Article

A High-Throughput Assay Using Liquid Chromatography-Tandem Mass Spectrometry for Simultaneous In Vivo Phenotyping of 5 Major Cytochrome P450 Enzymes in Patients

May 2009
Therapeutic Drug Monitoring 31(2):239-46

DOI:10.1097/FTD.0b013e318197e1bf

Source
PubMed

Authors:

Sussan Ghassabian

The University of Queensland

Manoranjenni Chetty

Simcyp ;

Show all 10 authorsHide

The phenotyping cocktail is a practical approach for phenotyping of cytochrome P450 (CYP) enzymes in vivo. In this study, a liquid chromatography-tandem mass spectrometry method using a dual-extraction approach was developed and validated to quantify 5 selective substrates and their metabolites for the simultaneous phenotyping CYPs 1A2, 2C19, 2C9, 2D6, and 3A4 in patient blood samples. The assay was applied in a pilot study of 11 patients with schizophrenia. Five blood samples were collected before and at 1, 2, 4, and 6 hours after administration of a phenotyping cocktail consisting of 100 mg caffeine, 20 mg omeprazole, 25 mg losartan, 30 mg dextromethorphan, and 2 mg midazolam. The method successfully quantitated the CYP enzyme activities without serious side effects in patients. The ratios of metabolite to parent area under the concentration-time curve values were calculated over the 6-hour postdosage to reflect CYP2D6, CYP3A4, and CYP2C9 activities. The ratios of metabolite to parent plasma concentrations were calculated at 4-hour postdosage for CYP1A2 and at 4- or 6-hour postdose for CYP2C19, respectively. The plasma concentration of midazolam at 4 hours was also estimated as another phenotyping index for CYP3A4 activity. The simultaneous assay of all these analytes in a single matrix (plasma) will increase the feasibility of CYP phenotyping in patients.

Critical Assessment of Phenotyping Cocktails for Clinical Use in an African Context

Article

Full-text available

Jul 2023

Interethnic and interindividual variability in in vivo cytochrome P450 (CYP450)-dependent metabolism and altered drug absorption via expressed transport channels such as P-glycoprotein (P-gp) contribute to the adverse drug reactions, drug–drug interaction and therapeutic failure seen in clinical practice. A cost-effective phenotyping approach could be advantageous in providing real-time information on in vivo phenotypes to assist clinicians with individualized drug therapy, especially in resource-constrained countries such as South Africa. A number of phenotyping cocktails have been developed and the aim of this study was to critically assess the feasibility of their use in a South African context. A literature search on library databases (including AccessMedicine, BMJ, ClinicalKey, MEDLINE (Ovid), PubMed, Scopus and TOXLINE) was limited to in vivo cocktails used in the human population to phenotype phase I metabolism and/or P-gp transport. The study found that the implementation of phenotyping in clinical practice is currently limited by multiple administration routes, the varying availability of probe drugs, therapeutic doses eliciting side effects, the interaction between probe drugs and extensive sampling procedures. Analytical challenges include complicated sample workup or extraction assays and impractical analytical procedures with low detection limits, analyte sensitivity and specificity. It was concluded that a single time point, non-invasive capillary sampling, combined with a low-dose probe drug cocktail, to simultaneously quantify in vivo drug and metabolite concentrations, would enhance the feasibility and cost-effectiveness of routine phenotyping in clinical practice; however, future research is needed to establish whether the quantitative bioanalysis of drugs in a capillary whole-blood matrix correlates with that of the standard plasma/serum matrixes used as a reference in the current clinical environment.

An improved LC–MS/MS method for simultaneous evaluation of CYP2C9, CYP2C19, CYP2D6 and CYP3A4 activity

Article

Sep 2018

Aim: To develop an LC-MS/MS assay to quantitate well-tolerated substrates; midazolam (CYP3A), omeprazole (CYP2C19), dextromethorphan (CYP2D6), losartan (CYP2C9) and their respective metabolites' concentrations in plasma samples. Patients & methods: A solid-phase extraction method was optimized to extract analytes of interest simultaneously from human plasma samples. The assay analyzed plasma samples collected from patients who received equal or lower than therapeutic doses of CYP substrates. Results: This assay was validated based on the European Medicines Agency guideline for bioanalytical method validation and was sensitive, linear, accurate and precise with acceptable recovery and matrix effects. Conclusion: Small sample volume and dose of cytochrome P450 substrates, short-run time, using stable isotope internal standards and being cost effective are the major advantages of the assay.

Multiplex Phenotyping for Systems Medicine: A One-Point Optimized Practical Sampling Strategy for Simultaneous Estimation of CYP1A2, CYP2C9, CYP2C19, and CYP2D6 Activities Using a Cocktail Approach

Article

Nov 2015

Phenotyping of the CYP450 enzyme activities contributes to personalized medicine, but the past phenotyping approaches have followed a piecemeal strategy measuring single enzyme activities in vivo. A barrier to phenotyping of populations in rural and remote areas is the limited time and resources for sample collection. The CEIBA cocktail approach allows metabolic capacity estimation of multiple CYP450 enzymes in a single sample analysis, but the attendant sample collection schemes for applications in diverse global settings are yet to be optimized. The present study aimed to select an optimal matrix to simultaneously analyze CYP450 enzyme activities so as to simplify the sampling schemes in the phenotyping protocol to enhance its throughput and feasibility in native populations or in remote and underserviced geographies and social contexts. We evaluated 13 Ecuadorians healthy volunteers for CYP1A2, CYP2C9, CYP2C19, and CYP2D6 genotypes and their metabolic phenotypes, including CYP3A4, in plasma and urine after administering one reduced dose of caffeine, losartan, omeprazole, and dextromethorphan. Pharmacokinetic analyses were performed, and the correlation between AUC parent/AUC metabolite and the ratio between concentrations of probe drugs and their corresponding metabolites at timepoints ranging from 0 to 12 hours post-dose were analyzed. A single sampling timepoint, 4 hours post-dose in plasma, was identified as optimal to reflect the metabolic activity of the attendant CYP450 enzymes. This study optimizes the CEIBA multiplexed phenotyping approach and offers new ways forward for integrated drug metabolism analyses, in the pursuit of global personalized medicine applications in resource-limited regions, be they in developed or developing countries.

A rapid and simple LC-MS/MS method for the simultaneous evaluation of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 hydroxylation capacity

Article

Full-text available

Mar 2014

Background: The analytical method here reported for the CEIBA cocktail approach has been developed and validated for the simultaneous determination of several probe drugs and their corresponding cytochrome P450 (CYP) enzyme-specific metabolites in just one analysis. This methodology has been proposed in order to overcome some drawbacks concerning the complexity and low throughput of analytical methodologies associated with previously proposed cocktail approaches. Methods & results: Caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6 and CYP3A4) and their corresponding metabolites were all analyzed in a single analytical run by gradient LC coupled to MS/MS. Sample preparation was conducted with solid-phase extraction. This method was fully validated and applied to CYP450 enzyme phenotyping of 20 healthy volunteers. Conclusion: This method constitutes a rapid and simplified analytical tool to be used with the CEIBA cocktail approach for the main CYP450 enzymes phenotyping.

Study Protocol for a Pilot, Open-Label, Prospective, and Observational Study to Evaluate the Pharmacokinetics of Drugs Administered to Patients during Extracorporeal Circulation; Potential of In Vivo Cytochrome P450 Phenotyping to Optimise Pharmacotherapy

Article

Full-text available

May 2019

Pharmacokinetic alterations of medications administered during surgeries involving cardiopulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO) have been reported. The impact of CPB on the cytochrome P450 (CYP) enzymes’ activity is the key factor. The metabolic rates of caffeine, dextromethorphan, midazolam, omeprazole, and Losartan to the CYP-specific metabolites are validated measures of in vivo CYP 1A2, 2D6, 3A4, 2C19, and 2C9 activities, respectively. The study aim is to assess the activities of major CYPs in patients on extracorporeal circulation (EC). This is a pilot, prospective, open-label, observational study in patients undergoing surgery using EC and patients undergoing laparoscopic cholecystectomy as a control group. CYP activities will be measured on the day, and 1–2 days pre-surgery/3–4 days post-surgery (cardiac surgery and Laparoscopic cholecystectomy) and 1–2 days after starting ECMO, 1–2 weeks after starting ECMO, and 1–2 days after discontinuation from ECMO. Aforementioned CYP substrates will be administered to the patient and blood samples will be collected at 0, 1, 2, 4, and 6 h post-dose. Major CYP enzymes’ activities will be compared in each participant on the day, and before/after surgery. The CYP activities will be compared in three study groups to investigate the impact of CYPs on EC.

