinflammatory activities on multiple CYP enzymes. Clinical DDI studies employing cocktail approach have been conducted for several TPs with proinflammatory activities and for those TPs with proinflammatory activities which had no clinical DDI study conducted, languages for potential DDI risk due to cytokine‐drug interaction were included in the label. Up to date drug cocktails including clinically validated and unvalidated for DDI assessment were summarized in this review. Most clinically validated cocktails focused either on CYP enzymes or transporters. Additional effort was needed to validate a cocktail to include both the major CYP enzymes and key transporters. In silico methods for assessment of the DDI for TPs with proinflammatory activities were also discussed.
... Participants were given a validated test [the Geneva phenotyping micro cocktail (Bosilkovska et al., 2016)] to evaluate the first step of monooxygenase metabolism, essentially CYP450 isoform activity, in the morning in a fasting condition. The metabolic ratio (metabolite concentration/substrate concentration) of each CYP-specific probe substrate was measured in plasma 2 h after micro cocktail intake. ...
... The enzymatic activity of cytochromes was evaluated through the metabolic ratio values (metabolite concentration/substrate concentration), their dispersion from the median, and their comparison to Geneva cocktail standard reference values (Bosilkovska et al., 2016). ...
Article
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Context The management of behavioral symptoms and rigidity in patients with dementia constitutes a significant challenge. Short-term studies suggest an interest in the use of medical cannabis, but long-term data are lacking. Objectives The objective of this study was to investigate the feasibility and long-term safety of administering tetrahydrocannabinol/cannabidiol (THC/CBD) treatment as an additional drug to a poly medicated population with severe dementia, evaluate clinical improvements, and collect information on the pharmacokinetics of cannabinoids and possible drug–drug interactions. Methods A prospective observational study of patients with severe dementia living in a long-term care home to whom the physicians had prescribed a medical cannabis treatment. Data were collected over 2 years. We assessed the changes in medical cannabis dosages, safety parameters, variations in neuropsychiatric problems, agitation, rigidity, the most invalidating daily activity, and disabling behavior trouble scores. We evaluated the pharmacokinetics of cannabinoids by measuring plasma levels and analyzing the enzymatic activity. Results We assessed 19 patients (81.4 years—17 women and two men) receiving an average of 12.4 mg THC/24.8 mg CBD per day for up to 13 months, with no reported problems related to the treatment and limited adverse drug reactions. Clinical scores showed a marked improvement that was stable over time, deprescription of other medications, and care facilitated. The pharmacokinetic evaluation showed an expected slight reduction in the enzymatic activity of CYP1A2 and CYP2C19. Conclusion A long-term THC/CBD (1:2) medication can be administered safely and with overall positive clinical improvement to poly medicated older adults with severe dementia and associated problems. The results must be confirmed in a randomized trial.
... The VAMS technology has been studied for the TDM of different classes of drugs such as antidepressants, antiepileptics, antibiotics, and analgesics, exploiting different biological matrices (25)(26)(27)(28). However, microfluidic-generated DBS (mfDBS) technologies have not been widely investigated for TDM purposes (29)(30)(31). ...
... Chromatographic conditions were developed and optimized, starting from those previously used to analyze CLZ and its metabolites by HPLC-ED and modifying mobile phase composition and detector parameters in order to optimize the separation of chromatographic peaks and increase the sensitivity of the method (30). The resulting mobile phase was composed of ACN (15%, V/V), MeOH (20%, V/V), 26.6 mM phosphate buffer, and 3.2 mM KCl, and it contained 0.4% of triethylamine (65%, V/V); the pH was adjusted to 2.50. ...
Article
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Clozapine is one of the most widely used second-generation antipsychotic drugs (SGAs) for the treatment of schizophrenia. Despite advantages over first-generation drugs, clozapine still shows significant side effects and interindividual variations in efficacy. In order to ensure frequent therapeutic drug monitoring (TDM) and improve the compliance of psychiatric patients undergoing clozapine treatment, two novel dried microsampling approaches based on whole blood and plasma volumetric absorptive microsampling (b-VAMS and p-VAMS) and microfluidic generated-dried blood spot technology (mfDBS) were developed and coupled to HPLC with electrochemical detection (ED). The proposed miniaturized strategies by means of VAMS and microfluidic channel-based devices provide several advantages in terms of collection, storage, and handling compared to classical blood and plasma processing. Satisfactory validation results were obtained for all microsampling platforms, with mean extraction yields >85.1%, precision as relative standard deviation (RSD) < 5.1%, and stability < 4.5% analyte loss after 30 days for p-VAMS; mean extraction yields > 83.4%, precision RSD < 5.4%, and stability < 4.6% analyte loss after 30 days for b-VAMS, and mean extraction yields > 74.0%, precision RSD < 5.6%, and stability < 4.9% analyte loss after 30 days for mfDBS. The original microsampling methodologies have been successfully applied to the blood and plasma collected from five psychiatric patients for the monitoring of the levels of clozapine and its main metabolites, providing robust and reliable quali-quantitative results. Comparisons between results of the two dried microsampling technologies with those obtained by classic fluid plasma analysis were in good agreement and have demonstrated that the proposed miniaturized approaches could be suitable for TDM purposes.
... Many phenotyping cocktails have been developed and used in recent years. [20][21][22][23][24][25] For this study, the compilation of the probe drugs was selected according to a validated phenotyping cocktail (Geneva cocktail 26,27 ), which also included a probe drug for P-gp (fexofenadine). To our knowledge, this is the first seven-probe drug cocktail interaction study investigating St. John's wort. ...
... The intra-subject coefficients of variation (CVs) for the AUCs of the administered cocktail probe drugs vary between 8% and 30% as reported by Bosilkovska. 27 A sample size of 16 participants allowed a rejection of each null hypothesis "interaction present induction/ inhibition" with α = 0.05 (one-sided) and a power of at least 99% for a tolerance zone of 0.50-2.0 assuming as a conservative approach that the intra-individual CVs exceed 30% and the true ratio of μ test / μ reference = 1.0. ...
Article
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Hypericum perforatum L. (St. John's wort) is used to treat mild‐to‐moderate depression. Its potential safety risks are pharmacokinetic drug interactions via cytochrome P450 enzymes and P‐glycoprotein, presumably caused by hyperforin. In a phase I, open‐label, non‐randomized, single‐sequence study, the low‐hyperforin Hypericum extract Ze 117 was investigated using a drugs cocktail in 20 healthy volunteers. No pharmacokinetic interactions of Ze 117 were observed for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP3A4 and P‐glycoprotein. AUC and Cmax of the used probe drugs showed 90%‐confidence intervals of the geometric mean ratios of the drugs taken together with Ze 117 vs. probe drug alone well within the predefined bioequivalence range of 80% to 125%. Though Ze 117 did not induce dextromethorphan metabolism by CYP2D6, it weakly increased dextromethorphan AUC ratio (mean 147.99, 95% CI 126.32‐173.39) but not the corresponding metabolic ratio. Ze 117 does not show clinically relevant pharmacokinetic interactions with important CYPs and P‐glycoprotein. This article is protected by copyright. All rights reserved.
... One low-hyperforin SJW extract approved in various countries for the short-term treatment of depression is Ze117. This formulation with the brand name Rebalance ® exhibits pharmacological efficacy (Friede et al., 2001;Schrader, 2000), but no relevant impact on the pharmacokinetics of substrates of drug metabolizing enzymes and P-gp (Bosilkovska et al., 2016;Zahner et al., 2019). Considering the availability of the SJW formulations of differing hyperforin content, we sought to directly compare their in vitro and in vivo effect on the PXR-target gene CYP3A23-3A1 in rats. ...
Article
The pregnane X receptor (PXR) is a ligand activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant® was used as it contains a high hyperforin content and Rebalance® because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant® and Rebalance® transactivated the CYP3A-reporter 3.8 fold and 2.8 fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared to control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant® we observed 1.8-times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount and a 1.6-fold increase in activity compared to control. For Rebalance® we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared to control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. Significance Statement Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the PXR-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro Importantly, CYP3A induction mimics findings in humans but our results suggest that another so far unknown constituent of SJW is responsible for the expression and function modifying effects in rat liver.
... This further verifies the cocktail's ability to predict normal and modified CYP activity accurately. Due to the combination of a straightforward, user-friendly capillary sample device [19], this low-dose, high-throughput concoction can be utilized as a phenotyping instrument in various settings, including hospitals, private clinical practice, and research in countries with limited resources. ...
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The inter-individual variability of CYP450s enzyme activity may be reduced by comparing the effects of bariatric surgery on CYP-mediated drug elimination in comparable patients before and after surgery. The current research will use a low-dose phenotyping cocktail to simultaneously evaluate the activities of six CYP isoforms and P-gp.The results showed that following weight reduction after surgery, the activity of all enzymes increased compared to the obese period, which was statistically significant in the case of CYP3A, CYP2B6, CYP2C9, and CYP1A2. Furthermore, the activity of P-gp after surgery decreased without reaching a statistical significance (p-value>0.05). Obese individuals had decreased CYP3A and CYP2D6 activity than the control group, albeit only CYP3A was statistically important. In addition, there was a trend to increase activity for CYP1A2, CYP2B6, CYP2C9, and CYP2C19 in obese patients compared to the control group, without reaching statistical insignificance (p-value≥0.05). After six months (at least), all enzymes and the P-gp pump activity were significantly higher than the control group except for CYP2D6.Ultimately, a greater comprehension of phenoconversion can aid in altering the patient's treatment. Further studies are required to confirm the changes in the metabolic ratios of probes after bariatric surgery to demonstrate the findings' clinical application. As a result, the effects of inflammation-induced phenoconversion on medication metabolism may differ greatly across persons and drug CYP pathways. It is essential to apply these results to the clinic to recommend dose adjustments.
... Given that givosiran introduces a second block in the pathway, it cannot be excluded that a further reduction in heme availability could hamper the activation of multiple biological processes that take place in the liver in situations of stress (antioxidant response, inflammation, hypoxia), important detoxification processes, or adequate energy supply for natural hepatocyte proliferation or liver regeneration. Therefore, reduced availability of heme in the liver could be associated with the reported AEs associated with ALAS-1 iRNA, such as bile acid disorders [50], reduced drug metabolization rates [51,52], or dysregulation of one-carbon metabolism [53,54]. Concomitant hypermethioninemia and hyperhomocysteinemia resembling classic homocystinuria have been associated with givosiran treatment. ...
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Rare diseases, especially monogenic diseases, which usually affect a single target protein, have attracted growing interest in drug research by encouraging pharmaceutical companies to design and develop therapeutic products to be tested in the clinical arena. Acute intermittent porphyria (AIP) is one of these rare diseases. AIP is characterized by haploinsufficiency in the third enzyme of the heme biosynthesis pathway. Identification of the liver as the target organ and a detailed molecular characterization have enabled the development and approval of several therapies to manage this disease, such as glucose infusions, heme replenishment, and, more recently, an siRNA strategy that aims to down-regulate the key limiting enzyme of heme synthesis. Given the involvement of hepatic hemoproteins in essential metabolic functions, important questions regarding energy supply, antioxidant and detoxifying responses, and glucose homeostasis remain to be elucidated. This review reports recent insights into the pathogenesis of acute attacks and provides an update on emerging treatments aimed at increasing the activity of the deficient enzyme in the liver and restoring the physiological regulation of the pathway. While further studies are needed to optimize gene therapy vectors or large-scale production of liver-targeted PBGD proteins, effective protection of PBGD mRNA against the acute attacks has already been successfully confirmed in mice and large animals, and mRNA transfer technology is being tested in several clinical trials for metabolic diseases.
... Phenotyping procedures took place in a psychiatric hospitalization unit or day hospital. CYP/P-gp activities were assessed using a cocktail approach previously described by Bosilkovska et al. [27,28] and called the "Geneva Cocktail". The cocktail approach consists of administering several probe drugs, each one being metabolized by a specific CYP or substrate of the P-gp. ...
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Altered cytochromes P450 enzymes (CYP) and P-glycoprotein transporter (P-gp) activity may explain variabilities in drug response. In this study, we analyzed four years of phenotypic assessments of CYP/P-gp activities to optimize pharmacotherapy in psychiatry. A low-dose probe cocktail was administered to evaluate CYP1A2, 2B6, 2D6, 2C9, 2C19, 3A4, and P-gp activities using the probe/metabolite concentration ratio in blood or the AUC. A therapeutic adjustment was suggested depending on the phenotyping results. From January 2017 to June 2021, we performed 32 phenotypings, 10 for adverse drug reaction, 6 for non-response, and 16 for both reasons. Depending on the CYP/P-gp evaluated, only 23% to 56% of patients had normal activity. Activity was decreased in up to 57% and increased in up to 60% of cases, depending on the CYP/P-gp evaluated. In 11/32 cases (34%), the therapeutic problem was attributable to the patient’s metabolic profile. In 10/32 cases (31%), phenotyping excluded the metabolic profile as the cause of the therapeutic problem. For all ten individuals for which we had follow-up information, phenotyping allowed us to clearly state or clearly exclude the metabolic profile as a possible cause of therapeutic failure. Among them, seven showed a clinical improvement after dosage adaptation, or drug or pharmacological class switching. Our study confirmed the interest of CYP and P-gp phenotyping for therapeutic optimization in psychiatry.
... The adverse events associated with givosiran were elevation of serum aminotransferases levels and changes in serum creatinine and estimated Glomerular Filtration Rate, suggesting kidney function issues (Balwani et al., 2020). Downregulation of ALAS1 combined with defective heme synthesis in severely affected patients could lower hepatic heme content and reduce the activity of heme-dependent hepatic proteins causing reduced drug metabolization rates (Bosilkovska et al., 2016) and aggravation of the dysregulation of one-carbon metabolism, resembling classic homocystinuria (Fontanellas et al., 2021;To-Figueras et al., 2021). For severe affected patients that do not respond to current treatments, liver transplantation is the only therapeutic option. ...
Chapter
Inborn errors of metabolism (IEM) encompass a group of monogenic diseases affecting both pediatric and adult populations and currently lack effective treatments. Some IEM such as familial hypercholesterolemia or X-linked protoporphyria are caused by gain of function mutations, while others are characterized by an impaired protein function, causing a metabolic pathway blockage. Pathophysiology classification includes intoxication, storage and energy-related metabolic disorders. Factors specific to each disease trigger acute metabolic decompensations. IEM require prompt and effective care, since therapeutic delay has been associated with the development of fatal events including severe metabolic acidosis, hyperammonemia, cerebral edema, and death. Rapid expression of therapeutic proteins can be achieved hours after the administration of messenger RNAs (mRNA), representing an etiological solution for acute decompensations. mRNA-based therapy relies on modified RNAs with enhanced stability and translatability into therapeutic proteins. The proteins produced in the ribosomes can be targeted to specific intracellular compartments, the cell membrane, or be secreted. Non-immunogenic lipid nanoparticle formulations have been optimized to prevent RNA degradation and to allow safe repetitive administrations depending on the disease physiopathology and clinical status of the patients, thus, mRNA could be also an effective chronic treatment for IEM. Given that the liver plays a key role in most of metabolic pathways or can be used as bioreactor for excretable proteins, this review focuses on the preclinical and clinical evidence that supports the implementation of mRNA technology as a promising personalized strategy for liver metabolic disorders such as acute intermittent porphyria, ornithine transcarbamylase deficiency or glycogen storage disease.
... A new generation of microsampling devices has been proposed to extend the volumetric DBS application to untrained people and make self-sampling at home possible. HemaSpot TM (Spot on Science, San Francisco, CA) [35], volumetric absorptive microsampling (VAMS) (Neoteryx, USA [23,[36][37][38], volumetric absorptive paper disc (VAPD) [39], and microfluidic-based systems, as HemaXis DB (DBS System SA, Gland, Switzerland) [40,41] and Capitainer-B (Capitainer AB, Stockholm, Sweden) [26,[42][43][44], are just a few illustrative user-friendly devices on the market [45]. Even when the analysis of the whole spot is performed, the influence of HCT variability on the extraction efficiency should be considered [28]. ...
Article
Dried Blood Spot (DBS) analysis is a well-established practice for monitoring newborns in neonatal units because of the non-invasive collection of a very limited blood volume. Unfortunately, the haematocrit level and the drop volume can adversely affect the quality of the analytical data. For this reason, we performed an in-depth comparison of non-volumetric and volumetric sampling (e.g. Minivette® POCT microcapillary, and the microfluidic-based systems HemaXisTM DB and Capitainer-B) for the DBS analysis of the isoprostanoids 8-iso prostaglandin F2α and 8-iso prostaglandin E2, and prostaglandin E2, which represent well recognized indexes of oxidative stress and inflammation. Results highlighted a significant impact of drop volume on the analytical performances for the non-volumetric approach due to the inhomogeneous distribution of the analytes across the DBS. The innovative addition of labelled internal standards (ISs) on the filter paper before sampling did not circumvent the problem, as ISs did not accurately mimic the spreading of analytes in the DBS. Differently from HemaXisTM DB and Capitainer-B, the Minivette® POCT device showed an almost quantitative recovery with an overall variability of about 15%, and resulted the best option for an easy and reliable DBS collection in the clinic. Our procedure was used for the determination of the three metabolites in preterm newborns suffering from Patent Ductus Arteriosus (PDA). During our longitudinal monitoring, most of the preterm newborns with PDA showed a marked decrease (40-60%) of target analytes upon ibuprofen therapy, suggesting the potential involvement of the isoprostanoids and prostanoids in promoting drug responsiveness and ductal closure.
... Dried blood spots were then stored at -20°C in a sealable plastic bag until analysis, as previously described 33 . No mutual drug-drug interactions were observed in the Geneva cocktail 34 . CYP2D6 was not modulated by bupropion because of the extreme low doses and time intervals used 34 . ...
Article
Full-text available
Coronavirus Disease 2019 (COVID‐19), caused by SARS‐CoV‐2 infection, is a severe acute respiratory syndrome with an underlying inflammatory state. We have previously demonstrated that acute inflammation modulates cytochromes P450 (CYP) activities in an isoform‐specific manner. We therefore hypothesized that COVID‐19 might also impact CYP activities, and thus aimed to evaluate the impact of acute inflammation in the context of SARS‐CoV‐2 infection on the six main human CYPs activity. This prospective observational study was conducted in 28 patients hospitalized at the Geneva University Hospitals (Switzerland) with a diagnosis of moderate to severe COVID‐19. They received the Geneva phenotyping cocktail orally during the first 72h of hospitalization and after three months. Capillary blood samples were collected 2h after cocktail administration to assess the metabolic ratios (MRs) of CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A. CRP, IL‐6 and TNF‐α levels were also measured in blood. CYP1A2, CYP2C19, and CYP3A MRs decreased by 52.6% (p=0.0001), 74.7% (p=0.0006) and 22.8% (p=0.045), respectively, in COVID‐19 patients. CYP2B6 and CYP2C9 MRs increased by 101.1% (p=0.009) and 55.8% (p=0.0006) respectively. CYP2D6 MRs variation did not reach statistical significance (p=0.072). As expected, COVID‐19 was a good acute inflammation model as mean serum levels of CRP, IL‐6 and TNF‐α were significantly (p<0.001) higher during SARS‐CoV‐2 infection. CYP activities are modulated in an isoform‐specific manner by SARS‐CoV‐2 infection. The pharmacokinetics of CYP substrates, whether used to treat the disease or as the usual treatment of patients, could be therefore clinically impacted.
... The selection of the probe drugs and doses used in the phenotyping cocktail was based on previous DDI studies conducted in healthy adult volunteers. [26][27][28][29][30][31][32][33][34][35][36][37] Napabucasin 240 or 480 mg was administered orally twice daily (every 12 hours); healthy volunteers received the morning dose following an overnight fast, remained fasted for 4 hours after dosing, and fasted 1 to 2 hours before administration of the evening dose. Potential DDIs were assessed on the basis of the napabucasin 240-mg dose only; DDIs were not assessed for the 480-mg dose because healthy volunteers did not receive the 480-mg twice-daily regimen in conjunction with the probe drugs (due to a protocol amendment on October 12, 2017, that adjusted the dose to 240 mg). ...
Article
Full-text available
Napabucasin is an orally administered reactive oxygen species generator that is bioactivated by the intracellular antioxidant nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1. Napabucasin induces cell death in cancer cells, including cancer stem cells. This phase 1 study (NCT03411122) evaluated napabucasin drug‐drug interaction potential for 7 cytochrome P450 (CYP) enzymes and the breast cancer resistance protein transporter/organic anion transporter 3. Healthy volunteers who tolerated napabucasin during period 1 received probe drugs during period 2, and in period 3 received napabucasin (240 mg twice daily; days 1‐11) plus a phenotyping cocktail containing omeprazole (CYP2C19), caffeine (CYP1A2), flurbiprofen (CYP2C9), bupropion (CYP2B6), dextromethorphan (CYP2D6), midazolam (CYP3A) (all oral; day 6), intravenous midazolam (day 7), repaglinide (CYP2C8; day 8), and rosuvastatin (breast cancer resistance protein/organic anion transporter 3; day 9). Drug‐drug interaction potential was evaluated in 17 of 30 enrolled volunteers. Napabucasin coadministration increased the area under the plasma concentration–time curve from time 0 extrapolated to infinity (geometric mean ratio [90% confidence interval]) of caffeine (124% [109.0%‐141.4%]), intravenous midazolam (118% [94.4%‐147.3%]), repaglinide (127% [104.7%‐153.3%]), and rosuvastatin (213% [42.5%‐1068.3%]) and decreased the area under the plasma concentration–time curve from time 0 extrapolated to infinity of dextromethorphan (71% [47.1%‐108.3%]), bupropion (79% [64.6%‐97.0%]), and hydroxybupropion (45% [15.7%‐129.6%]). No serious adverse events/deaths were reported. Generally, napabucasin is not expected to induce/inhibit drug clearance to a clinically meaningful degree.
... After filling the capillary channel, the device was manually closed, allowing the outlet of the capillary to be in contact with the card, resulting in the transfer of a defined volume of blood (10 µL) to the DBS card, which takes about 5 s. [68][69][70] After completion of the transfer, the capillary channel should be completely empty, which can their own blood. The kit was classified as "very easy "or "easy" to use by 83.6% of participants who mailed the kit to the lab. ...
Article
Dried blood spots (DBS) have been utilized in newborn screening programs for several years. More recently, there has been growing interest in using DBS as a home sampling tool for the quantitative determination of analytes. However, this presents challenges, mainly due to the well-known hematocrit effect and other DBS-specific parameters, including spotted volume and punch site, which could add to the method uncertainty. Therefore, new microsampling devices that quantitatively collect capillary dried blood are continuously being developed.In this review, we provided an overview of devices that are commercially available or under development that allow the quantitative (volumetric) collection of dried blood(-based) microsamples, and are meant to be used for home or remote sampling. Considering the field of therapeutic drug monitoring (TDM), we examined different aspects that are important for a device to be implemented in clinical practice, including ease of patient use, technical performance, and ease of integration in the workflow of a clinical laboratory. Costs related to microsampling devices are briefly discussed, as this additionally plays an important role in the decision-making process.Although the added-value of home sampling for TDM and the willingness of patients to perform home sampling has been demonstrated in some studies, real clinical implementation is progressing at a slower pace. More extensive evaluation of these newly developed devices, not only analytically but also clinically, is needed to demonstrate their real-life applicability, which is a prerequisite for their use in the field of TDM.
... The absence of mutual drug-drug interactions within the Geneva cocktail was previously demonstrated and bupropion is used at such a low dose that no effect on CYP2D6 activity is demonstrated. 19 The cocktail was also previously validated using dried blood spots as a sampling method. 20 Capillary blood samples were collected 2 hours after drug administration in a fasting patient and dried blood spots were stored at -20°C in a sealable plastic bag until analysis, as previously described. ...
Article
Full-text available
Cytochromes P450 (CYP) are subject to important interindividual variability in their activity due to genetic and environmental factors and some diseases. Limited human data support the idea that inflammation down‐regulates CYP activities. Our study aimed to evaluate the impact of orthopedic surgery (acute inflammation model) on the activity of six human CYP. This prospective observational study was conducted in thirty patients who underwent elective hip surgery at the Geneva University Hospitals in Switzerland. The Geneva phenotyping cocktail containing caffeine, bupropion, flurbiprofen, omeprazole, dextromethorphan and midazolam as probe drugs respectively assessing CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A activities was administered orally before surgery, day 1 (D1) and 3 (D3) post‐surgery and at discharge. Capillary blood samples were collected 2h after cocktail intake to assess metabolic ratios (MR). Serum inflammatory markers (CRP, IL‐6, IL‐1β, TNF‐α, IFN‐γ) were also measured in blood. CYP1A2 MR decreased by 53% (p<0.0001) between baseline and the nadir at D1. CYP2C19 and CYP3A activities (MRs) decreased by 57% (p=0.0002) and 61% (p<0.0001) respectively, with the nadir at D3. CYP2B6 and CYP2C9 MRs increased by 120% (p<0.0001) and 79% (p=0.018) respectively and peaked at D1. Surgery did not have a significant impact on CYP2D6 MR. Hip surgery was a good acute inflammation model as CRP, IL‐6 and TNF‐α peak levels were reached between D1 and day 2 (D2). Acute inflammation modulated CYP activity in an isoform‐specific manner, with different magnitudes and kinetics. Acute inflammation may thus have a clinically relevant impact on the pharmacokinetics of these CYP substrates.