Simultaneous Determination of Cytochrome P450 Oxidation Capacity in Humans: A Review on the Phenotyping Cocktail Approach

Article

Sep 2016
CURR PHARM BIOTECHNO

Intra- and/or inter-individual variability in drug response is mainly a result of either sub-therapeutic or supratherapeutic plasma levels of the active drugs and their metabolites, with this variability mainly being influenced by differences in the rate of drug metabolism. Indeed, drug metabolism is largely determined by genetic polymorphism in the CYP enzymes, which are responsible for approximately 85% of the drug metabolism process. However, this genetic heterogeneity can accurately predict actual drug metabolizing capacity (oxidation phenotype) for some individuals: poor metabolizers (PMs), who cannot produce the drug metabolizing enzymes, and 20% of ultra-rapid metabolizers. According to EMA recommendations, phenotyping procedures for drug interaction studies and clinical research are therefore required to obtain actual data on the main CYP enzymes. With this purpose, cocktail phenotyping approaches give information on the activity of different CYPs in just one experiment. In this review, the issues related to the phenotyping of the main CYP enzymes are reviewed, and the current in vivo phenotyping cocktails are analysed: the sampling procedures, probe drugs utilized, analytical techniques and main applications are also discussed. Based on this analysis, a fully validated cocktail approach to measure the metabolic activity of the main CYP enzymes and drug transporters is still required. This novel approach should fulfil certain conditions: a faster and simpler analytical methodology to obtain information on several CYPs in one experiment, minimal sample amounts, and minimal doses of optimal probe drugs.

Pharmakokinetik und Metabolismus von delta9-Tetrahydrocannabinolsäure A im Menschen

Thesis

Jan 2012

Ariane Wohlfarth

Ziel der vorliegenden Arbeit war es, die pharmakokinetischen Eigenschaften von delta9-Tetrahydrocannabinolsäure A (THCA) und ihren Metabolismus im Menschen aufzuklären. Ausgangspunkt war die These, dass THCA – die nicht psychoaktive, biogenetische Vorläufersubstanz von THC in der Cannabispflanze – ein Marker für kurz zurückliegenden Cannabiskonsum sein könnte. Ein solcher Marker wäre hilfreich, um einen akuten Cannabiseffekt anhand von Blut- oder Urinproben nachzuweisen, was bis heute ein Problem in der forensischen Praxis darstellt. Kernstück der Arbeit war eine Humanstudie mit 16 Probanden, die THCA oral und intravenös erhielten. Dafür waren im Vorfeld einige Vorarbeiten nötig: Um auf Enzymaktivitäten beruhende Unterschiede in der Pharmakokinetik zu erkennen, wurde als erstes eine Cocktail-Phänotypisierungsmethode für die fünf CYP-Isoenzyme 1A2, 2C9, 2C19, 2D6 und 3A4 entwickelt. Neben der Auswahl der Testsubstanzen, der Phänotypisierungsindices und eines Probennahmeschemas umfasste dieser Schritt auch die Entwicklung und Validierung einer effektiven Aufbereitungsmethode und einer empfindlichen Analysenmethode. Die für die Humanstudie benötigte THCA wurde mittels Flash-Chromatographie aus Cannabisrohmaterial isoliert. Mit zwei voneinander unabhängigen Systemen konnte schließlich hochreine THCA (Reinheit > 98,5%) gewonnen werden, die zur Herstellung der Prüfpräparate diente. Als Grundlage für die intravenöse Applikation der lipophilen und thermoinstabilen THCA erwies sich eine parenterale Fettemulsion als geeignet. Letzter Schritt war die Entwicklung einer ESI-LC-MS/MS-Methode zum empfindlichen Nachweis von THCA und ihren Metaboliten sowie die Validierung der Quantifizierungsmethode für THCA. Im Fokus standen die Optimierung der Probenaufbereitung mittels Proteinfällung und eine kurze Analysendauer. Die Humanstudie selbst war in zwei Teile unterteilt: Im ersten Abschnitt erhielten die Probanden 10 mg THCA oral, im zweiten 5 mg intravenös und gaben jeweils über 96 h Blut- und Urinproben ab. Die Phänotypisierung erfolgte zwischen beiden Abschnitten. Mit einer Nachweisgrenze von 0,1 ng/ml stellte sich bei der Messung der Proben schließlich heraus, dass THCA bei den meisten Probanden bis zur letzten Serumprobe nachweisbar ist. Im Urin fanden sich nur minimale THCA-Mengen, eine Umwandlung in vivo von THCA zu THC im Körper fand nicht statt. Es erfolgte eine pharmakokinetische Analyse gemäß den Prinzipien der „compartmental“ und der „non-compartmental analysis“, mit der erstmals grundlegende pharmakokinetische Parameter der THCA (AUC, Clearance, Verteilungsvolumina, Halbwertzeiten sowie Makro- und Mikrokonstanten des Kompartimentmodells) bestimmt wurden. Dabei zeigte sich, dass die Pharmakokinetik von THCA am besten durch ein Drei-Kompartimentmodell beschrieben werden kann, was auf die Existenz eines tiefen Kompartiments deutet. Die meisten Parameter ähnelten prinzipiell denen von THC. Der einzige fundamentale Unterschied zu THC war die Plasmaclearance, die für THCA zehnfach geringer ist als für THC. Die orale Bioverfügbarkeit lag bei ~ 40 %. Es bestätigte sich, dass der Metabolismus von THCA analog zu THC verläuft. Hauptmetabolite im Serum sind 11-OH-THCA, THCA-8-on und THCA-COOH-Glucuronid, THCA-COOH sowie 9,10 Bis-OH-Hexahydrocannabinolsäure A (HHCA). Wie zu erwarten war, sind die Hauptmetabolite im Urin vor allem Glucuronide, wobei THCA-COOH-Glucuronid, 11-OH-THCA-Glucuronid, 8- und 4’-OH-THCA-COOH-Glucuronid die intensivsten Signale lieferten. Oft fand sich bis zur letzten abgegebenen Urinprobe wie auch im Serum unglucuronidierte 9,10-Bis-OH-HHCA. Zusätzlich wurde ein in vitro Experiment mit isolierten CYP-Isoenzymen durchgeführt, in dem sich herausstellte, dass vor allem die drei CYP 450-Isoenzyme 2C9, 3A4 und 2C19 beim Abbau von THCA eine Rolle spielen. In einer statistischen Analyse wurden schließlich die aus der Phänotypisierungsstudie ermittelten Indices mit den THCA-Clearances korreliert und untersucht, ob sich weitere Hinweise auf die abbauenden Isoenzyme ergeben. Für CYP 3A4 ließ sich ein statistisch signifikanter Zusammenhang zwischen Enzymaktivität und Clearance bestätigen, für alle anderen Kombinationen war dies jedoch nicht möglich. Die Hypothese, THCA könnte als Marker für kurzzeitig zurückliegenden Cannabiskonsum dienen, bestätigte sich nicht. Dieser Anwendung steht die Umverteilung in ein tiefes Kompartiment entgegen, was zu Akkumulation und langer Nachweisbarkeit führt. Hilfreich für ein anderes Konzept könnten jedoch einige THCA-Metaboliten werden. Dieses Konzept verfolgt den Ansatz, verschiedene Cannabisinhaltsstoffe und nachrangig gebildete Metaboliten zu screenen und daraus Schlüsse über Konsumzeitpunkt und letztendlich den Effekt zu ziehen. In dieses Substanzspektrum könnten Metaboliten wie THCA-Glucuronid, 8alpha- bzw. 8beta-OH-THCA, 8,11-Bis-OH-THCA sowie 8- bzw. 4’-OH-THCA-COOH-Glucuronid, die sich bei allen Probanden nur kurz nach der Applikation fanden, eingeschlossen werden.

Pharmakokinetik und Metabolismus von Δ9-Tetrahydrocannabinolsäure A im Menschen

Article

Phenotyping Indices of CYP450 and P-Glycoprotein in Human Volunteers and in Patients Treated with Painkillers or Psychotropic Drugs

Article

Full-text available

Mar 2023

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.

A Cocktail Interaction Study Evaluating the Drug-Drug Interaction Potential of the Perpetrator Drug ASP8477 at Multiple Ascending Dose Levels

Article

Full-text available

Feb 2019

ASP8477 (molecular weight 325.36 g/mol) is a fatty acid amide hydrolase inhibitor intended for the treatment of neuropathic pain. Results from in vitro studies indicated that ASP8477 is a direct inhibitor of cytochrome P450 (CYP) 2C8, 2C9, 2C19, 2D6, and 3A4 enzymes at expected efficacious concentrations, with the strongest effect on CYP2C19; a phase 1 study confirmed ASP8477 to be a CYP2C19 inhibitor. To further evaluate the interaction potential of ASP8477, a cocktail interaction study was performed using the probe substrates of the validated Inje cocktail containing losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4). Because ASP8477 shows nonlinear pharmacokinetics, 3 doses (20, 60, and 100 mg) were evaluated. This study revealed changes in exposure (area under the concentration‐time curve) of the probe substrates after treatment with 20, 60, and 100 mg ASP8477, respectively, compared with substrates alone with geometric mean ratios of: midazolam, 119%, 151%, and 158%; losartan, 107%, 144%, and 190%; omeprazole, 213%, 456%, and 610%; and dextromethorphan, 138%, 340%, and 555% (with increasing doses, respectively). Overall, ASP8477 was a weak inhibitor for CYP3A4 and CYP2C9, a moderate to strong inhibitor for CYP2C19, and a weak to strong inhibitor for CYP2D6, with doses from 20 to 100 mg. This study confirmed that the Inje cocktail approach was able to detect relevant drug‐drug interactions impacting further development of ASP8477 and future therapeutic use. With the approach used here, the inhibiting effect of a perpetrator drug on different CYP enzymes can be evaluated, and at different doses, thereby supporting dose recommendations for potential interactions.

In vivo Phenotyping Methods: Cytochrome P450 Probes with Emphasis on the Cocktail Approach

Article

Full-text available

Feb 2017
CURR PHARM DESIGN

Background: Differences in drug response among patients are common. Most major drugs are effective in only 25 to 60 percent of the patients, in part due to the CYP enzymes, whose activity vary up to 50-fold between individuals for some index metabolic reactions. Several factors affect CYP activity, among which genetic polymorphisms have been studied as the major cause for long time. Age, gender, disease states, and environmental influences such as smoking, concomitant drug treatment or exposure to environmental chemicals are also important. Methods: This article reviews the available literature on multiple phenotypes assessment as an important tool to predict possible therapeutic failures or toxic reactions to conventional drug doses during patient evaluation. Results: Probe drugs can be used in various combinations allowing for the in vivo assessment of multiple pathways of drug metabolism in a single experiment, configuring a new tool known as phenotyping "cocktails". There are several drug cocktails with different advantages and disadvantages. Most of them have sufficient clinical evidence and data validation to support their use in clinical setting as a surrogate for the risk of adverse reaction in the course of therapy, leading to a better balance between efficacy and safety. Conclusion: Probes characteristics and metabolic ratio measurements are important in the evaluation of phenotyping cocktails as near-future applications.

CombiCap, a Novel Drug Formulation for the Basel Phenotyping Cocktail

Article

Aug 2016
INT J PHARMACEUT

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.

358 Psikiyatride sitokrom P450 fenotiplemesinin kullanımı Psikiyatride sitokrom P450 fenotiplemesinin kullanımı The use of cytochrome P450 phenotyping in psychiatry

Article

Full-text available

Jan 2014

The Use of Cytochrome P450 Phenotyping in Psychiatry

Article

Full-text available

Jan 2014

Individualization of pharmacotherapy is essential in order to optimize efficacy and minimize toxicity, especially for compounds with narrow therapeutic index. Cytochrome P450 phenotyping has been a valuable research tool and a way of assessing the genetic basis of metabolic capacity. Technique depends on estimating metabolic capacity based on conclusions drawn from another probe drug. Phenotyping allows estimation of the total influence of drug interactions, genetic polymorphisms, hepatic diseases and other factors altering pharmacokinetics. This requires the use of selective substrates for specific cytochrome enzymes. Recently some phenotyping methods are becoming widely used especially for the in vivo evaluation of multiple cytochrome enzymes by using probe cocktails.

Evaluation of drug-metabolizing enzyme hydroxylation phenotypes in Hispanic populations: the CEIBA cocktail

Article

Jun 2013
Drug Metabol Drug Interact

Abstract Interindividual differences in response to drug treatments are mainly caused by differences in drug metabolism, in which cytochrome P450 (CYP450) enzymes are involved. Genetic polymorphisms of these enzymes have a key role in this variability. However, environmental factors, endogenous metabolism and disease states also have a great influence on the actual drug metabolism rate (metabolic phenotype). Consequently, the genotype does not always correlate with the actual drug hydroxylation phenotype. In this sense, in vivo phenotyping strategies represent an alternative to evaluate the interindividual variability in drug metabolism. Therefore, the 'cocktail' approach is considered as an advantageous strategy to obtain actual and reliable information on several CYP activities in just one experiment. As reviewed, phenotyping studies on Latin-American populations, which comprise about 400 million people, are scarce, and only selective phenotyping methods were applied. Therefore, a novel cocktail approach is here proposed as a phenotyping tool to evaluate the relationship between genotype and phenotype of major CYP enzymes in Hispanic populations. This determination will allow adaptation of drug therapies to these populations and consequently to benefit from the application of pharmacogenetics in the reduction of drug adverse effects and in the improvement of therapeutic responses.

Recent Progress on Bioinformatics, Functional Genomics, and Metabolomics Research of Cytochrome P450 and its Impact on Drug Discovery

Article

Jun 2012
CURR TOP MED CHEM

The cytochrome P450 superfamily is responsible primarily for human drug metabolism, which is of critical importance for the drug discovery and development. Rapid advancement of bioinformatics, functional genomics and metabolomics has been made over the last decade. These disciplines are essential in target identification, lead discovery and optimization. In this review, we summarize the recent progress on cytochrome P450 and its role on drug metabolism in the context of bioinformatics, functional genomics and metabolomics. Data are integrated into various databases and web-based platforms on cytochrome P450. These research tools and resources are playing an increasingly important role in drug discovery, and are helping in achieving the ultimate goal of personalized medicine, that is, to prescribe personalized drugs according to each person's genetic makeup, metabolic level, and drug disposition.

Cocktail Approach for In Vivo Phenotyping of 5 Major CYP450 Isoenzymes: Development of an Effective Sampling, Extraction, and Analytical Procedure and Pilot Study With Comparative Genotyping

Article

Sep 2011
J CLIN PHARMACOL

In this study, the authors developed a phenotyping method for CYP1A2, 2C9, 2C19, 2D6, and 3A4 using a cocktail of 100 mg caffeine, 125 mg tolbutamide, 20 mg omeprazole, 30 mg dextromethorphan, and 2 mg midazolam. A simple sampling scheme was established collecting 3 blood samples at 0, 4, and 24 hours followed by solid-phase extraction and liquid chromatography/tandem mass spectrometry analysis. After addition of 8 deuterated internal standards and extraction, the analytes were separated using gradient elution with ammonium acetate and methanol. Data acquisition was performed on a triple quadrupole linear ion trap mass spectrometer in multiple-reaction monitoring mode with positive electrospray ionization. The assay was validated according to international guidelines: limits of quantification (LOQs) were between 0.25 and 1.0 ng/mL for all analytes, except for paraxanthine and caffeine (20 ng/mL). Extraction efficiencies ranged between 77% and 103% and matrix effects between 23% and 95%; precision and accuracy data fulfilled accepted criteria. Calibration curves from LOQ to 1000 ng/mL were established for undiluted and 1:10 diluted plasma (r > 0.998). The method was tested in a pilot study with 14 volunteers. Additional genotyping of the probands generally demonstrated good accordance with the measured phenotyping indices but also disclosed certain contradictory results.

Cardiac Arrest and Therapeutic Hypothermia Decrease Isoform-Specific Cytochrome P450 Drug Metabolism

Article

Aug 2011
DRUG METAB DISPOS

Mild therapeutic hypothermia is emerging clinically as a neuroprotection therapy for individuals experiencing cardiac arrest (CA); however, its effects combined with disease pathogenesis on drug disposition and response have not been fully elucidated. We determined the activities of four major hepatic-metabolizing enzymes (CYP3A, CYP2C, CYP2D, and CYP2E) during hypothermia after experimental CA in rats by evaluating the pharmacokinetics of their probe drugs as a function of altered body temperature. Animals were randomized into sham normothermia (37.5-38°C), CA normothermia, sham hypothermia (32.5-33°C), and CA hypothermia groups. Probe drugs (midazolam, diclofenac, dextromethorphan, and chlorzoxazone) were given simultaneously by intravenous bolus after temperature stabilization. Multiple blood samples were collected between 0 and 8 h after drug administration. Pharmacokinetic (PK) analysis was conducted using a noncompartmental approach and population PK modeling. Noncompartmental analysis showed that the clearance of midazolam (CYP3A) in CA hypothermia was reduced from sham normothermia rats (681.6 ± 190.0 versus 1268.8 ± 348.9 ml · h(-1) · kg(-1), p < 0.05). The clearance of chlorzoxazone (CYP2E) in CA hypothermia was also reduced from sham normothermia rats (229.6 ± 75.6 versus 561.89 ± 215.9 ml · h(-1) · kg(-1), p < 0.05). Population PK analysis further demonstrated the decreased clearance of midazolam (CYP3A) was associated with CA injury (p < 0.05). The decreased clearance of chlorzoxazone (CYP2E1) was also associated with CA injury (p < 0.01). Hypothermia was found to be associated with the decreased volume of distribution of midazolam (V(1)), dextromethorphan (V(1)), and peripheral compartment for chlorzoxazone (V(2)) (p < 0.05, p < 0.05, and p < 0.01, respectively). Our data indicate that hypothermia, CA, and their interaction alter cytochrome P450-isoform specific activities in an isoform-specific manner.

Pharmacokinetics of Caffeine: A Systematic Analysis of Reported Data for Application in Metabolic Phenotyping and Liver Function Testing

Article

Full-text available

Apr 2022

Caffeine is by far the most ubiquitous psychostimulant worldwide found in tea, coffee, cocoa, energy drinks, and many other beverages and food. Caffeine is almost exclusively metabolized in the liver by the cytochrome P-450 enzyme system to the main product paraxanthine and the additional products theobromine and theophylline. Besides its stimulating properties, two important applications of caffeine are metabolic phenotyping of cytochrome P450 1A2 (CYP1A2) and liver function testing. An open challenge in this context is to identify underlying causes of the large inter-individual variability in caffeine pharmacokinetics. Data is urgently needed to understand and quantify confounding factors such as lifestyle (e.g., smoking), the effects of drug-caffeine interactions (e.g., medication metabolized via CYP1A2), and the effect of disease. Here we report the first integrative and systematic analysis of data on caffeine pharmacokinetics from 141 publications and provide a comprehensive high-quality data set on the pharmacokinetics of caffeine, caffeine metabolites, and their metabolic ratios in human adults. The data set is enriched by meta-data on the characteristics of studied patient cohorts and subjects (e.g., age, body weight, smoking status, health status), the applied interventions (e.g., dosing, substance, route of application), measured pharmacokinetic time-courses, and pharmacokinetic parameters (e.g., clearance, half-life, area under the curve). We demonstrate via multiple applications how the data set can be used to solidify existing knowledge and gain new insights relevant for metabolic phenotyping and liver function testing based on caffeine. Specifically, we analyzed 1) the alteration of caffeine pharmacokinetics with smoking and use of oral contraceptives; 2) drug-drug interactions with caffeine as possible confounding factors of caffeine pharmacokinetics or source of adverse effects; 3) alteration of caffeine pharmacokinetics in disease; and 4) the applicability of caffeine as a salivary test substance by comparison of plasma and saliva data. In conclusion, our data set and analyses provide important resources which could enable more accurate caffeine-based metabolic phenotyping and liver function testing.