... The clinical approach to estimation of metabolic capacity, i.e. injection of innocuous probes and 84 monitoring their pharmacokinetic curves [14], is impossible to apply with postmortem cases. 85 ...
Preprint
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Metabolic capacity, an estimation of the rate at which xenobiotics are transformed by drug metabolizing enzymes in the human body, can be a relevant piece of information for forensic toxicologists, allowing for example differentiation between a medical error and an accidental intoxication. Metabolic capacity is typically estimated postmortem via enzyme genotyping. However, this classification of individuals according to their metabolizer type suffers from poor correlation with the actual metabolic capacity due to several factors affecting enzyme expression. As an alternative to the genomics based estimation, we describe a proteomics approach to characterization and quantification of cytochrome P450 (CYP) enzymes which has the potential to yield a more accurate estimation of metabolic capacity. CYP-rich liver microsomes were isolated by mechanical homogenization followed by ultracentrifugation. The microsomal pellet was resuspended, reduced, alkylated and digested with trypsin. The resulting tryptic digest was purified by solid phase extraction, evaporated and reconstituted for analysis on a liquid chromatography system coupled to tandem mass spectrometry (LC-MS/MS). Method parameters were optimized by design of experiments to maximize sensitivity. Prevalent and deleterious CYP 2D6 and CYP 3A4 polymorphisms were monitored via analysis of the peptides expressing the genetic mutations. Simultaneously, the enzyme expression level in the tissue was estimated by quantification of selected CYP 2D6 and CYP 3A4 peptides. Combination of the activity and expression components should allow for a more accurate estimation of metabolic capacity. While application of this method on polymorphic human liver microsome samples show results matching the expected pattern, sample complexity remains a barrier to full validation and wide application in the forensic toxicology field.
... First of all, a perpetrator drug can affect transporter-mediated uptake and/or efflux of a victim drug across cell membranes of tissues, as well as its excretion from the tissue (e.g., to the blood or outside of the body). Also, a perpetrator drug is able to induce or inhibit metabolic enzymes and consequently change the enzyme-mediated elimination of a victim drug [25,[35][36][37]. A transporter-mediated metabolite and/or parent form of a victim drug excretion can be altered by a perpetrator drug as well in DDIs. ...
Article
Full-text available
Systemic exposure of a drug is generally associated with its pharmacodynamic (PD) effect (e.g., efficacy and toxicity). In this regard, the change in area under the plasma concentration-time curve (AUC) of a drug, representing its systemic exposure, has been mainly considered in evaluation of drug-drug interactions (DDIs). Besides the systemic exposure, the drug concentration in the tissues has emerged as a factor to alter the PD effects. In this review, the status of systemic exposure, and/or tissue exposure changes in DDIs, were discussed based on the recent reports dealing with transporters and/or metabolic enzymes mediating DDIs. Particularly, the tissue concentration in the intestine, liver and kidney were referred to as important factors of PK-based DDIs.
... These studies are performed even with two times lower than the lowest commercialized doses of probes in the administered cocktail, which minimizes the probability of interactions. The further benefits of the cocktail demonstrates using limited blood sampling as phenotyping metrics instead of some blood and urine collections [15,24]. ...
Article
Full-text available
Cytochrome P450s (CYP450) family is one of the most critical factors in the metabolism process. Hence, the present study aims to characterize the activity of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, and P-glycoprotein (P-gp) pump in patients with type 2 diabetes (T2DM). This characterization was performed before and after good glycemic control versus non-diabetic subjects following the administration of a substrate probe drug cocktail. This single-center clinical study proposes the characterization of T2DM impacts on major CYP450 drug-metabolizing enzyme and P-glycoprotein (P-gp) activities. The main propose of the present study is evaluating any alternation in major CYP450 enzymes and P- gp activities in patients with T2DM, before (A1C>7%) and after (A1C≤7%) good glycemic control along with comparing the activities versus non-diabetic subjects. The phenotypes will be assessed following the oral administration of a drug cocktail containing caffeine (CYP1A2), bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4/5), and fexofenadine (P-gp) as probe substrates. Besides, the effect of some variables such as blood glucose levels and inflammation on the metabolism process will be examined. Furthermore, the influence of variables such as glycemia, genetic polymorphisms, and inflammation will be evaluated. The first patient has entered the study in Dec 2018.
... None of the subjects reported any side effects after single drug or cocktail administration. Detailed results of drug-drug interactions between the CYP probe substrates contained in the cocktail are described elsewhere [14]. An unexpected interaction occurred between fexofenadine and CYP probe drugs. ...
Article
Full-text available
Drug metabolic enzymes and transporters are responsible for an important variability in drug disposition. The cocktail approach is a sound strategy for the simultaneous evaluation of several enzyme and transporter activities for a personalized dosage of medications. Recently, we have demonstrated the reliability of the Geneva cocktail, combining the use of dried blood spots (DBS) and reduced dose of phenotyping drugs for the evaluation of the activity of six cytochromes and P-glycoprotein (P-gp). As part of a study evaluating potential drug–drug interactions between probe drugs of the Geneva cocktail, the present paper focuses on the impact of cytochromes (CYP) probe drugs on the disposition of fexofenadine, a P-gp test drug. In a randomized four-way Latin-square crossover study, 30 healthy volunteers (15 men and 15 women) received caffeine 50 mg, bupropion 20 mg, flurbiprofen 10 mg, omeprazole 10 mg, dextromethorphan 10 mg, midazolam 1 mg, and fexofenadine 25 mg alone (or as part of a previously validated combination) and all together (Geneva cocktail). The determination of drug concentrations was performed in DBS samples and pharmacokinetic parameters were calculated. Fexofenadine AUC0–8 h and Cmax decreased by 43% (geometric mean ratio: 0.57; CI 90: 0.50–0.65; p < 0.001) and 49% (geometric mean ratio: 0.51; CI 90: 0.44–0.59; p < 0.001), respectively, when fexofenadine was administered as part of the Geneva cocktail in comparison to fexofenadine alone. Consequently, the apparent oral clearance (Cl/F) increased 1.7-fold (CI 90: 1.49–1.93; p < 0.001). There was no interaction between the remaining probes. In conclusion, an unexpected interaction occurred between fexofenadine and one or several of the following substances: caffeine, bupropion, flurbiprofen, omeprazole, dextromethorphan, and midazolam. Further studies are necessary to elucidate the mechanism of this interaction.
... In clinical practice, phenotyping can be used primarily for two purposes: prospectively to identify patients at risk of such events, or retrospectively to explain adverse effects or therapeutic failure [13]. For instance, the Geneva Cocktail, which includes 100 mg of caffeine (CYP1A2), 25 mg of bupropion (CYP2B6), 25 mg of flurbiprofen (CYP2C9), 5 mg of omeprazole (CYP2C19), 10 mg of dextromethorphan (DEM) (CYP2D6) and 1 mg of midazolam (MDZ) (CYP3A), was used to explain severe neuropathic pain developed in a 21-year-old patient during vincristine therapy [14,15]. Indeed, the reduction in CYP3A activity observed in the phenotyping test suggests a decrease in the metabolic capacity of vincristine. ...
Article
Full-text available
Drug response is subject to an important within- and between-individual variability owing, mainly, to pharmacokinetic and pharmacodynamic factors. Pharmacokinetics includes metabolism by cytochrome P450 (CYP450), major enzymes of phase I reactions that are responsible for the biotransformation of around 60% of the currently approved drugs. CYP450 activity and/or expression are influenced by multiple intrinsic and extrinsic factors, such as drug–drug interactions or genetic polymorphisms. Present phenotyping strategies with xenobiotics used to assess CYP450 activity could be replaced by less invasive procedures using endogenous CYP450 biomarkers. In this work, we review existing knowledge on endobiotics and their ability to characterise variability of the CYP1A2, CYP2C19, CYP2D6 and CYP3A enzymes in humans. To date, it appears that there is a lack of clinical data for the majority of the endogenous compounds described in the literature or some important limitations to allow their use in clinical practice. Additional studies are needed to fill the gap or to identify new candidates, in particular through the use of metabolomics. The use of multivariate models is also a very promising approach to enhance prediction by combining several endogenous phenotyping metrics and other covariates.
... In addition, in some situations, especially concerning problematic and vulnerable patient population, only limited volumes of blood are available [19]. To bypass those issues, the use of cellulose paper cards has been mentioned [20]. The first use of dried blood spot (DBS) has been reported by Guthrie and Susie more than 50 years ago, for paediatric purpose [21]. ...
Article
Background: Hyphenation of liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) offers the potential to develop broad-spectrum screening procedures from low volumes of biological matrices. In parallel, dried blood spot (DBS) has become a valuable tool in the bioanalysis landscape to overcome conventional blood collection issues. Herein, we demonstrated the applicability of DBS as micro-sampling procedure for broad-spectrum toxicological screening. Methods: A method was developed on a HRMS system in data dependant acquisition (DDA) mode using an extensive inclusion list to promote collection of relevant data. 104 real toxicology cases were analysed, and the results were cross-validated with one published and one commercial screening procedures. Quantitative MRM analyses were also performed on identified substances on a triple quadrupole instrument as a complementary confirmation procedure. Results: The method showed limits of identification (LOIs) in appropriateness with therapeutic ranges for all the classes of interest. Applying the three screening approaches on 104 real cases, 271 identifications were performed including 14 and 6 classes of prescribed and illicit drugs, respectively. Among the detected substances, 23% were only detected by the proposed method. Based on confirmatory analyses, we demonstrated that the use of blood micro-samples did not impair the sensitivity allowing more identifications in the low concentration ranges. Conclusion: A LC-HRMS assay was successfully developed for toxicological screening of blood microsamples demonstrating a high identification power at low concentration ranges. The validation procedure and the analysis of real cases demonstrated the potential of this assay by supplementing screening approaches of reference.
... To our knowledge, the present study was the first to apply the HemaXis ™ DBS sampling device for TDM purposes in a clinical setting. The device has previously been applied for cytochrome P450 phenotyping in healthy volunteers [51] and is currently being used in a venlafaxine trial [52]. Additionally, this is one of the first studies to validate clinically a multi-analyte DBS immunosuppressant assay by direct comparison of DBS and WB tacrolimus samples [17,[20][21][22][23][24], and the third for mycophenolic acid [21,53]. ...
Article
Aims: Tacrolimus and mycophenolic acid dosing after renal transplantation is individualized through therapeutic drug monitoring (TDM). Home-based dried blood spot (DBS) sampling has potential to replace conventional TDM sampling at the clinic. A liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed to quantify tacrolimus and mycophenolic acid in DBS and clinically validated for abbreviated area under the concentration-time curve (AUC) monitoring using an innovative volumetric DBS sampling device. Methods: Clinical validation was performed by direct comparison of paired DBS and whole blood (WB) (tacrolimus) and plasma (mycophenolic acid) concentrations and AUCs. Agreement was evaluated with Passing-Bablok regression, Bland-Altman analysis and DBS-to-WB predictive performance. TDM dosing recommendations based on both methods were compared to assess clinical impact. Results: Paired tacrolimus (n=200) and mycophenolic acid (n=192) DBS and WB samples were collected from 65 kidney (pancreas) transplant recipients. Differences for tacrolimus and mycophenolic acid were within ±20% for 84.5% and 76.6% of concentrations and 90.5% and 90.7% of AUCs. Tacrolimus and mycophenolic acid dosing recommendation differences occurred on 44.4% and 4.7% of occasions. Tacrolimus DBS dosing recommendations were 0.35±0.14 mg higher than for WB and 8±3% of the initial dose. Mycophenolic acid DBS dosing recommendations were 23.3±31.9 mg lower than for plasma and 2±3.5% of the initial dose. Conclusions: Tacrolimus and mycophenolic acid TDM for outpatient renal transplant recipients, based on abbreviated AUC collected with a DBS sampling device, is comparable to conventional TDM based on WB sampling. Patient training and guidance on good blood-spotting practices is essential to ensure method feasibility.
... To this end, individuals need to remain in an institution for at least several hours, which is often not feasible for patients or in epidemiological studies. Therefore, limited sampling strategies (LSSs) have been tested for many probe drugs (1,21,(107)(108)(109)(110), where a few blood samples or even a single sample is taken, and an estimate of an appropriate metric describing the PKP is derived based on drug concentrations or metabolic metabolite/parent ratios. However, the current DDI guidelines ask for coverage of the complete concentration profile for a reason: Otherwise, effects on different PKPs may no longer be discernible. ...