Pharmacokinetics of Caffeine: A Systematic Analysis of Reported Data for Application in Metabolic Phenotyping and Liver Function Testing

Article

Full-text available

Feb 2022

Pharmacokinetics of caffeine: A systematic analysis of reported data for application in metabolic phenotyping and liver function testing

Preprint

Full-text available

Jul 2021

Caffeine is by far the most ubiquitous psychostimulant worldwide found in tea, coffee, cocoa, energy drinks, and many other beverages. Caffeine is almost completely metabolized in the liver by the cytochrome P-450 enzyme system mainly to paraxanthine and the additional products theobromine and theophylline. Besides its stimulating properties, two important applications of caffeine are metabolic phenotyping of cytochrome P450 1A2 (CYP1A2) and liver function testing. An open challenge in this context is to identify underlying causes of the large inter-individual variability in caffeine pharmacokinetics. Data is urgently needed to understand and quantify confounding factors such as lifestyle (e.g. smoking), the effects of drug-caffeine interactions (e.g. medication metabolized via CYP1A2), and the effect of disease. Here we report the first integrative and systematic analysis of data on caffeine pharmacokinetics from 147 publications and provide a comprehensive high-quality data set on the pharmacokinetics of caffeine, caffeine metabolites, and their metabolic ratios in human adults. The data set is enriched by meta-data on the characteristics of studied patient cohorts and subjects (e.g. age, body weight, smoking status, health status), the applied interventions (e.g. dosing, substance, route of application), measured pharmacokinetic time-courses, and pharmacokinetic parameters (e.g. clearance, half-life, area under the curve). We demonstrate via multiple applications how the data set can be used to solidify existing knowledge and gain new insights relevant for metabolic phenotyping and liver function testing based on caffeine. Specifically, we analyzed (i) the alteration of caffeine pharmacokinetics with smoking and oral contraceptive use; (ii) drug-drug interactions with caffeine as possible confounding factors of caffeine pharmacokinetics or source of adverse effects; (iii) alteration of caffeine pharmacokinetics in disease; and (iv) the applicability of caffeine as a salivary test substance by comparison of plasma and saliva data. In conclusion, our data set and analyses provide important resources which could enable more accurate caffeine-based metabolic phenotyping and liver function testing.

Differential inhibition of human CYP2C8 and molecular docking interactions elicited by sorafenib and its major N-oxide metabolite

Article

Feb 2021
CHEM-BIOL INTERACT

The tyrosine kinase inhibitor sorafenib (SOR) is being used increasingly in combination with other anticancer agents like paclitaxel, but this increases the potential for drug toxicity. SOR inhibits several human CYPs, including CYP2C8, which is a major enzyme in the elimination of oncology drugs like paclitaxel and imatinib. It has been reported that CYP2C8 inhibition by SOR in human liver microsomes is potentiated by NADPH-dependent biotransformation. This implicates a SOR metabolite in enhanced inhibition, although the identity of that metabolite is presently unclear. The present study evaluated the capacity of the major N-oxide metabolite of SOR (SNO) to inhibit CYP2C8-dependent paclitaxel 6α-hydroxylation. The IC50 of SNO against CYP2C8 activity was found to be 3.7-fold lower than that for the parent drug (14 μM versus 51 μM). In molecular docking studies, both SOR and SNO interacted with active site residues in CYP2C8, but four additional major hydrogen and halogen bonding interactions were identified between SNO and amino acids in the B-B’ loop region and helixes F’ and I that comprise the catalytic region of the enzyme. In contrast, the binding of both SOR and SNO to active site residues in the closely related human CYP2C9 enzyme was similar, as were the IC50s determined against CYP2C9-mediated losartan oxidation. These findings suggest that the active metabolite SNO could impair the elimination of coadministered drugs that are substrates for CYP2C8, and mediate toxic adverse events, perhaps in those individuals in whom SNO is formed extensively.

An improved cytochrome P450-phenotyping cocktail with a simplified and highly sensitive UHPLC-MS/MS assay in human plasma

Article

Feb 2020
J CHROMATOGR B

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.

Inhibition of Hepatic CYP2D6 by the Active N-Oxide Metabolite of Sorafenib

Article

Nov 2019
AAPS J

The multikinase inhibitor sorafenib (SOR) is used to treat patients with hepatocellular and renal carcinomas. SOR undergoes CYP-mediated biotransformation to a pharmacologically active N-oxide metabolite (SNO) that has been shown to accumulate to varying extents in individuals. Kinase inhibitors like SOR are frequently coadministered with a range of other drugs to improve the efficacy of anticancer drug therapy and to treat comorbidities. Recent evidence has suggested that SNO is more effective than SOR as an inhibitor of CYP3A4-mediated midazolam 1′-hydroxylation. CYP2D6 is also reportedly inhibited by SOR. The present study assessed the possibility that SNO might contribute to CYP2D6 inhibition. The inhibition kinetics of CYP2D6-mediated dextromethorphan O-demethylation were analyzed in human hepatic microsomes, with SNO found to be ~ 19-fold more active than SOR (Kis 1.8 ± 0.3 μM and 34 ± 11 μM, respectively). Molecular docking studies of SOR and SNO were undertaken using multiple crystal structures of CYP2D6. Both molecules mediated interactions with key amino acid residues in putative substrate recognition sites of CYP2D6. However, a larger number of H-bonding interactions was noted between the N-oxide moiety of SNO and active site residues that account for its greater inhibition potency. These findings suggest that SNO has the potential to contribute to pharmacokinetic interactions involving SOR, perhaps in those individuals in whom SNO accumulates.

Sorafenib N-Oxide Is an Inhibitor of Human Hepatic CYP3A4

Article

Mar 2019
AAPS J

The multi-kinase inhibitor sorafenib (SOR) is clinically important in the treatment of hepatocellular and renal cancers and undergoes CYP3A4-dependent oxidation in liver to the pharmacologically active N-oxide metabolite (SNO). There have been reports that kinase inhibitors such as SOR may precipitate pharmacokinetic interactions with coadministered drugs that compete for CYP3A4-mediated biotransformation, but these occur non-uniformly in patients. Clinical evidence also indicates that SNO accumulates in serum of some patients during prolonged SOR therapy. In this study undertaken in hepatic microsomes from individual donors, we assessed the possibility that SNO might contribute to pharmacokinetic interactions mediated by SOR. Enzyme kinetics of CYP3A4-mediated midazolam 1′-hydroxylation in individual human hepatic microsomes were analyzed by non-linear regression and appropriate replots. Thus, SNO and SOR were linear-mixed inhibitors of microsomal CYP3A4 activity (Kis 15 ± 4 and 33 ± 14 μM, respectively). To assess these findings, further molecular docking studies of SOR and SNO with the 1TQN crystal structure of CYP3A4 were undertaken. SNO elicited a larger number of interactions with key amino acid residues located in substrate recognition sequences of the enzyme. In the optimal docking pose, the N-oxide moiety of SNO was also found to interact directly with the heme moiety of CYP3A4. These findings suggest that SNO could contribute to pharmacokinetic interactions involving SOR, perhaps in individuals who produce high circulating concentrations of the metabolite.

Use of Cocktail Probe Drugs for Indexing Cytochrome P450 Enzymes in Clinical Pharmacology Studies – Review of Case Studies

Article

Nov 2018
Drug Metabol Lett

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.

Post-modern Medicolegal and Forensic Toxicology

Chapter

Jan 2017

Hans Maurer

The ongoing progress in analytical and life sciences is opening up new perspectives in post-modern medico-legal and forensic toxicology. In times of personalized medicine, the interpretation of analytical results in clinical and forensic cases should also be based on genetic aspects, pharmacogenomics in particular. Pharmacologic and toxic effects of drugs or poisons may be influenced by the genotype and phenotype of an individual, but also by the isoenzymes involved in their metabolism and membrane transport. Further individual factors such as body mass, age, sex, kidney and liver function, and drug-drug (food-drug) interactions may have an impact. Detailed knowledge of all these factors is a prerequisite for evidence-based case interpretation. In this chapter, the current knowledge of possible risks in variations of the effects of relevant therapeutic drugs, herbal drugs, and drugs of abuse will be presented. A critical discussion of the impact on the interpretation of analytical results in clinical and forensic toxicology will follow.

Impact of Tetrahydropalmatine on the Pharmacokinetics of Probe Drugs for CYP1A2, 2D6 and 3A Isoenzymes in Beagle Dogs

Article

Mar 2016
PHYTOTHER RES

Tetrahydropalmatine (Tet) exhibit multiple pharmacological activities and is used frequently by clinical practitioners. In this study, we evaluate the in vivo effects of single and repeated oral Tet administrations on CYP1A2, 2D6 and 3A activities in six beagle dogs in a randomized, controlled, open-label, crossover study. A cocktail approach, with dosages of the probe drugs caffeine (3.0 mg/kg), metoprolol (2.33 mg/kg) and midazolam (0.45 mg/kg), was used to measure cytochrome P450 (CYP) metabolic activities. The cocktail was administered orally as a single dose (12 mg/kg) 1 day prior to and 4 days after repeated oral Tet administrations (12 mg/kg three times daily). The probe drugs and their metabolites in plasma were quantified simultaneously by a validated HPLC technique, and non-compartmental parameters were used to evaluate metabolic variables for assessment of CYP inhibition or induction. Tet had no or minor impact on the pharmacokinetics and metabolism of the probe drugs caffeine and metoprolol, CYP1A2 and CYP2D6 substrates, respectively. However, Tet increased AUC0-24 h and decreased AUCratio(0-24 h) (1-hydroxymidazolam/midazolam ratio) for midazolam statistically significant, both in single or multiple dosing of Tet, with up to 39 or 57% increase for AUC0-24 h and 29% or 22 decrease for AUCratio(0-24 h), respectively, in line with previous in vitro findings for its CYP3A4 inhibition. The extensive use of Tet and herbal medicines containing Tet makes Tet a candidate for further evaluation of CYP3A-mediated herb-drug interactions. Copyright © 2016 John Wiley & Sons, Ltd.

Chronic administration of caderofloxacin, a new fluoroquinolone, increases hepatic CYP2E1 expression and activity in rats

Article

Feb 2016

Aim: Caderofloxacin is a new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of caderofloxacin interacting with co-administered drugs. Methods: Male rats were treated with caderofloxacin (9 mg/kg, ig) once or twice daily for 14 consecutive days. The effects of caderofloxacin on CYP3A, 2D6, 2C19, 1A2, 2E1 and 2C9 were evaluated using a "cocktail" of 6 probes (midazolam, dextromethorphan, omeprazole, theophylline, chlorzoxazone and diclofenac) injected on d 0 (prior to caderofloxacin exposure) and d 15 (after caderofloxacin exposure). Hepatic microsomes from the caderofloxacin-treated rats were used to assess CYP2E1 activity and chlorzoxazone metabolism. The expression of CYP2E1 mRNA and protein in hepatic microsomes was analyzed with RT-PCR and Western blotting, respectively. Results: Fourteen-day administration of caderofloxacin significantly increased the activity of hepatic CYP2E1, leading to enhanced metabolism of chlorzoxazone. In vitro microsomal study confirmed that CYP2E1 was a major metabolic enzyme involved in chlorzoxazone metabolism, and the 14-d administration of caderofloxacin significantly increased the activity of CYP2E1 in hepatic microsomes, resulting in increased formation of 6-hydroxychlorzoxazone. Furthermore, the 14-d administration of caderofloxacin significantly increased the expression of CYP2E1 mRNA and protein in liver microsomes, which was consistent with the pharmacokinetic results. Conclusion: Fourteen-day administration of caderofloxacin can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a potential drug-drug interaction mediated by CYP2E1 induction should be considered.

Evaluation of the effects of therapeutic hypothermia and cardiac arrest on specific cytochrome P450 isoform activity

Article

Sep 2011

Jiangquan Zhou

Therapeutic hypothermia (TH, 32-35C) mediated neuroprotection after brain ischemia has been demonstrated by both preclinical and clinical studies. Studies to identify the effects of mild hypothermia on CYP450 metabolism in humans are limited and the translational significance of the observations in the rat model remains to be identified. The goal of this study is to evaluate the effects of therapeutic hypothermia and relevant disease model cardiac arrest (CA) on hepatic drug metabolism. Specifically, this study evaluated the effects of therapeutic hypothermia on specific CYP450-mediated drug metabolism in preclinical and in translational clinical studies. There are several conclusions can be made based on our study results. Mild hypothermia and cardiac arrest alter CYP activity in an isoform specific manner. Magnitude of the reduction is likely temperature, and extraction ratio specific. In animal model, the combination of hypothermia (33C) and CA was most likely to be associated with isoform specific decrease of enzyme activities with greater changes observed for CYP3A and CYP2E1. Hypothermia decreased the volume of distribution of multiple probe substrates. In healthy volunteers, we found significant correlation between temperature and the clearance of CYP3A probe drug midazolam. Short duration hypothermia studies with hepatically eliminated drugs suggest ~11% reduction in clearance per C change. Microdosed cocktail probes are likely to be very useful in PK study design in critically ill patients due to the potential linearity of PK of probe drugs and no drug-drug interaction in the cocktail combination. In addition, effect of cooling on receptor dynamic response is unknown on the current data. In conclusion, our results have shown the effect of CA and hypothermia with interaction on isoform specific activity. Given the prominent role of mild hypothermia in the management of patients with CA, further translational studies using clinically relevant drugs and pharmacokinetics-pharmacodynamics modeling are vital for validation and prediction of drug dosing in different disease and temperature states.

Metabolism of Drugs and Other Xenobiotics

Chapter

Jun 2012

Michael Murray

Simultaneous In Vivo Phenotyping of CYP Enzymes

Article

Mar 2013

As major determinants of the duration of drug action the CYP enzymes strongly influence drug efficacy and toxicity. In vivo phenotyping for CYP activities using cocktails of well-tolerated CYP-specific substrates may be valuable in the development of personalized medicine protocols, particularly for drugs that have significant toxicity profiles. However, the use of the cocktail approach in the clinic is dependent on the rapid provision of patient-specific information to the clinician. Here we describe the application of liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the simultaneous phenotyping of five major drug-metabolizing CYPs in patients within a 5-min assay.

High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC-MS/MS

Article

Jul 2012

A rapid LC-MS/MS assay method for simultaneous quantification of morphine, fentanyl, midazolam and their major metabolites: morphine-3-β-D-glucuronide (M3G), morphine-6-β-D-glucuronide (M6G), norfentanyl, 1'-hydroxymidazolam (1-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) in samples of human plasma has been developed and validated. Robotic on-line solid phase extraction (SPE) instrumentation was used to elute the eight analytes of interest from polymeric SPE cartridges to which had been added aliquots (150 μL) of human plasma and aliquots (150 μL) of a mixture of two internal standards, viz. morphine-d3 (200 ng/mL) and 1'-hydroxymidazolam-d5 (50 ng/mL) in 50 mM ammonium acetate buffer (pH 9.25). Cartridges were washed using 10% methanol in ammonium acetate buffer, pH 9.25 (1 mL, 2 mL/min) before elution with mobile phase comprising 0.1% formic acid in water (A) and acetonitrile (B) with a flow rate of 0.6 mL/min using an 11.5 min run time. The analytes were separated on a C18 X-Terra® analytical column. The linear concentration ranges were 0.5-100 ng/mL for fentanyl, norfentanyl and midazolam; 1-200 ng/mL for 4-hydroxymidazolam, 2.5-500 ng/mL for 1'-hydroxymidazolam and 3.5-700 ng/mL for morphine, M3G, and M6G. The method showed acceptable within-run and between-run precision (relative standard deviation (RSD) and accuracy <20%) for quality control (QC) samples spiked at concentrations of 80% and 50% of the ULOQ, 3 times higher than the LLOQ, and also at the LLOQ. Furthermore, analytes were stable in samples (after mixing with internal standard) for at least 48 h in the autosampler (except for 4-hydroxymidazolam which decreased by 22% after 24 h), 5 h at room temperature and after three cycles of freeze and thaw. No autosampler carry-over was observed and the absolute recovery (the area ratio of analyte in plasma relative to that in ammonium acetate buffer 50 mM, pH 9.25) was in the range 40% (midazolam) to 110% (morphine). The assay was applied successfully to the measurement of the analytes of interest in plasma samples from patients on extracorporeal membrane oxygenation (ECMO).

High-sensitivity liquid chromatography-tandem mass spectrometry for the simultaneous determination of five drugs and their cytochrome P450-specific probe metabolites in human plasma

Article

Mar 2012

A sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method with electrospray ionization was developed for the simultaneous quantitation of five probe drugs and their metabolites in human plasma for assessing the in vivo activities of cytochrome P450 (CYP). CYP isoform specific substrates and their metabolites of CYP1A2 (caffeine), CYP2C9 (losartan), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan) and CYP3A (midazolam) were all simultaneously analyzed using LC-MS/MS after administration of a mixture of five drugs (i.e., a "cocktail approach") to healthy volunteers. The assay uses propranolol as an internal standard; dual liquid extraction; a Xbridge MS C(18) (100 mm × 2.1mm, 3.5 μm) column; a gradient mobile phase of 0.1% formic acid/acetonitrile (7/3→3/7); mass spectrometric detection in positive ion mode. The method was validated from 5 to 500 ng/mL for caffeine and paraxanthine, 0.1-40 ng/mL for losartan and EXP3174, 0.05-20 ng/mL for omeprazole and 5-hydroxyomeprazole, 0.008-0.8 ng/mL for dextromethorphan and dextrorphan, 0.01-1.0 ng/mL for midazolam, and 0.04-4 ng/mL for 1'-hydroxymidazolam. The intra- and inter-day precision over the concentration ranges for all analytes were lower than 12.5% and 13.8% (relative standard deviation, %RSD), and accuracy was between 86.5% and 108.4% and between 87.0% and 107.0%, respectively. This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.