Article
Pharmacokinetic parameters of selective probe substrates are used to quantify the activity of an individual pharmacokinetic process (PKP) and the effect of perpetrator drugs thereon in clinical drug-drug interaction (DDI) studies. For instance, oral caffeine is used to quantify hepatic CYP1A2 activity, and oral dagibatran etexilate for intestinal P-glycoprotein (P-gp) activity. However, no probe substrate depends exclusively on the PKP it is meant to quantify. Lack of selectivity for a given enzyme/transporter and expression of the respective enzyme/transporter at several sites in the human body are the main challenges. Thus, a detailed understanding of the role of individual PKPs for the pharmacokinetics of any probe substrate is essential to allocate the effect of a perpetrator drug to a specific PKP; this is a prerequisite for reliably informed pharmacokinetic models that will allow for the quantitative prediction of perpetrator effects on therapeutic drugs, also in respective patient populations not included in DDI studies. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 59 is January 6, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The use of cocktail of drug substrates to confirm the drug-drug interaction potential specific to various cytochrome (CYP) P450 enzymes in human subjects is now increasingly practised during clinical Phase 1 development. Table 1 summarizes the various options that are currently used for cocktail of drug substrates that target specific CYP P450 enzymes [1][2][3]. One of the advantages of such a practise is that the risk of relevant clinical drug-drug interaction can be potentially identified early in development. ...
Article
This communication attempts to provide introspection and perspectives on a recently reported work that has suggested that a short term fasting may have the potential to impact the pharmacokinetics of certain drug substrates which are metabolized by cytochrome P450 (CYP) enzymes. In particular, drug substrates that undergo metabolism via CYP1A2, CYP2C9 and CYP2D6 enzymes appear to show altered pharmacokinetics. Based on the assessment, although the magnitude of change in exposure values due to short term fasting was not alarming, it may be prudent to interpret the obtained pharmacokinetic data with caution when a longer duration fasting is provided in the clinical study design.
... Dextromethorphan is one of the gold standard probe drug for CYP2D6 and low dosing may be used to minimize the potential therapeutic effects of the probes and drug-drug interactions. In adults, a 10 mg dextromethorphan dose (i.e., at least two times lower than the standard therapeutic dose) is used (Bosilkovska et al., 2014(Bosilkovska et al., , 2016. In children a dose of 0.15 mg/kg (i.e., two to four times lower than the standard therapeutic dose) has been successfully evaluated in our laboratory. ...
Article
Full-text available
Children represent a vulnerable population in which management of nociceptive pain is complex. Drug responses in children differ from adults due to age-related differences. Moreover, therapeutic choices are limited by the lack of indication for a number of analgesic drugs due to the challenge of conducting clinical trials in children. Furthermore the assessment of efficacy as well as tolerance may be complicated by children’s inability to communicate properly. According to the World Health Organization, weak opioids such as tramadol and codeine, may be used in addition to paracetamol and ibuprofen for moderate nociceptive pain in both children and adults. However, codeine prescription has been restricted for the last 5 years in children because of the risk of fatal overdoses linked to the variable activity of cytochrome P450 (CYP) 2D6 which bioactivates codeine. Even though tramadol has been considered a safe alternative to codeine, it is well established that tramadol pharmacodynamic opioid effects, efficacy and safety, are also largely influenced by CYP2D6 activity. For this reason, the US Food and Drug Administration recently released a boxed warning regarding the use of tramadol in children. To provide safe and effective tramadol prescription in children, a personalized approach, with dose adaptation according to CYP2D6 activity, would certainly be the safest method. We therefore recommend this approach in children requiring chronic or recurrent nociceptive pain treatment with tramadol. In case of acute inpatients nociceptive pain management, prescribing tramadol at the minimal effective dose, in a child appropriate dosage form and after clear instructions are given to the parents, remains reasonable based on current data. In all other situations, morphine should be preferred for moderate to severe nociceptive pain conditions.
... Fig. 4 displays the concentration ratios of the 6 probe drugs and corresponding metabolites obtained with the CombiCap and the marketed products (A). The data for marketed products, except from the FLU data that were published from the Geneva Cocktail by Bosilkovska et al. (2016), were obtained from the clinical trial (n = 16) described by Derungs et al. (2015). The comparison was done for specific time points defined in the article that are 2 h for MID, 4 h for CAF and OME and 6 h for EFA and MET. ...
Article
Phenotyping of cytochrome P450 isoenzymes is used for metabolic profiling. Phenotyping cocktails are usually administered as individual marketed products, which are not designed for diagnostic applications. Therefore, a formulation strategy was developed, which can be applied to any phenotyping cocktail. The formulation was validated in vitro and in vivo in human volunteers using caffeine, efavirenz, flurbiprofen, metoprolol, midazolam, and omeprazole (Basel Cocktail). Spray dried di-calcium phosphate particles (Fujicalin) were used as an inert drug carrier for probe drugs. All drugs were successfully loaded into Fujicalin by a solvent evaporation method. Mini-tablets were produced and demonstrated good physical characteristics, expected drug content and immediate release profiles for all drug formulations. Mini-tablets were introduced into a capsule (CombiCap) and used for a pilot study in human volunteers. Plasma samples were collected and analyzed by liquid chromatography and mass spectrometry. Plasma concentration ratios between the parent drugs and the respective metabolites were equivalent for the novel CombiCap formulation and individually dosed Basel Cocktail drugs. We conclude that the CombiCap formulation platform can be easily adopted for different types of phenotyping cocktails due to its scalable and modular design, which allows a simple and convenient combination of variable doses of different probe drugs.
... Automated handling of DBS samples (by flow-through desorption of DBS or direct extraction from the surface of the card) promises to be a valuable tool eliminating tedious manual sample preparation procedures 4,5,[7][8][9][10][11][12][13] . Next to automated DBS methodologies, some practical micro sampling devices (partially) overcoming HCT issues have recently been developed [14][15][16][17][18] . Despite continuous evolution, the field of DBS still lacks a convincing all-round approach providing affordable, practical and reliable practices in sample collection, shipping, storage and analysis. ...
Article
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A workflow overcoming micro sample collection issues and hematocrit (HCT)-related bias would facilitate more widespread use of dried blood spots (DBS). This report describes comparative results between the use of a pipette and a microfluidic-based sampling device for the creation of volumetric DBS. Both approaches were successfully coupled to HCT-independent, fully automated sample preparation and on-line liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis allowing quantitative determination of five stimulants in finger prick blood. Reproducible, selective, accurate and precise responses meeting generally accepted regulated bioanalysis guidelines were observed over the range of 5 to 1000 ng/mL whole blood. The applied heated flow-through solvent desorption of the entire spot and on-line solid phase extraction (SPE) procedure were unaffected by the blood’s HCT value within the tested range of 28.0% to 61.5% HCT. Enhanced stability for mephedrone on DBS compared to liquid whole blood was observed. Finger prick blood samples were collected using both volumetric sampling approaches over a time course of 25 hours after intake of a single oral dose of phentermine. A pharmacokinetic curve for the incurred phentermine was successfully produced using the described validated method. These results suggest that either volumetric sample collection method may be amenable to field-use followed by fully automated, HCT-independent DBS-SPE-LC-MS/MS bioanalysis for the quantitation of these representative controlled substances. Analytical data from DBS prepared with a pipette and microfluidic-based sampling devices were comparable, but the latter is easier to operate, making this approach more suitable for sample collection by unskilled persons.
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Pritelivir is a novel viral helicase‐primase inhibitor active against herpes simplex virus. In vitro drug‐drug interaction studies indicated that pritelivir has the potential for clinically relevant interactions on the cytochrome P450 (CYP) enzymes 2C8, 2C9, 3A4, and 2B6, and intestinal uptake transporter organic anion transporting polypeptide (OATP) 2B1 and efflux transporter breast cancer resistance protein (BCRP). This was evaluated in 2 clinical trials. In 1 trial the substrates flurbiprofen (CYP2C9), bupropion (CYP2B6), and midazolam (CYP3A4) were administered simultaneously as part of the Geneva cocktail, while the substrate celiprolol (OAPT2B1) was administered separately. In another trial, the substrates repaglinide (CYP2C8) and rosuvastatin (BCRP) were administered separately. Exposure parameters of the substrates and their metabolites (flurbiprofen and bupropion only) were compared after administration with or without pritelivir under therapeutic concentrations. The results of these trials indicated that pritelivir has no clinically relevant effect on the exposure of substrates for the intestinal uptake transporter OATP2B1 and the CYP enzymes 3A4, 2B6, 2C9, and 2C8, and has a weak inhibitory effect on the intestinal efflux transporter BCRP. In summary, the results suggest that pritelivir has a low drug‐drug interaction potential.
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As a selective and potent inhibitor targeting the isocitrate dehydrogenase‐2 (IDH2) mutant protein, enasidenib obtained approval from the US Food and Drug Administration (FDA) in 2017 for adult patients with acute myeloid leukemia (AML) with an IDH2 mutation. In vitro investigations demonstrated that enasidenib affects various drug metabolic enzymes and transporters. This current investigation aimed to assess enasidenib on the pharmacokinetics (PKs) of CYP substrates, including dextromethorphan (CYP2D6 probe drug), flurbiprofen (CYP2C9 probe drug), midazolam (CYP3A4 probe drug), omeprazole (CYP2C19 probe drug), and pioglitazone (CYP2C8 probe drug), in patients with AML or myelodysplastic syndrome. Results showed that following the co‐administration of enasidenib (100 mg, once daily) for 28 days, the PK parameters AUC (0‐∞) and C max of dextromethorphan increased by 1.37 (90% confidence interval (CI): 0.96, 1.96) and 1.24 (90% CI: 0.94, 1.65)‐fold, respectively, compared to dextromethorphan alone. For flurbiprofen, these parameters increased by 1.14 (90%CI: 1.01, 1.29) and 0.97 (90% CI 0.86, 1.08)‐fold, respectively, when compared to flurbiprofen alone. Conversely, midazolam exhibited decreases to 0.57 (90% CI 0.34, 0.97) and 0.77 (90% CI 0.39, 1.53)‐fold, respectively, in comparison to midazolam alone. The parameters for omeprazole increased by 1.86 (90% CI: 1.33, 2.60) and 1.47 (0.93, 2.31)‐fold, respectively, compared to omeprazole alone, while those for pioglitazone decreased to 0.80 (90% CI: 0.62, 1.03) and 0.87 (90% CI: 0.65, 1.16)‐fold, respectively, in comparison to pioglitazone alone. These findings provide valuable insights into dose recommendations concerning drugs acting as substrates of CYP2D6, CYP2C9, CYP3A4, CYP2C19, and CYP2C8 when administered concurrently with enasidenib.
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As the first‐in‐class, selective, and potent inhibitor of the isocitrate dehydrogenase‐2 (IDH2) mutant protein, enasidenib was approved by the US Food and Drug Administration (FDA) in 2017 for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with an IDH2 mutation. Known for its interactions with various cytochrome P450 (CYP) enzymes and transporters in vitro, a clinical pharmacokinetics (PK) trial was initiated to assess the impact of multiple doses of enasidenib on the single‐dose PK of sensitive probe substrates of several cytochrome P450 enzymes and transporters. In this study, a population pharmacokinetic analysis approach was employed to address challenges posed by high, nonzero baseline caffeine concentrations. Moreover, we integrated full Bayesian inference into this approach innovatively for a more detailed understanding of parameter uncertainty and greater modeling flexibility, alongside Student's t‐distribution for robust error modeling in handling the abnormal outlier caffeine concentration data observed in this trial. Our analyses demonstrated that multiple doses of enasidenib altered caffeine clearance to a clinically meaningful extent, as evidenced by an approximate 8‐fold decrease. This finding led to a specific recommendation in the package insert to avoid the concurrent use of certain CYP1A2 substrates with enasidenib, unless directed otherwise in the prescribing information. Furthermore, this research underlines the technical benefits of integrating full Bayesian inference and incorporating Student's t‐distribution for residual error modeling in the PK field.
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Pharmacogenomics (PGx) enables personalized treatment for the prediction of drug response and to avoid adverse drug reactions. Currently, PGx mainly relies on the genetic information of absorption, distribution, metabolism, and excretion (ADME) targets such as drug-metabolizing enzymes or transporters to predict differences in the patient's phenotype. However, there is evidence that the phenotype-genotype concordance is limited. Thus, we discuss different phenotyping strategies using exogenous xenobiotics (e.g., drug cocktails) or endogenous compounds for phenotype prediction. In particular, minimally invasive approaches focusing on liquid biopsies offer great potential to preemptively determine metabolic and transport capacities. Early studies indicate that ADME phenotyping using exosomes released from the liver is reliable. In addition, pharmacometric modeling and artificial intelligence improve phenotype prediction. However, further prospective studies are needed to demonstrate the clinical utility of individualized treatment based on phenotyping strategies, not only relying on genetics. The present review summarizes current knowledge and limitations. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 64 is January 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Drug-metabolizing enzymes and drug transporters are key determinants of drug pharmacokinetics and response. The cocktail-based cytochrome P450 (CYP) and drug transporter phenotyping approach consists in the administration of multiple CYP or transporter-specific probe drugs to determine their activities simultaneously. Several drug cocktails have been developed over the past two decades in order to assess CYP450 activity in human subjects. However, phenotyping indices were mostly established for healthy volunteers. In this study, we first performed a literature review of 27 clinical pharmacokinetic studies using drug phenotypic cocktails in order to determine 95%,95% tolerance intervals of phenotyping indices in healthy volunteers. Then, we applied these phenotypic indices to 46 phenotypic assessments processed in patients having therapeutic issues when treated with painkillers or psychotropic drugs. Patients were given the complete phenotypic cocktail in order to explore the phenotypic activity of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A, and P-glycoprotein (P-gp). P-gp activity was evaluated by determining AUC0–6h for plasma concentrations over time of fexofenadine, a well-known substrate of P-gp. CYP metabolic activities were assessed by measuring the CYP-specific metabolite/parent drug probe plasma concentrations, yielding single-point metabolic ratios at 2 h, 3 h, and 6 h or AUC0–6h ratio after oral administration of the cocktail. The amplitude of phenotyping indices observed in our patients was much wider than those observed in the literature for healthy volunteers. Our study helps define the range of phenotyping indices with “normal” activities in human volunteers and allows classification of patients for further clinical studies regarding CYP and P-gp activities.
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La population pédiatrique est considérée comme vulnérable, et la prise en charge de la douleur nociceptive y est parfois complexe mais essentielle. Depuis 2013, la prescription de codéine est restreinte chez les enfants en raison du risque de dépression respiratoire parfois fatale lié à l’activité variable du cytochrome P450 (CYP) 2D6 qui bioactive la codéine en morphine. Les options thérapeutiques approuvées chez l’enfant sont limitées, et pour nombre de prescripteurs, le tramadol est devenu l’alternative de choix à la codéine. Le tramadol est cependant, comme la codéine, un promédicament opioïde qui doit être bioactivé par le CYP2D6. Il est donc également soumis à une importante variabilité de sa réponse et expose les enfants aux mêmes risques de complications respiratoires. La décision de traiter par tramadol doit prendre en compte les comédications, les comorbidités du patient, le type de douleur et les conditions de surveillance. Les soignants et les parents doivent être informés des risques liés à l’administration du tramadol, notamment la variabilité interindividuelle, les risques d’interactions médicamenteuses et les signes de surdosage. Dans les situations de douleurs récurrentes, une approche personnalisée, avec adaptation des doses et sélection du médicament antalgique en fonction de l’activité du CYP2D6, est certainement la méthode la plus sûre. Lorsque l’activité du CYP2D6 n’est pas connue, la prescription de tramadol reste envisageable si le traitement est initié à la dose minimale efficace, titré sous surveillance et administré sous une forme posologique adaptée à l’enfant. Chez l’enfant de moins de 12 ans et en présence de facteurs de risque de dépression respiratoire, la morphine reste une option prudente, puisque son métabolisme ne dépend pas du CYP2D6.
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A specific ultra‐high performance liquid chromatography/quadrupole time‐of‐flightmass spectrometry (UHPLC‐Q‐TOF‐MS/MS) method has been described for the simultaneous determination of the metabolites of tacrine, bupropion, diclofenac, dextromethorphan and midazolam, which are the five probe drugs of the five cytochrome P450 (CYP450) isoforms CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A4. The inhibition degree is determined by calculating IC50. The chromatographic separation was performed on a C18 column with a mobile phase consisted of 0.1% formic acid and acetonitrile. The mass spectrometric analysis was conducted in a positive electrospray ionization mode. IC50 values of CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A were 113.4 μmol·L‐1, 83.78 μmol·L‐1, 22.50 μmol·L‐1, 9.081 μmol·L‐1 and 52.76 μmol·L‐1, respectively. The in vitro results demonstrated that vindoline could inhibit CYP2D1 activity in rats, and weak inhibitory effect on CYP2C11 and CYP3A, but had no obvious effects on CYP1A2 and CYP2B.
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As a first-in-class, selective, potent inhibitor of the isocitrate dehydrogenase-2 (IDH2) mutant protein, enasidenib was approved by the US Food and Drug Administration (FDA) in 2017 for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with an isocitrate dehydrogenase-2 (IDH2) mutation. An in vitro study showed that enasidenib at clinical relevant concentrations, has effects on multiple drug metabolic enzymes and transporters, including inhibition of P-gp, BCRP, OATP1B1 and OATP1B3 transporters. Therefore, a drug-drug interaction (DDI) study was conducted to assess the impact of enasidenib at steady state on the pharmacokinetics (PK) of several probe compounds in patients with relapsed or refractory AML or myelodysplastic syndrome (MDS), including the probes herein described in this article, digoxin and rosuvastatin. Results from eight patients (all Asian) with a mean age of 67.1 years showed that following co-administration of enasidenib (100 mg, 28-day QD schedule) for 28 days (at steady-state), digoxin (0.25 mg) AUC0-30 was 1.2-fold (90% CI:0.9, 1.6), compared with digoxin alone. Following co-administration of enasidenib (100 mg, 28-day QD schedule) for 28 days (at steady-state), rosuvastatin (10 mg) AUC0-inf was 3.4-fold (90% CI: 2.6, 4.5) compared with rosuvastatin alone. These results should serve as the basis for dose recommendations for drugs which are substrates of P-gp, BCRP, OATP1B1 and OATP1B3 transporters, when used concomitantly with enasidenib. This article is protected by copyright. All rights reserved.
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Personalized therapy suggests the appropriate drug at the right dose for the first time through genotype-based individualized therapy, instead of prescribing medicines by the traditional one-size-fits-all manner, thereby claiming that it will make medicines safer and more effective. Accordingly, polymorphisms of drug metabolizing enzymes (DMEs), which induce inter-individual variability in the pharmacokinetics of a drug, have attracted great interest in the context of personalized medicine.Obesity is one of the most common chronic diseases in the world, including Iran, and the prevalence is increasing according to predictions. The remarkable role of P450 cytochromes has been verified in the metabolism of numerous drugs, toxins, carcinogen compounds, and the synthesis of some intrinsic compounds, such as steroid hormones. Thus, evaluating the activity of these enzymes is of great importance because any functionality variation can lead to failure in the treatment or unwanted side effects of some drugs.Therefore, any change in the activity of these enzymes in obese patients can also be problematic in the treatment process of these patients in comparison to normal weighted ones. Since only a few human studies have examined the role of inflammation in altering the function of these enzymes, it seems to be necessary to investigate the effect of obesity on the expression and activity of these enzymes; in which the role of inflammatory processes has been proven.Most importantly, it is worth evaluating changes in the activity levels of cytochrome P450 (CYP450) and the inflammatory cytokines after a course of post-surgical treatment and weight loss.To evaluate the activity of CYPs, a multi-drug cocktail is prescribed to obese patients before and after obesity surgery, as well as to healthy volunteers, to provide simultaneous evaluation of different isoforms.A complete demographic data, medical examinations, laboratory tests, and the CYPs genotype of all participants can be extremely important during this investigation.
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The noradrenaline and dopamine reuptake inhibitor bupropion is metabolized by CYP2B6 and recommended by the FDA as the only sensitive substrate for clinical CYP2B6 drug–drug interaction (DDI) studies. The aim of this study was to build a whole-body physiologically based pharmacokinetic (PBPK) model of bupropion including its DDI-relevant metabolites, and to qualify the model using clinical drug–gene interaction (DGI) and DDI data. The model was built in PK-Sim® applying clinical data of 67 studies. It incorporates CYP2B6-mediated hydroxylation of bupropion, metabolism via CYP2C19 and 11β-HSD, as well as binding to pharmacological targets. The impact of CYP2B6 polymorphisms is described for normal, poor, intermediate, and rapid metabolizers, with various allele combinations of the genetic variants CYP2B6*1, *4, *5 and *6. DDI model performance was evaluated by prediction of clinical studies with rifampicin (CYP2B6 and CYP2C19 inducer), fluvoxamine (CYP2C19 inhibitor) and voriconazole (CYP2B6 and CYP2C19 inhibitor). Model performance quantification showed 20/20 DGI ratios of hydroxybupropion to bupropion AUC ratios (DGI AUCHBup/Bup ratios), 12/13 DDI AUCHBup/Bup ratios, and 7/7 DDGI AUCHBup/Bup ratios within 2-fold of observed values. The developed model is freely available in the Open Systems Pharmacology model repository.
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Measuring in vivo changes in the drug metabolizing activity of cytochrome P450 (CYP) enzymes is critical to understanding and assessing drug-drug, drug-diet and drug-disease interactions. The sensitivity and specificity of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) makes it an ideal tool for analyzing drugs and their metabolites in biological matrices, and has demonstrated utility in CYP phenotyping across varied applications. Published CYP phenotyping cocktail assays often require large plasma sample volumes (0.5-1 mL), have relatively low sensitivity and multi-step complex sample preparation and extraction procedures. Further, variability exists in the way that recovery and matrix effects are investigated and reported, and some studies fail to report these data altogether. Therefore, the aim of this study was to develop, validate and optimize a simplified assay for the probe drugs caffeine (metabolized by CYP1A2), omeprazole (CYP2C19), losartan (CYP2C9), dextromethorphan (CYP2D6), midazolam (CYP3A4) and their respective enzyme-specific metabolites in small volumes (100 μL) of human plasma, that addresses the issues noted. Analyte extraction involved protein precipitation with acetonitrile and solid-phase extraction (SPE). Samples were analyzed using an Agilent 1290 infinity LC system in tandem with 6460A triple quadrupole mass spectrometers. The assay met FDA guideline-recommended requirements for specificity, sensitivity (analyte LLOQs 0.78-23.4 ng/mL), accuracy (intra-day RE% nominal concentration 90.7-110.2%; inter-day RE% 87.0-110.5%) and precision (intra-day analyte RSD% 0.46-11.4%; inter-day RSD% 1.36-11.2%). Recovery and matrix effects were thoroughly investigated and excluded as potential interferers with assay performance. This assay has been used successfully to phenotype CYP activity in a human clinical trial participant. Importantly, the authors provide a contemporary commentary on commonly found issues in the CYP phenotyping cocktail assay literature, and make recommendations concerning best-practice approaches and the standardization of data reporting in this area.
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Aims Bioequivalence (BE) trials aim to demonstrate that the 90% confidence interval of the T/R‐ratio of the pharmacokinetic metrics between two formulations (test [T] and reference [R]) of a drug is fully included in the acceptance interval [0.80, 1.25]. Traditionally, the sample size of BE trials is based on a power calculation based on the intrasubject variability coefficient of variation (CV) and the T/R‐ratio of the metrics. Since the exact value of the T/R‐ratio is not known prior to the trial, it is often assumed that the difference between the treatments does not exceed 5%. Hence, uncertainty about the T/R‐ratio is expressed by using a fixed value for the sample size calculation. We propose to characterise the uncertainty about the T/R‐ratio by a (normal) distribution for the log(T/R‐ratio), with an assumed mean of log θ = 0.00 (i.e. θ = 1.00) and a standard deviation σu, which quantifies the uncertainty. Evaluating this distribution leads to the statistical assurance of the BE trial. Methods The assurance of a clinical trial can be derived by integrating the power over the distribution of the input parameters, in this case, the assumed distribution of the log(T/R)‐ratio. Because it is an average power, the assurance can be interpreted as a measure of the probability of success that does not depend on a specific assumed value for the log(T/R)‐ratio. The relationship between power and assurance will be analysed by comparing the numerical outcomes. Results Using the assurance concept, values of the standard deviation for the distribution of potential log(T/R)‐ratios can be chosen to reflect the magnitude of uncertainty. For most practical cases (i.e. when 0.95 ≤ θ ≤ 1.05), the sample size is not, or only slightly, changed when σ = |log(θ)|. Conclusion The advantage of deriving the assurance for BE trials is that uncertainty is directly expressed as a parameter of variability.