Quantification of femtomolar concentrations of the CYP3A substrate midazolam and its main metabolite 1'-hydroxymidazolam in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry

Article

Mar 2012
ANAL BIOANAL CHEM

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1′-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 μm UPLC® column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1′-hydroxymidazolam were quantified using deuterium- and 13C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05–250 pg/mL) and 1′-hydroxymidazolam (0.25–125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1′-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1–100 μg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1′-hydroxymidazolam for at least 10 h, even after the administration of only 1 μg of midazolam. Figure UPLC/MS/MS quantification of femtomolar midazolam plasma concentrations

Repeated administration of berberine inhibits cytochromes P450 in humans

Article

Aug 2011
EUR J CLIN PHARMACOL

Berberine is a plant alkaloid that is widely used to treat gastrointestinal infections, diabetes, hypertension, and hypercholesterolemia. Many studies have reported interactions between berberine-containing products and cytochromes P450 (CYPs), but little is known about whether berberine alters CYP activities in humans, especially after repeated doses. A two-phase randomized-crossover clinical study in healthy male subjects was performed. After 2 weeks of berberine (300 mg, t.i.d., p.o.) administration, midazolam, omeprazole, dextromethorphan, losartan, and caffeine were used to evaluate enzyme activities of CYP3A4, 2C19, 2D6, 2C9, and CYP1A2, respectively. A decrease in CYP2D6 activity was observed as the 0-8 h urinary dextromethorphan/dextrorphan increased ninefold (P < 0.01). In addition, losartan/E-3174 ratio doubled (P < 0.01) after BBR administration, indicating a decrease in CYP2C9 activity. CYP3A4 activity was also inhibited, as the C(max), AUC(0-∞), and AUC(0-12) of midazolam were increased 38% (P < 0.05), 40% (P < 0.01), and 37% (P < 0.05) after BBR treatment, respectively. Compared with the placebo period, the T(max) and T(1/2) of midazolam during BBR administration were prolonged from 3.03 ± 0.27 to 3.66 ± 0.37 h and 0.66 ± 0.08 to 0.99 ± 0.09 h, respectively; the oral clearance of midazolam was decreased 27% (P < 0.05); and the phenotypic indices of 1 h midazolam/1'-hydroxymidazolam increased 59% (P < 0.01). There were no statistically significant differences in the pharmacokinetic parameters of the other probe drugs between placebo and the BBR-treated group. Repeated administration of berberine (300 mg, t.i.d., p.o.) decreased CYP2D6, 2C9, and CYP3A4 activities. Drug-drug interactions should be considered when berberine is administered.

Simultaneous quantification of dextromethorphan and its metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in human plasma by ultra performance liquid chromatography/tandem triple-quadrupole mass spectrometry

Article

Sep 2010

A rapid and sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the simultaneous quantitative determination of dextromethorphan (DM) and its metabolites dextrorphan (DX), 3-methoxymorphinan (3MM) and 3-hydroxymorphinan (3HM), in human lithium heparinized plasma. The extraction involved a simple liquid-liquid extraction with 1 ml n-butylchloride from 200μl aliquots of plasma, after the addition of 20 μl 4% (v/v) ammonium hydroxide and 100 μl stable labeled isotopic internal standards in acetonitrile. Chromatographic separations were achieved on an Aquity UPLC(®) BEH C(18) 1.7 μm 2.1 mm x 100mm column eluted at a flow-rate of 0.250 ml/min on a gradient of acetonitrile. The overall cycle time of the method was 7 min, with elution times of 1.3min for DX and 3HM, 2.8 min for 3MM and 2.9min for DM. The multiple reaction monitoring transitions were set at 272>215 (m/z), at 258>133 (m/z), at 258>213 (m/z) and at 244>157 (m/z) for DM, DX, 3MM and 3HM, respectively. The calibration curves were linear (r²≥0.995) over the range of 0.500-100 nM with the lower limit of quantitation validated at 0.500 nM for all compounds, which is equivalent to 136, 129, 129 and 122 pg/ml for DM, DX, 3MM and 3HM, respectively. Extraction recoveries were constant, but ranged from 39% for DM to 83% for DX. The within-run and between-run precisions were within 11.6%, while the accuracy ranged from 92.7 to 110.6%. The applicability of the bioanalytical method was demonstrated and is currently implemented in a clinical trial to study DM as probe-drug for individualized tamoxifen treatment in breast cancer patients.

The Participation of Cytochrome P450 3A4 in Clozapine Biotransformation Is Detected in People With Schizophrenia by High-Throughput In Vivo Phenotyping

Article

Oct 2010
J CLIN PSYCHOPHARM

Effect of propiverine on cytochrome P450 enzymes: A cocktail interaction study in healthy volunteers

Article

Full-text available

Jan 2006

The present study was conducted to assess a possible in vivo effect of propiverine, an anticholinergic drug to treat urinary incontinence and related disorders, on the activity of intestinal CYP3A4 and of hepatic CYP3A4, CYP2C9, CYP2C19, and CYP1A2. The activity of the respective cytochromes P450 was measured using the following metrics of selective substrates given as a tailored low-dose phenotyping cocktail: intestinal availability of midazolam (2 mg orally), clearance of midazolam (1 mg i.v.), apparent clearance of tolbutamide (125 mg orally), urinary excretion of 4'-hydroxymephenytoin 0 to 8 h postdose (50 mg of mephenytoin orally), and the paraxanthine/caffeine plasma ratio 6 h postdose (150 mg of caffeine orally). These metrics were determined in 16 healthy young men at the end of 7 days of treatment with 15 mg of propiverine (test) or placebo (reference) twice daily. All phenotyping drugs were quantified by liquid chromatography-tandem mass spectrometry. Chronic propiverine treatment reduced hepatic and intestinal CYP3A4 activity slightly to 0.89-fold and 0.80-fold, respectively [90% confidence interval (CI) for test/reference ratios 0.85-0.93 and 0.72-0.89], with the combined effect resulting in a 1.46-fold increase in area under the curve of oral midazolam (90% CI 1.36-1.57). Propiverine had no relevant effect on CYP2C9, CYP2C19, and CYP1A2 (90% CI for test/reference ratios 0.93-1.00, 0.84-0.96, and 0.97-1.07, respectively). All study drugs were well tolerated. In conclusion, propiverine has a minor potential to cause drug-drug interactions.

Maribavir pharmacokinetics and the effects of multiple-dose maribavir on cytochrome P450 (CYP) 1A2, CYP 2C9, CYP 2C19, CYP 2D6, CYP 3A, N-acetyltransferase-2, and xanthine oxidase activities in healthy adults

Article

Full-text available

May 2006

Maribavir (1263W94, VP-41263) is an oral anticytomegalovirus agent under clinical development. The pharmacokinetics and safety of maribavir and the effects of maribavir on the activities of cytochrome P450 (CYP) 1A2, CYP 2C9, CYP 2C19, CYP 2D6, CYP 3A, N-acetyltransferase-2 (NAT-2), and xanthine oxidase (XO) were evaluated in a randomized, double-blind, placebo-controlled study. Twenty healthy subjects received a five-drug phenotyping cocktail of caffeine (CYP 1A2, NAT-2, XO), warfarin plus vitamin K (CYP 2C9), omeprazole (CYP 2C19), dextromethorphan (CYP 2D6), and midazolam (CYP 3A) 4 days before and after 7 days of treatment with maribavir at 400 mg twice daily (16 subjects) or placebo (4 subjects) for 10 days. Maribavir did not affect the CYP 1A2, CYP 2C9, CYP 3A, NAT-2, or XO activities. Bioequivalence was not demonstrated for CYP 2C19 and CYP 2D6, suggesting a decrease or inhibition of CYP 2C19 and CYP 2D6 activities. The pharmacokinetics of maribavir following a single dose and after 10 days of treatment were similar, with minimal accumulation at steady state. Maribavir was safe and well tolerated. Taste disturbance was the most frequently reported adverse event. These results will further guide evaluation of the drug interaction potential and clinical development of maribavir.

Limited sampling strategy to predict AUC of the CYP3A phenotyping probe midazolam in adults: Application to various assay techniques

Article

Jan 2002

Midazolam clearance is used to phenotype hepatic CYP3A activity but requires multiple plasma samples following a single intravenous dose. The authors evaluated the use of a limited sampling scheme, using different assay techniques to determine the reproducibility of such a strategy in estimating midazolam AUC. Seventy-three healthy adults received midazolam as a single intravenous bolus dose. At least eight plasma samples were collected from each subject and were assayed using either LC/MS/MS or electron capture gas chromatography. Eleven subjects were randomly selected for the training set using stepwise linear regression to determine relationships between midazolam plasma concentrations and AUC. Validation of the predictive equations was done using the remaining 62 subjects. Mean percent error (MPE), mean absolute error (MAE), and root mean square error (RMSE) were calculated to determine bias and precision. Based on the training set, five models were generated with coefficients of determination ranging from 0.87 to 0.95. Validation showed that MPE, MAE, and RMSE values were acceptable for three of the models. Intrasubject reproducibility was good. In addition, training set data from one institution were able to predict data from the other two institutions using other assay techniques. Minimized plasma sampling may provide a simpler method for estimating midazolam AUC for CYP3A phenotyping. A limited sampling strategy is more convenient and cost-effective than standard sampling strategies and is applicable to more than one assay technique.