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Background The cocktail approach of probing drug metabolizing enzymes, in particular cytochrome P450 (CYP) enzymes, is a cornerstone in clinical pharmacology studies. The first report of the famous “Pittsburg cocktail” has led the way for the availability of numerous cocktail substrate mixtures that provide options for indexing of CYP enzymes and/or evaluating the perpetrator capacity of the drug. Objective The key objectives were: 1) To collate, tabulate, and discuss the various cocktail substrates to determine specific CYP enzyme activity in clinical pharmacology studies with specific case studies; 2) To introspect on how the cocktail approach has withstood the test of time and evolved for enabling key decision(s); 3) To provide some futuristic views on the use of cocktail in drug discovery and development. Method The review was compiled after consultation with databases such as PubMed (NCBI database) and Google scholar to source various published literature on cocktail approaches in drug development. Results In the reviewed case studies, CYP indexing was achieved using a single time point (differing for specific CYP enzyme) plasma determination of the metabolite to parent ratio for all CYP enzymes with the exception of CYP3A4/5, where multiple time points were required for exposure measurement of midazolam and its metabolite. Likewise, a single void of urine, for a specific time duration, has been utilized for the recovery measurements of parent and metabolite for CYP indexing purposes. Conclusion The review provides a comprehensive list of various types of cocktail approaches and discusses some key considerations including the evolution of the cocktail approaches over time, perspectives and futuristic views for the use of probe drugs to aid the execution of clinical pharmacology studies and data interpretation.
Chapter
Multiple diseases have a strong metabolic component, and metabolomics as a powerful phenotyping technology, in combination with orthogonal biological and clinical approaches, will undoubtedly play a determinant role in accelerating the understanding of mechanisms that underlie these complex diseases determined by a set of genetic, lifestyle, and environmental exposure factors. Here, we provide several examples of valuable findings from metabolomics-led studies in diabetes and obesity metabolism, neurodegenerative disorders, and cancer metabolism and offer a longer term vision toward personalized approach to medicine, from population-based studies to pharmacometabolomics.
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Volumetric absorptive microsampling (VAMS) is a novel sampling technique that allows the straightforward collection of an accurate volume of blood (approximately 10μL) from a drop or pool of blood by dipping an absorbent polymeric tip into it. The resulting blood microsample is dried and analyzed as a whole. The aim of this study was to evaluate the potential of VAMS to overcome the hematocrit bias, an important issue in the analysis of dried blood microsamples. An LC-MS/MS method for analysis of the model compounds caffeine and paraxanthine in VAMS samples was fully validated and fulfilled all pre-established criteria. In conjunction with previously validated procedures for dried blood spots (DBS) and blood, this allowed us to set up a meticulous comparative study in which both compounds were determined in over 80 corresponding VAMS, DBS and liquid whole blood samples. These originated from authentic human patient samples, covering a wide hematocrit range (0.21-0.50). By calculating the differences with reference whole blood concentrations, we found that analyte concentrations in VAMS samples were not affected by a bias that changed over the evaluated hematocrit range, in contrast to DBS results. However, VAMS concentrations tend to overestimate whole blood concentrations, as a consistent positive bias was observed. A different behavior of VAMS samples prepared from incurred and spiked blood, combined with a somewhat reduced recovery of caffeine and paraxanthine from VAMS tips at high hematocrit values, an effect that was not observed for DBS using a very similar extraction procedure, was found to be at the basis of the observed VAMS-whole blood deviations. Based on this study, being the first in which the validity and robustness of VAMS is evaluated by analyzing incurred human samples, it can be concluded that VAMS effectively assists in eliminating the effect of hematocrit. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
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The capillary dried blood spot (DBS) sampling method suitability was assessed for simultaneous cytochromes P450 (CYP) and P-glycoprotein (P-gp) phenotyping using a cocktail approach.Ten volunteers received an oral cocktail capsule containing low dose probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A) and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), with CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pre-treatment with inducer rifampicin. Probes/metabolites concentrations were determined in DBS and plasma using a single LC-MS/MS method.The drugs pharmacokinetic profiles were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in presence of inhibitors and inducer. Minimally invasive 1-point and 3-point (at 2, 3 and 6h) DBS sampling methods were found to reliably reflect CYPs and P-gp activities at each session.Clinical Pharmacology & Therapeutics (2014); Accepted article preview online 10 April 2014 doi:10.1038/clpt.2014.83.
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Background: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. Results: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. Conclusion: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.
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A four-drug cytochrome P450 (CYP) phenotyping cocktail was developed to rapidly and safely determine CYP2D6, CYP2C19, CYP2C9 and CYP1A2 enzyme activity and phenotype. The cocktail consisted of the single CYP phenotyping probes of 50 mg tramadol (CYP2D6), 20 mg omeprazole (CYP2C19), 25 mg losartan (CYP2C9) and 200 mg caffeine (CYP1A2) and was administered as a single oral dose. For enzyme activity measurements, urine was collected as 8 h post-administration and blood was sampled at 4 h. The enzyme activity was determined by metabolic ratios of molar concentrations of the drugs and their enzyme catalyzed metabolites and was correlated to the relevant genotypes. In a pilot study in 12 healthy male volunteers the CYP genotype-phenotype correlation and robustness of the cocktail was successfully determined without detection of any adverse drug reactions. In the subsequent population study, four female volunteers experienced unexpected and unacceptable moderate and severe adverse reactions (ARs) of headache, dizziness, nausea, vomiting, blue fingers, nails and lips and difficulties in urinating, which led to the study being prematurely terminated after inclusion of only 25 subjects (17 males, 7 females). Attention must be paid to adverse reactions when designing new combinations of phenotype cocktails regardless of the doses and drugs involved. We specifically warn against the combination of tramadol, omeprazole, losartan and caffeine.
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We investigated whether a single blood measurement using the minimally invasive technique of a finger prick to draw a blood sample of 5 µl (to yield a dried blood spot (DBS)) is suitable for the assessment of flurbiprofen (FLB) metabolic ratio (MR). Ten healthy volunteers who had been genotyped for CYP2C9 were recruited as subjects. They received FLB alone in session 1 and FLB with fluconazole in session 2. In session 3, the subjects were pretreated for 4 days with rifampicin and received FLB with the last dose of rifampicin on day 5. Plasma and DBS samples were obtained between 0 and 8 h after FLB administration, and urine was collected during the 8 h after administration. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. FLB's apparent clearance values decreased by 35% in plasma and DBS during session 2 and increased by 75% in plasma and by 30% in DBS during session 3. Good correlations were observed between MRs calculated from urine, plasma, and DBS samples.
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Dextromethorphan urinary metabolic ratio is widely used to determine the CYP2D6 phenotype, but its utility to reflect subtle differences in catalytic activity is unclear. We evaluated the capability of dextromethorphan urinary metabolic ratio to predict dextromethorphan oral clearance as a measure of CYP2D6 activity. Data from 10 healthy extensive metabolizers of CYP2D6 were given 30 mg of dextromethorphan hydrobromide orally on two occasions. Blood and urine samples were collected for 72 h. Dextromethorphan and dextrorphan were determined in urine by high-performance liquid chromatography with fluorescence detection and in serum by liquid chromatography-mass spectrometry. The urinary metabolic ratio was very weakly correlated with dextromethorphan oral clearance (r = 0.24; p = 0.04). In contrast, the dextromethorphan oral clearance was highly correlated with the dextromethorphan to dextrorphan area under the concentration-time curve ratio (r = 0.84; p = 0.005) and the 3-h (r = 0.60; p = 0.003), 4-h (r = 0.72, p < 0.001), 6-h (r = 0.67; p < 0.001), and 8-h (r = 0.74; p < 0.001) dextromethorphan to dextrorphan serum ratios. Assuming an effect size of 30%, the number of volunteers required for crossover and cross-sectional studies using the urinary metabolic ratio as the CYP2D6 index was calculated to be 56 and 524, respectively, whereas 14 and 60 subjects are needed if oral clearance is used. Considering the required sample size and the low correlation with oral clearance, urinary metabolic ratio is not recommended as the primary outcome variable in studies requiring the detection of modest changes in CYP2D6 activity.
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Polymorphisms of the cytochrome P450 2D6 (CYP2D6) gene affecting enzyme activity are involved in interindividual variability in drug efficiency/toxicity. Four phenotypic groups are found in the general population: ultra rapid (UM), extensive (EM), intermediate (IM) and poor (PM) metabolizers. The AmpliChip CYP450 test is the first genotyping array allowing simultaneous analysis of 33 CYP2D6 alleles. The main aim of this study was to evaluate the performance of this test in CYP2D6 phenotype prediction. We first verified the AmpliChip CYP450 test genotyping accuracy for five CYP2D6 alleles routinely analysed in our laboratory (alleles 3,4,5,6, x N; n=100). Results confirmed those obtained by real-time PCR. Major improvements using the array are the detection of CYP2D6 intermediate alleles and identification of the duplicated alleles. CYP2D6 phenotype was determined by assessing urinary elimination of dextromethorphan and its metabolite dextrorphan and compared to the array prediction (n=165). Although a low sensitivity of UM prediction by genotyping was observed, phenotype prediction was optimal for PM and satisfying for EM and IM.
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Interindividual variability in the expression and function of drug metabolizing cytochrome P (CYP) 450 enzymes, determined by a combination of genetic, non-genetic and environmental parameters, is a major source of variable drug response. Phenotyping by administration of a selective enzyme substrate, followed by the determination of a specific phenotyping metric, is an appropriate approach to assess the in vivo activity of CYP450 enzymes as it takes into account all influencing factors. A phenotyping protocol should be as simple and convenient as possible. Typically, phenotyping metrics are determined in traditional matrices, such as blood, plasma or urine. Several sampling strategies have been proposed as an alternative for these traditional sampling techniques. In this review, we provide a comprehensive overview of available methods using dried blood spots (DBS), hair, oral fluid, exhaled breath and sweat for in vivo CYP450 phenotyping. We discuss the relation between phenotyping metrics measured in these samples and those in conventional matrices, along with the advantages and limitations of the alternative sampling techniques. Reliable phenotyping procedures for several clinically relevant CYP450 enzymes, including CYP1A2, CYP2C19 and CYP2D6, are currently available for oral fluid, breath or DBS, while additional studies are needed for other CYP450 isoforms, such as CYP3A4. The role of hair analysis for this purpose remains to be established. Being non- or minimally invasive, these sampling strategies provide convenient and patient-friendly alternatives for classical phenotyping procedures, which may contribute to the implementation of CYP450 phenotyping in clinical practice.
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Cytochrome P450 1A2 (CYP1A2) plays an important role in drug metabolism. Caffeine (CAF) is converted into paraxanthine (PX) by this enzyme and is used as a xenobiotic substrate to determine the CYP1A2 phenotype in humans. A method for the quantification of CAF and PX in saliva was developed using liquid-liquid extraction with ethyl acetate and analysis with ultra-high-performance liquid chromatography. Peaks from CAF, PX and internal standard were resolved within 6min. The method was validated from 0.05 to 5μgmL(-1) CAF and 0.025-2.5μgmL(-1) PX. Inter- and intra-day accuracies ranged from 91.2 to 107.2% with precisions <13.5%. The limits of detection were 0.16 and 0.63 ngmL(-1) for PX and CAF, respectively. PX/CAF concentration ratios from volunteers were 0.26-1.09 with mean ratios of 0.78±0.26 and 0.38±0.10 for regular and light/non-coffee drinkers, respectively. Copyright © 2015. Published by Elsevier B.V.
Article
Cytochromes P450 (CYP) constitute the major drug-metabolizing enzyme system in humans. The activity of these enzymes is subjected to a great inter-individual variability which can cause interindividual differences in plasma drug concentrations and result in therapeutic failure or side effects. To avoid these problems, it is of great importance to evaluate the in vivo CYP activity (phenotyping). In the beginning of the 1990s, a 'cocktail' approach was developed, aiming to simultaneously assess the activity of multiple CYPs by the administration of a cocktail of CYP-specific probe drugs [1]. Several cocktails using different probe drugs have been developed in the past years [2-7]. Few of these cocktails use well-validated probes such as caffeine, omeprazole, dextromethorphan and midazolam for the phenotyping of CYP1A2, CYP2C19, CYP2D6 and CYP3A, respectively [2, 4, 5]. These drugs have no mutual interactions as previously demonstrated [5]. Two of these cocktails also include losartan [4] or warfarin [2] as probes for CYP2C9 activity. These drugs, as well as tolbutamide [8] and phenytoin [9], have been proposed as potentially useful CYP2C9 probes. However, all of the proposed CYP2C9 substrates are associated with limitations which make them less than ideal as probes for this enzyme [10]. Recently, flurbiprofen has been validated as a reliable probe for CYP2C9 phenotyping both in urine [10] and single point plasma samples [11]. Flurbiprofen has also been incorporated in the Pittsburgh cocktail and was shown not to interact with the other probes. [7]. In this study, we aimed to validate the incorporation of flurbiprofen as a CYP2C9 probe to a cocktail composed of caffeine, omeprazole, dextromethorphan and midazolam and verify the effect these drugs have on flurbiprofen metabolic ratio. This article is protected by copyright. All rights reserved.
Article
Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms.
Article
Background: We have previously shown that flurbiprofen (F) metabolism to 4'-hydroxy-flurbiprofen (OH-F) provides an in vivo measure of CYP2C9 activity. The fractional metabolic clearance to OH-F is closely associated with the flurbiprofen recovery ratio (FRR= OH-F/OH-F+F)(r=0.8488, p<0.0001, N=12). This study evaluates the possibility of incorporating flurbiprofen to the currently validated five-drug Pittsburgh cocktail.Methods: In a three-way cross-over design with randomized order of drug administration, 12 healthy subjects (age SD, 29.9 6.6) were enrolled. Each subject received F (50mg) and the Pittsburgh five-drug cocktail (caffeine 100mg, mephenytoin 100mg, debrisoquine 10mg, chlorzoxazone 250mg, dapsone 100mg) separately and in combination on three occasions over five weeks. Urine was collected from 0 to 8 hours, and plasma was obtained at 4 and 8 hours after drug administration. Parent drug and metabolite concentrations were measured to determine phenotypic indices for the metabolizing enzymes.Results: There were no statistically significant differences in any of the indices, whether assessed as part of the five-drug cocktail, six-drug cocktail, or in the case of flurbiprofen, individually. The percentage change of the trait measures observed is illustrated below. The six-drug cocktail was well tolerated.Conclusion: The results show that flurbiprofen can be administered within the validated Pittsburgh cocktail without interaction to provide a measure of CYP2C9 activity.
Article
Objectives: To test whether the multiple phenotype and genotype relationships established using therapeutic dose, can be reproduced following oral microdosing using substrates of CYP2C9 (warfarin and glibenclamide), CYP2C19 (lansoprazole), CYP2D6 (dextromethorphan), and OATPs (glibenclamide). Methods: A cocktail of test drugs was administered orally under the microdose in liquid or capsule form, and then a therapeutic dose of dextromethorphan was administered to 17 healthy subjects whose genotypes for CYPs and OATPs had been prescreened. Concentrations of the drugs and their metabolites were measured by LC-MS/MS. Results: The AUC and t1/2 of glibenclamide following the microdosing tended to be higher and longer, respectively, in CYP2C9*1/*3 than CYP2C9*1/*1 subjects. In contrast, there were no significant differences in any of the pharmacokinetic parameters for warfarin between the two genotypes. For CYP2D6 following the therapeutic dose, there was good concordance between genotype and phenotype; however, such relationships disappeared after microdosing. For CYP2C19 following the microdosing, there were significant differences between EMs and PMs in the pharmacokinetic parameters of lansoprazole. The relative AUC0-12 ratio of lansoprazole in EMs and PMs was 1:3.3 - 4.3. Among test drugs, phenotypic measurements of lansoprazole accorded well with the CYP2C19 genotype at the microdose as well as therapeutic dose. Conclusions: The present study suggests that 1) the sampling strategy should be optimized according to pharmacokinetic profiles of the test drugs following oral microdosing, and 2) microdosing can be applied to the pharmacogenomic study of CYP-specific drugs.
Article
The aim of this crossover human male volunteer study was to investigate the utility of microdosing in the investigation of drug-drug interactions. A mixture of midazolam, tolbutamide, caffeine and fexofenadine were administered as a microdose (25 μg each) before and after administration of a combined pharmacological dose of ketoconazole (400 mg) and fluvoxamine (100 mg) to inhibit P-glycoprotein and metabolism by cytochrome P450 (CYP) 1A2, CYP3A4 and CYP2C9. When administered alone, pharmacokinetics for all four microdosed compounds scaled well with those reported for therapeutic doses and with previously performed microdose studies. The pharmacokinetics of each compound administered as a microdose were significantly altered after the administration of ketoconazole and fluvoxamine, showing statistically significant (p < 0.01) 12.8-, 8.1- and 3.2-fold increases in the area under the plasma concentration-time curve from time zero to infinity (AUC(∞)) for midazolam, caffeine and fexofenadine, respectively. A 1.8-fold increase (not statistically significant) in AUC(∞) was observed for tolbutamide. The changes in pharmacokinetics mediated by ketoconazole and fluvoxamine were quantitatively consistent with previously reported, non-microdose, drug-drug interaction data from studies including the same compounds. The initial data reported here demonstrate the utility of microdosing to investigate the risk of development drugs being victims of drug-drug interactions.
Article
* Numerous cocktails using concurrent administration of several cytochrome P450 (CYP) isoform-selective probe drugs have been reported to investigate drug-drug interactions in vivo. * This approach has several advantages: characterize the inhibitory or induction potential of compounds in development toward the CYP enzymes identified in vitro in an in vivo situation, assess several enzymes in the same trial, and have complete in vivo information about potential CYP-based drug interactions. * This study describes a new cocktail containing five probe drugs that has never been published. * This cocktail can be used to test the effects of a new chemical entity on multiple CYP isoforms in a single clinical study: CYP1A2 (caffeine), CYP2C9 (warfarin), CYP2C19 (omeprazole), CYP2D6 (metoprolol), and CYP3A (midazolam) and was designed to overcome potential liabilities of other reported cocktails. To assess the pharmacokinetics (PK) of selective substrates of CYP1A2 (caffeine), CYP2C9 (S-warfarin), CYP2C19 (omeprazole), CYP2D6 (metoprolol) and CYP3A (midazolam) when administered orally and concurrently as a cocktail relative to the drugs administered alone. This was an open-label, single-dose, randomized, six-treatment six-period six-sequence William's design study with a wash-out of 7 or 14 days. Thirty healthy male subjects received 100 mg caffeine, 100 mg metoprolol, 0.03 mg kg(-1) midazolam, 20 mg omeprazole and 10 mg warfarin individually and in combination (cocktail). Poor metabolizers of CYP2C9, 2C19 and 2D6 were excluded. Plasma samples were obtained up to 48 h for caffeine, metoprolol and omeprazole, 12 h for midazolam, 312 h for warfarin and the cocktail. Three different validated liquid chromatography tandem mass spectrometry methods were used. Noncompartmental PK parameters were calculated. Log-transformed C(max), AUC(last) and AUC for each analyte were analysed with a linear mixed effects model with fixed term for treatment, sequence and period, and random term for subject within sequence. Point estimates (90% CI) for treatment ratios (individual/cocktail) were computed for each analyte C(max), AUC(last) and AUC. There was no PK interaction between the probe drugs when administered in combination as a cocktail, relative to the probes administered alone, as the 90% CI of the PK parameters was within the prespecified bioequivalence limits of 0.80, 1.25. The lack of interaction between probes indicates that this cocktail could be used to evaluate the potential for multiple drug-drug interactions in vivo.
Article
Human CYP2B6 has been thought to account for a minor portion (<1%) of total hepatic cytochrome P450 (CYP) content and to have a minor function in human drug metabolism. Recent studies, however, indicate that the average relative contribution of CYP2B6 to total hepatic CYP content ranges from 2% to 10%. An increased interest in CYP2B6 research has been stimulated by the identification of an ever-increasing substrate list for this enzyme, polymorphic and ethnic variations in expression levels, and evidence for cross-regulation with CYP3A4, UGT1A1 and several hepatic drug transporters by the nuclear receptors pregnane X receptor and constitutive androstane receptor. Moreover, 20- to 250-fold interindividual variation in CYP2B6 expression has been demonstrated, presumably due to transcriptional regulation and polymorphisms. These individual differences may result in variable systemic exposure to drugs metabolized by CYP2B6, including the antineoplastics cyclophosphamide and ifosfamide, the antiretrovirals nevirapine and efavirenz, the anesthetics propofol and ketamine, the synthetic opioid methadone, and the anti-Parkinsonian selegiline. The potential clinical significance of CYP2B6 further enforces the need for a comprehensive review of this xenobiotic metabolizing enzyme. This communication summarizes recent advances in our understanding of this traditionally neglected enzyme and provides an overall picture of CYP2B6 with respect to expression, localization, substrate-specificity, inhibition, regulation, polymorphisms and clinical significance. Emphasis is given to nuclear receptor mediated transcriptional regulation, genetic polymorphisms, and their clinical significance.
Article
In a controlled clinical trial, the elimination of caffeine was examined in 20 healthy women prior to and during one cycle of treatment with either of two oral contraceptive formulations, one containing 0.075 mg gestodene and 0.03 mg ethinylestradiol and one containing 0.125 mg levonorgestrel and 0.03 mg ethinylestradiol. In addition, caffeine clearance was determined 1 month after the last intake of the oral contraceptives. Compared with pretreatment values, the clearance of caffeine was reduced by about 54% and 55% after one treatment cycle with gestodene- and the levonorgestrel-containing oral contraceptive, respectively. Other pharmacokinetic parameters of caffeine, such as t max and C max, were not affected. Clearance values returned to pretreatment values 1 month after the last administration of the oral contraceptives. There was no difference in the reduction of caffeine clearance between contraceptive formulations. A small, but significant difference in the AUC(0–24 h) values of ethinylestradiol was noted between both preparations. There was no correlation between the AUC(model) values of caffeine and the AUC(0–24 h) values of ethinylestradiol. In the present study, a somewhat more pronounced effect on the elimination of caffeine was observed than in previous investigations, where several contraceptive steroids were administered only for a period of 2 weeks.
Article
Cytochrome P450 phenotyping provides valuable information about real-time activity of these important drug-metabolizing enzymes through the use of specific probe drugs. Despite more than 20 years of research, few conclusions regarding optimal phenotyping methods have been reached. Caffeine offers many advantages for CYP1A2 phenotyping, but the widely used caffeine urinary metabolic ratios may not be the optimal method of measuring CYP1A2 activity. Several probes of CYP2C9 activity have been suggested, but little information exists regarding their use, largely due to the narrow therapeutic index of most CYP2C9 probes. Mephenytoin has long been considered the standard CYP2C19 phenotyping probe, but problems such as sample stability and adverse effects have prompted the investigation of potential alternatives, such as omeprazole. Several well-validated CYP2D6 probes are available, including dextromethorphan, debrisoquin and sparteine, but, in most cases, dextromethorphan may be preferred due to its wide safety margin and availability. Chlorzoxazone remains the only CYP2E1 probe that has received much study. However, questions concerning phenotyping method and involvement of other enzymes have impaired its acceptance as a suitable CYP2E1 phenotyping probe. CYP3A phenotyping has been the subject of numerous investigations, reviews and commentaries. Nevertheless, much controversy regarding the selection of an ideal CYP3A probe remains. Of all the proposed methods, midazolam plasma clearance and the erythromycin breath test have been the most rigorously studied and appear to be the most reliable of the available methods. Despite the limitations of many currently available probes, with continued research, phenotyping will become an even more valuable research and clinical resource.
Article
Although it is known that the use of oral contraceptives inhibits oxidative drug metabolism, there is little information regarding their effect on CYP2C19 activity. Moreover, earlier reports suggest that there may be differences in CYP2C19 activity between men and women. We sought to assess the effect of sex and intake of oral contraceptives on CYP2C19 activity as measured by the probe drugs mephenytoin and omeprazole. To determine CYP2C19 activity in white Swedish subjects, 644 subjects previously phenotyped with mephenytoin and 175 subjects phenotyped with omeprazole were investigated. The 8-hour urinary mephenytoin S/R ratio after ingestion of 100 mg mephenytoin and the plasma concentration ratio of omeprazole/hydroxyomeprazole at 3 hours after ingestion of 20 mg omeprazole were used as measures of CYP2C19 activity. Differences in these ratios and in their frequency distributions were then examined among women with and without oral contraceptives and men. In addition, nearly all subjects in the omeprazole group had been genotyped with regard to the CYP2C19*2 (ml) allele. Subjects homozygous for the CYP2C19*2 allele were excluded from the study. The median mephenytoin S/R ratio was 2.5-fold higher in the subgroup of women taking oral contraceptives compared with either women not taking oral contraceptives (P < .001) or men (P < .001). Similarly, the mean omeprazole/hydroxyomeprazole ratio was twice as high in the oral contraceptive group compared with women not taking oral contraceptives (P < .001) or men (P < .001). However, no differences were evident between women not taking oral contraceptives and men in either the mephenytoin group (P = .48) or the omeprazole group (P = .77). The oral contraceptive-induced inhibitory effect on CYP2C19 activity was similar between the CYP2C19*1/*1 and *1/*2 genotypes, and they were independent of age. Intake of oral contraceptives significantly inhibits CYP2C19 activity, but there is no true sex-related difference in CYP2C19 activity in healthy, white, Swedish subjects.
Article
The human cytochrome P450 (CYP) superfamily comprises 57 genes. These genes code for enzymes that can have a role in: metabolism of drugs, foreign chemicals, arachidonic acid and eicosanoids; cholesterol metabolism and bile-acid biosynthesis; steroid synthesis and metabolism; vitamin D(3) synthesis and metabolism; retinoic acid hydroxylation; and those of still unknown function. Cytochrome P450 was once believed to be mainly a hepatic drug detoxication system, but is now understood to include a myriad of enzymic reactions implicated in important life processes. Mutations in many CYP genes cause inborn errors of metabolism and contribute to many clinically relevant diseases.