PII-37Validation of incorporating flurbiprofen into the Pittsburgh cocktail

Article

Feb 2006

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.

Combined phenotypic assessment of CYP1A2, CYP2C19, CYP2D6, CYP3A, N-acetyltransferase-2, and xanthine oxidase with the "Cooperstown cocktail"

Article

Oct 2000

Background: Simultaneous administration of several probes enhances the utility of phenotyping, but poor specificity, side effects, and use of drugs not approved by the Food and Drug Administration limit the usefulness of prior phenotyping cocktails. Objectives: To evaluate potential drug-drug interactions associated with use of a cocktail of caffeine, omeprazole, dextromethorphan, and midazolam for simultaneous phenotyping of CYP1A2, CYP2C19, CYP2D6, CYP3A, N-acetyltransferase-2, and xanthine oxidase. Methods: Twelve subjects received caffeine + dextromethorphan, omeprazole, and midazolam (each alone), and a cocktail of caffeine + dextromethorphan + omeprazole + midazolam. Blood samples were collected at 120 minutes for omeprazole and 5/-hydroxyomeprazole, and at 0, 5, 30, 60, 120, 240, 300, and 360 minutes for midazolam. Twelve-hour urine samples were collected for analysis of dextromethorphan, caffeine, and metabolites. Results: The median CYP1A2 metabolic ratio after administration of caffeine + dextromethorphan was not significantly different from that obtained with the cocktail (P = .84). Likewise, the median N-acetyltransferase-2, xanthine oxidase, and CYP2D6 metabolic ratios were not significantly different after cocktail administration (P = .977 for each N-acetyltransferase-2; P = .795 for xanthine oxidase; P = .75 for CYP2D6). The median CYP2C19 metabolic ratio after cocktail administration was not significantly different from that obtained after omeprazole administered alone (P = 1.000). Also, midazolam plasma clearance was not significantly different after cocktail administration compared with that after administration of midazolam alone (P = .708). The only side effect was sedation, which was associated with intravenous midazolam and occurred to a similar extent after both individual and cocktail phenotyping. Conclusions: These results indicate no pharmacokinetic or pharmacodynamic interactions that would limit the utility of this phenotyping cocktail for simultaneous measurement of the activity of multiple drug-metabolizing enzymes.

Interaction between two probes used for phenotyping cytochromes P4501A2 (caffeine) and P4502E1 (chlorzoxazone) in humans

Article

Apr 1995
PHARMACOGENET GENOM

The first steps in the metabolism of caffeine and chlorzoxazone are primarily catalysed by CYP1A2 and CYP2E1, respectively. Accordingly, these compounds have been developed as metabolic probes for non-invasive phenotyping of these two P450s. Their specificities, however, have been shown to overlap. In this study, 140 mg of caffeine and 500 mg of chlorzoxazone were administered alone or together in 16 healthy subjects under standardized conditions. The metabolites of these two probes were measured in the blood and also in the urine for caffeine. CYP1A2 activity was determined either by the paraxanthine/caffeine ratio in the blood or by the usual caffeine metabolic ratio in the urine. The CYP2E1 activity was determined by the 6-OH-chlorzoxazone/chlorzoxazone ratio in blood. CYP1A2 activities measured in blood and urine were highly significantly correlated. CYP2E1 activity was not modified when chlorzoxazone was given together with caffeine. In contrast, an inhibition of CYP1A2 by chlorzoxazone was demonstrated by a 16% decrease in the caffeine metabolic ratio in urine when both caffeine and chlorzoxazone were given together. Under the same conditions, the paraxanthine/caffeine ratio in plasma also decreased by about 20%. These results were confirmed in vitro by the incubation of 1 mM caffeine with human hepatic liver microsomes in the presence of 0.4 mM chlorzoxazone. The overall metabolism of caffeine decreased by 38% compared to controls incubated without chlorzoxazone. As all three JV-demethylations of caffeine were inhibited by chlorzoxazone with an apparent K4 of 0.18, 0.3 and 0.5 mM for N-3-, N-l- and N-7-demethylations respectively, it is suggested that chlorzoxazone is metabolized by CYP1A2 and, as a result, is a competitive inhibitor of caffeine. (C) Lippincott-Raven Publishers.

Interaction between two probes used for phenotyping cytochromes P4501A2 (Caffeine) and P4502E1 (chlorzoxazone) in humans

Article

May 1995
Pharmacogenetics

Population pharmacokinetics of intravenous caffeine in neonates

Article

Jul 1997

The study the population pharmacokinetics of caffeine after intravenous administration to premature infants with apnea. A prospective, blinded parallel study in which daily caffeine citrate doses of 30, 15, and 3 mg/kg were administered over 7 days by intermittent intravenous infusion. Arterial blood samples (three to six per patient) were assayed for caffeine content by means of HPLC. Population pharmacokinetic modeling was performed with NONMEM. Clearance (L/hr) = (0.00000399 . current weight [grams]) + (0.000128 . postnatal age [days]). For gestational age > 28 weeks, volume of distribution (L) = (0.000764 . weight [grams] + (0.0468 . postnatal age [days]); for gestational age < or = 28 weeks, volume of distribution (L) = (0.000755 . weight [grams]) + (0.0224. postnatal age [days]). Interpatient variability (coefficient of variation, in percent) was approximately 25% for clearance and approximately 11% for volume of distribution. Intrapatient error (standard deviation) was 3.9 mg/L. There was insignificant bias between observed and model-predicated serum caffeine concentrations in a separate group of 30 infants. Caffeine was well tolerated at all doses. Clearance was markedly lower and volume of distribution was higher than the values reported previously for term infants and adults. Both parameters were significantly influenced by postnatal age and current body weight, whereas volume of distribution in infants > 28 weeks' gestational age was higher than that in more premature babies. The predictive performance and the clinical application of the derived population models was satisfactorily shown.

Determination of drug-metabolizing enzyme activity in vivo: Pharmacokinetic and statistical issues

Article

Jan 1999

ethnic dia erences in the in vivo metabolism of drugs and other xenobiotics. The key issues in assessing drug-metabolizing enzyme activity in vivo include separation (with the aid of genotyping) of risk and exposure; choice of the best experimental index to use, the selection and number of subjects for study, and the use of objective criteria for assessing multimodality in frequency distributions of in vivo data. To avoid misinterpretation and overinterpretation of data, it is essential to have a good understanding of the pharmacokinetic basis of indirect measures of in vivo drugmetabolizing activity, and of appropriate statistical procedures for the analysis of frequency distributions. Pharmacokinetic basis for using indirect induces of drug metabolism Partial intrinsic clearance In theory, the closest in vivo measure of the activity of the gene product, that is the enzyme, ea ecting a particular metabolic pathway, i, of a drug is its intrinsic clearance down that route. This partial intrinsic clearance is de® ned in terms of unbound drug concentration in plasma ( CLu int, mi ), and can be viewed as the quotient of the apparent V max and K m of the drug with respect to the enzyme (Wilkinson and Shand 1975). Calculation of partial intrinsic clearance after oral drug administration requires measurement of the urinary recovery of the metabolite [Ae(mi) po ], the AUC (total area under the plasma drug concentration± time curve), plasma binding (indicated by fu, the free fraction) and renal clearance ( CL R ) of the

Integrated cytochrome P450 reaction phenotyping - Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes

Article

Mar 1999
BIOCHEM PHARMACOL

AD Rodrigues

With the increased availability of human liver tissue, recombinant (cDNA-expressed) cytochrome P450 proteins (rCYPs), and knowledge of the human CYP pool (e.g. immunoquantitated levels of each CYP form in native liver microsomes), it is now possible to carry out in vitro "CYP reaction phenotyping" in an integrated manner. Reaction phenotyping allows one to identify which CYP form(s) is (are) involved in the metabolism of a given drug, using a combination of data obtained with native human liver microsomes and rCYP proteins. The following describes how one can attempt to integrate such data. A total of ten drugs are included in the analysis, represented by twelve reactions (six hydroxylations, two O-demethylations, one N-demethylation, one O-deethylation, and two sulfoxidations) that are largely catalyzed (> or =20%) by various combinations of CYPs (CYP3A4, CYP2C9, CYP1A2, and CYP2D6), and characterized by a wide range of apparent Km values (12-820 microM). Briefly, reaction rates measured with individual rCYPs are normalized with respect to the nominal specific content of the corresponding CYP in native human liver microsomes. In turn, the normalized rates for each rCYP are summed, yielding a "total normalized rate" (TNR), and the normalized rate for each rCYP is expressed as a percent of the TNR (% TNR). Finally, % TNR is related to inhibition (percent inhibition in the presence of CYP form selective chemical inhibitors; % I) and univariate regression analysis (r > or = 0.63; P < or = 0.05; N > or = 10 different livers) data obtained with native human liver microsomes. Therefore, the reaction phenotype of a drug is assigned by integrating all three data sets (r, % TNR, and % I).