Article
Our objectives were (1) to determine whether the drugs caffeine, losartan, omeprazole, debrisoquin (INN, debrisoquine), and quinine can be given simultaneously in low doses as a cocktail for the phenotyping of cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4, respectively, and (2) to design an administration schedule to give as few sampling occasions as possible. Twenty-four subjects were given oral doses of 100 mg caffeine, 25 mg losartan, 20 mg omeprazole, 10 mg debrisoquin, and 250 mg quinine on separate days. After a washout period of at least 4 days, all drugs were given simultaneously except for quinine, which was given 8 hours after the other drugs. Blood and urine samples were collected to determine parent drug and metabolite concentrations for assessment of phenotyping indices. Any difference between both single and cocktail doses was tested on a log-normal distribution. The phenotypic indices of CYP1A2 (paraxanthine/caffeine in 4-hour plasma), CYP2C9 (losartan/E-3174 [metabolite of losartan] in 0- to 8-hour urine), CYP2C19 (omeprazole/5-hydroxyomeprazole in 3-hour plasma), and CYP3A4 (quinine/3-hydroxyquinine in 16-hour plasma) were not significantly changed when probe drugs were administered alone compared with together, although a tendency toward higher concentrations of losartan was seen during simultaneous administration (95% confidence interval, 0.51-1.002; P =.051). The CYP2D6 phenotypic index (debrisoquin/4-hydroxydebrisoquin in 0- to 8-hour urine) was significantly changed when drugs were given together (95% confidence interval, 0.45-0.87; P =.007), indicating an inhibition of the debrisoquin metabolism. The within-subject coefficients of variation (8%-25%) were much lower than the between-subject coefficients of variation (34%-79%). The administration of drugs together suggests an inhibition of debrisoquin metabolism caused by the concurrent drugs given. By separating debrisoquin from the other cocktail drugs, this method is likely to be used as a tool to phenotype the enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 with only 2 urinary collections and 2 blood-sampling occasions.
Article
Previously, we have validated a 4-drug phenotyping cocktail, the "Cooperstown cocktail," using caffeine (cytochrome p450 [CYP] 1A2, N-acetyltransferase-2 [NAT2], and xanthine oxidase [XO]), dextromethorphan (CYP2D6), omeprazole (CYP2C19), and intravenous midazolam (hepatic CYP3A). Data suggest that warfarin can be used as a safe and accurate biomarker for CYP2C9, and if warfarin is administered with vitamin K, the pharmacodynamic effect is ablated. Twelve subjects received the Cooperstown cocktail, warfarin plus vitamin K, and both sets of biomarkers (Cooperstown 5+1 cocktail) in a randomized crossover fashion. On the basis of log-transformed data and a paired t test, no significant difference was seen for S-warfarin area under the serum concentration-time curve from time 0 to infinity (P =.09), omeprazole metabolic ratio (P =.374), caffeine metabolic ratio (P =.169 for CYP1A2 activity), midazolam plasma clearance (P =.573), or dextromethorphan metabolic ratio (P =.747) with the Cooperstown cocktail, warfarin plus vitamin K alone, or the Cooperstown 5+1 cocktail. During drug administration, the only side effect was mild and short-lived sedation after intravenous midazolam administration. Phenotypic measurements were in concordance with the subject's CYP2C9, CYP2C19, and CYP2D6 genotypes. The Cooperstown 5+1 cocktail may be used to simultaneously assess the activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A, NAT2, and XO.
Article
Cytochrome P450s (CYPs) are an important family of enzymes in the metabolism of many therapeutic agents and endogenous metabolic reactions. The CYP3A subfamily is especially prominent in these metabolic activities. This review article focuses on how the factors of age and sex may influence the in vivo activity of human CYP3A. The functional activity of CYP3A varies based on issues such as interaction with one or more substrates and between individuals and/or localisation. For CYP3A substrates, intrinsic clearance is the component of total clearance that is contributed by the enzymes. Depending on the route of administration and the contribution of hepatic blood flow to overall clearance, sensitivities to changes in CYP3A activities may differ. Additionally, age may influence the hepatic blood flow and, in turn, affect CYP3A activity. A review of the literature regarding age influences on the clearance of CYP3A substrates does suggest that age can affect the clearance of certain CYP3A substrates. CYP3A is responsible for a large number of endogenous metabolic reactions involving steroid hormones, and enzyme activity has been reported to be induced and/or inhibited in the presence of some sex steroids. Based on published studies for most CYP3A substrates, sex does not appear to influence clearance; however, with certain substrates significant sex-related differences are found. In such cases, women primarily have higher clearance than men.
Article
The purpose of this study was to assess the effect of bupropion on cytochrome P450 2D6 (CYP2D6) activity. Twenty-one subjects completed this repeated-measures study in which dextromethorphan (30-mg oral dose) was administered to smokers at baseline and after 17 days of treatment with either bupropion sustained-release (150 mg twice daily) or matching placebo. Subjects quit smoking 3 days before the second dextromethorphan administration. To assess CYP2D6 activity, urinary dextromethorphan/dextrorphan metabolic ratios were calculated after an 8-hour urine collection. Thirteen subjects received bupropion, and 8 received placebo. In those receiving active medication, the dextromethorphan/dextrorphan ratio increased significantly at the second assessment relative to the first (0.012 +/- 0.012 vs. 0.418 +/- 0.302; P < 0.0004). No such change was observed in those randomized to placebo (0.009 +/- 0.010 vs. 0.017 +/- 0.015; P = NS). At baseline, all subjects were phenotypically extensive CYP2D6 metabolizers (metabolic ratio <0.3); after treatment, 6 of 13 subjects receiving bupropion, but none of those receiving placebo, had metabolic ratios consistent with poor CYP2D6 metabolizers. Bupropion is therefore a potent inhibitor of CYP2D6 activity, and care should be exercised when initiating or discontinuing bupropion use in patients taking drugs metabolized by CYP2D6.
Article
The effects of a common oral contraceptive preparation on the activity of 7 drug-metabolizing enzymes were investigated using the validated Cooperstown 5+1 Cocktail. In a randomized crossover fashion, 10 premenopausal women received caffeine, dextromethorphan, omeprazole, intravenous midazolam, and warfarin + vitamin K with and without a triphasic oral contraceptive (ethinyl estradiol 35 microg) and varying doses of daily norgestimate (0.18, 0.215, and 0.25 mg). Bioequivalence testing showed nonequivalence in drug versus no-drug treatment on the activity of drug-metabolizing enzymes (as reflected by metabolite ratios following probe drug administration); the activity of CYP1A2, CYP2C19, and NAT-2 decreased following the oral contraceptive, whereas the activity of CYP2C9 and CYP2D6 increased. No effects on xanthine oxidase or hepatic CYP3A were seen. Application of a non-parametric statistical testing approach revealed a significant difference only for CYP1A2 and CYP2C19. This triphasic oral contraceptive may have a clinically significant effect on the activity of some drug-metabolizing enzymes.
Article
Background: Major depressive disorder (MDD) is a common psychiatric condition, with 6.6% of the adult population in the United States experiencing a major depressive episode during any given year. Depressed patients must receive adequate treatment to maximize the likelihood of clinical success. Bupropion hydrochloride, a noradrenergic/dopaminergic antidepressant, is available in 3 oral formulations: immediate release (IR) (given TID), sustained release (SR) (given BID), and extended release (XL) (given QD). Understanding the pharmacokinetic (PK) properties and formulations of bupropion can help optimize clinical use. Objectives: : The aims of this article were to provide a review of the PK properties of bupropion and identify its various formulations and clinical applications to help optimize treatment of MDD. Methods: : In this review, data concerning PK trials/reports were collected from articles identified using a PubMed search. The search was conducted without date limitations and using the search terms bupropion, bupropion SR, bupropion XL, bupropion pharmacokinetics, bupropion metabolism, and bupropion drug interactions. Additional reports were selected from references that appeared in articles identified in the original search. In addition, data from studies summarized in product information and labeling were obtained. All available information, concentrating on studies in humans, pertinent to bupropion PK properties and/or formulations was included. Results: : Bupropion is extensively metabolized by the liver (t(1/2), approximately 21 hours). Hydroxybupropion, the primary active metabolite (t(1/2), approximately 20 hours), is formed by cytochrome P450 (CYP) 2B6. At steady state, C(max) of hydroxybupropion is 4- to 7-fold higher, and the AUC is approximately 10-fold greater, compared with those of the parent drug. Threohydrobupropion and erythrohydrobupropion (mean [SD] t(1/2) values, approximately 37 [13] and approximately 33 [10] hours, respectively), the other active metabolites of bupropion, are formed via nonmicrosomal pathways. Relative to bupropion, the C(max) values are approximately 5-fold greater for threohydrobupropion and similar for erythrohydrobupropion. Based on a mouse antitetrabenazine model, hydroxybupropion is approximately 50% as active as bupropion, and threohydrobupropion and erythrohydrobupropion are approximately 20% as active as bupropion. Bupropion lowers the seizure threshold and, therefore, concurrent administration with other agents that lower the seizure threshold should be undertaken cautiously. Potential interactions with other agents that are metabolized by CYP2B6 should be considered. In addition, bupropion inhibits CYP2D6 and may reduce clearance of agents metabolized by this enzyme. Absorption of the XL formulation is prolonged compared with the IR and SR formulations (T(max), approximately 5 hours vs approximately 1.5 and approximately 3 hours, respectively). Bupropion is dosed without regard to food. Conclusions: : Understanding the PK profile and formulations of bupropion can help optimize clinical use. Bupropion is metabolized extensively, resulting in 3 active metabolites. This metabolic profile, various patient factors (eg, age, medical illnesses), and potential drug interactions should be considered when prescribing bupropion. The 3 formulations-bupropion, bupropion SR, and bupropion XL-are bioequivalent and offer options to optimize treatment for patients with MDD.
Article
The aim of this study was to investigate the activity of the drug-metabolizing enzyme cytochrome P450 (CYP) 2B6 before and after in vivo induction by rifampin (INN, rifampicin) in white subjects and Chinese subjects by use of the probe drug bupropion (INN, amfebutamone). Healthy male white subjects (n = 9) and Chinese subjects (n = 9) (age range, 19-34 years) of known CYP2B6 genotype received orally administered bupropion (Zyban SR, 150 mg) alone and during daily treatment with rifampin (600 mg). Blood samples were taken for up to 72 hours after each bupropion dose, and plasma concentrations of bupropion and its active metabolites, hydroxybupropion, threohydrobupropion, and erythrohydrobupropion, were measured by HPLC. The subjects' CYP2B6 genotype was determined by use of a matrix-assisted laser desorption /ionization-time of flight (MALDI-TOF) mass spectrometry assay. Rifampin treatment increased the apparent clearance of bupropion in Chinese subjects and white subjects combined (n = 16) from 2.6 L x h(-1) x kg(-1) (95% confidence interval [CI], 2.3-3.0 L x h(-1) x kg(-1)) after bupropion alone to 7.9 L x h(-1) x kg(-1) (95% CI, 6.8-10.1 L x h(-1) x kg(-1)) during rifampin treatment. Rifampin treatment decreased the half-life of bupropion from 15.9 hours (95% CI, 13.5-20.4 hours) to 8.2 hours (95% CI, 6.7-12.4 hours). Rifampin treatment increased the hydroxybupropion maximum concentration from 395 ng/mL (95% CI, 341-497 ng/mL) to 548 ng/mL (95% CI, 490-638 ng/mL), decreased the area under the concentration-time curve extrapolated to infinity of hydroxybupropion from 14.7 microg x h/mL (95% CI, 12.7-18.4 microg x h/mL) to 8.4 microg x h/mL (95% CI, 7.4-10.2 microg x h/mL), and reduced the elimination half-life of hydroxybupropion from 21.9 hours (95% CI, 20.3-24.0 hours) to 10.7 hours (95% CI, 8.6-14.5 hours). There was no significant difference in the pharmacokinetics of bupropion or hydroxybupropion between white subjects and Chinese subjects before and after treatment with rifampin, once corrected for body weight. Rifampin significantly induces CYP2B6 activity in vivo, and the clinical consequences of potential interactions between rifampin and CYP2B6 substrates deserve further investigation. Rifampin appears to induce the elimination of hydroxybupropion. Differences in bupropion pharmacokinetics that were observed between white subjects and Chinese subjects can be attributed to differences in body weight, suggesting that, for a given subject weight, CYP2B6 activity is similar in white subjects and Chinese subjects.