Determination of a ‘GW cocktail’ of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry

Article

Dec 1999

A mass spectrometry based method for the simultaneous determination of an in vivo Greenford-Ware or 'GW cocktail' of CYP450 probe substrates and their metabolites in both human plasma and urine is described. The probe substrates, caffeine, diclofenac, mephenytoin, debrisoquine, chlorzoxazone and midazolam, together with their respective metabolites and stable isotope labelled internal standards, are simultaneously extracted from the biological matrix using solid phase extraction in 96-well microtitre plate format, automated by means of a custom built Zymark robotic system. The extracts are analysed by fast gradient high performance liquid chromatography (HPLC) with detection by tandem mass spectrometry (MS/MS) using thermally and pneumatically assisted electrospray ionisation in both positive and negative ion modes and selected reaction monitoring. The methods are specific, accurate and precise with intra- and inter-assay precision (%CV) of less than 15% for all analytes.

Phenotyping of drug-metabolizing enzymes in adults: A review of in-vivo cytochrome P450 phenotyping probes

Article

May 2000
Pharmacogenetics

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.

In-vivo phenotyping for CYP3A by a single-point determination of midazolam plasma concentration

Article

Jan 2002
Pharmacogenetics

We investigated whether a single plasma midazolam concentration could serve as an accurate predictor of total midazolam clearance, an established in-vivo probe measure of cytochrome P450 3A (CYP3A) activity. In a retrospective analysis of data from 224 healthy volunteers, non-compartmental pharmacokinetic parameters were estimated from plasma concentration-time curves following intravenous (IV) and/or oral administration. Based on statistical moment theory, the concentration at the mean residence time (MRT) should be the best predictor of the total area under the curve (AUC). Following IV or oral midazolam administration, the average MRT was found to be approximately 3.5 h, suggesting that the optimal single sampling time to predict AUC was between 3 and 4 h. Since a 4-h data point was common to all studies incorporated into this analysis, we selected this time point for further investigation. The concentrations of midazolam measured 4 h after an IV or oral dose explained 80 and 91% of the constitutive interindividual variability in midazolam AUC, respectively. The 4-h midazolam measurement was also an excellent predictor of drug-drug interactions involving CYP3A induction and inhibition. Compared with baseline values, the direction and magnitude of change in midazolam AUC and the 4-h concentration were completely concordant for all study subjects. We conclude that a single 4-h midazolam concentration following IV or oral administration represents an accurate marker of CYP3A phenotype under constitutive and modified states. Moreover, the single-point approach offers an efficient means to phenotype and identify individuals with important genetic polymorphisms that affect CYP3A activity.

Limited Sampling Strategy to Predict AUC of the CYP3A Phenotyping Probe Midazolam in Adults: Application to Various Assay Techniques

Article

May 2002

Midazolam clearance is used to phenotype hepatic CYP3A activity but requires multiple plasma samples following a single intravenous dose. The authors evaluated the use of a limited sampling scheme, using different assay techniques to determine the reproducibility of such a strategy in estimating midazolam AUC. Seventy-three healthy adults received midazolam as a single intravenous bolus dose. At least eight plasma samples were collected from each subject and were assayed using either LC/MS/MS or electron capture gas chromatography. Eleven subjects were randomly selected for the training set using stepwise linear regression to determine relationships between midazolam plasma concentrations and AUC. Validation of the predictive equations was done using the remaining 62 subjects. Mean percent error (MPE), mean absolute error (MAE), and root mean square error (RMSE) were calculated to determine bias and precision. Based on the training set, five models were generated with coefficients of determination ranging from 0.87 to 0.95. Validation showed that MPE, MAE, and RMSE values were acceptable for three of the models. Intrasubject reproducibility was good. In addition, training set datafrom one institution were able to predict data from the other two institutions using other assay techniques. Minimized plasma sampling mayprovide a simpler method for estimating midazolam AUC for CYP3A phenotyping. A limited sampling strategy is more convenient and cost-effective than standard sampling strategies and is applicable to more than one assay technique.

Clinical importance of the cytochromes P450

Article

Nov 2002

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.

The Karolinska cocktail for phenotyping of five human cytochrome P450 enzymes

Article

Jul 2003

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.

Combined phenotypic assessment of cytochrome P450 1A2, 2C9, 2C19, 2D6, and 3A, N-acetyltransferase-2, and xanthine oxidase activities with the "Cooperstown 5+1 cocktail"

Article

Dec 2003

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.

CYP3A5 Genotype and Midazolam Clearance in Australian Patients Receiving Chemotherapy

Article

Jul 2004

Cytochrome P450 (CYP) 3A enzymes are key metabolizing enzymes for many chemotherapeutic agents, and detection of functionally significant CYP3A genetic variants may be useful in predicting interpatient variation of drug clearance. We have examined the significance of CYP3A5*3 single-nucleotide polymorphism to overall CYP3A activity in vivo in a predominantly Caucasian Australian cancer population. Screening for wild-type CYP3A5*1 and CYP3A5*3 single nucleotide polymorphism by use of Taqman MGB probe allelic discrimination was performed in 67 patients with cancer (58 Caucasian patients). CYP3A activity was documented via clearance of either oral or intravenous midazolam in 64 patients. All patients had at least 1 CYP3A5*3 allele, and 9 (13%) patients were heterozygous for CYP3A5*3 and CYP3A5*1. Within the subset of Caucasian patients, 6 of 58 (10%) were CYP3A5*1/*3 heterozygotes. Mean midazolam clearance was 1.7 times higher in CYP3A5*1/*3 subjects than in CYP3A5*3/*3 subjects (95% confidence interval, 1.15-2.51; P =.01, 2-way ANOVA). Overall CYP3A activity is related to CYP3A5 genotype. CYP3A5 genotyping may be helpful in predicting the drug-metabolizing capability of individual cancer patients who are predominantly Caucasian in origin.

Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: Application to cytochrome P450 phenotyping studies

Article

Dec 2004

A liquid chromatography/mass spectrometry method, for rapid determination of five cytochrome P450 (CYP) probe drugs and their relevant metabolites in human plasma and urine, is described. The five specific probe substrates/metabolites, caffeine/paraxanthine (CYP1A2), tolbutamide/4-hydroxytolbutamide/carboxytolbutamide (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), debrisoquine/5-hydroxydebrisoquine (CYP2D6) and midazolam/1'-hydroxymidazolam (CYP3A), together with the internal standards (phenacetin and paracetamol), in plasma and urine, were extracted using solid-phase extraction. The chromatography was performed using a C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% formic acid in water (70:30). The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and multiple reaction monitoring was used for quantification. The method was validated over the concentration ranges 0.05-5 microg/mL for caffeine and paraxanthine, 0.02-2 microg/mL for tolbutamide, 0.1-20 microg/mL for 4-hydroxytolbutamide, carboxytolbutamide, debrisoquine and 5-hydroxydebrisoquine, 5-2500 ng/mL for omeprazole and 5-hydroxyomeprazole, and 1-100 ng/mL for midazolam and 1'-hydroxymidazolam. The intra- and inter-day precision were 0.3-13.7% and 1.9-14.3%, respectively, and the accuracy ranged from 93.5-107.2%. The lower limit of quantification varied between 1 and 100 ng/mL. The present method provides a robust, fast and sensitive analytical tool for the five-probe drug cocktail, and has been successfully applied to a clinical phenotyping study in 16 subjects.

Evaluation of the Drug Interaction Potential of Aplaviroc, a Novel Human Immunodeficiency Virus Entry Inhibitor, Using a Modified Cooperstown 5 + 1 Cocktail

Article

Jun 2006

Aplaviroc is a novel CCR5 antagonist, a class of compounds under investigation as viral entry inhibitors for the treatment of human immunodeficiency virus infection. A modified Cooperstown 5+1 cocktail was used to assess the drug interaction potential of aplaviroc. Fifteen healthy subjects were administered single oral doses of caffeine (CYP1A2), warfarin (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A) alone (reference treatment) and during steady-state administration of aplaviroc (400 mg every 12 hours, test treatment). Metabolite-to-parent area under the plasma concentration versus time curve (AUC) ratios (paraxanthine/caffeine and 5-hydroxyomeprazole/omeprazole), oral clearance (S-warfarin), AUC (midazolam), and metabolite-to-parent urinary excretion ratio (dextrorphan/dextromethorphan) were determined. The test-to-reference treatment ratios (geometric mean ratio and 90% confidence interval) were caffeine, 1.06 (0.97-1.17); S-warfarin, 0.93 (0.76-1.15); omeprazole, 1.07 (0.98-1.16); dextromethorphan, 1.17 (0.97-1.42); midazolam, 1.30 (1.04-1.63). No significant inhibition of CYP1A2, CYP2C9, CYP2C19, or CYP2D6 enzyme activity was observed. Mild inhibition of CYP3A isozymes should not preclude the use of concomitant CYP3A substrates in future clinical studies with aplaviroc.

PII-37Validation of incorporating flurbiprofen into the Pittsburgh cocktail

Article

Oct 2006

We have previously shown that flurbiprofen metabolism to 4'-hydroxyflurbiprofen provides an in vivo measure of cytochrome P450 (CYP) 2C9 activity. This study evaluated the possibility of incorporating flurbiprofen into the current 5-drug Pittsburgh cocktail. In a randomized, 3-way, Latin-square, crossover-design study, 24 healthy subjects (mean age [+/-SD], 47.8 +/- 15.1 years) received flurbiprofen (50 mg) and the Pittsburgh 5-drug cocktail (100 mg caffeine, 100 mg mephenytoin, 10 mg debrisoquin [INN, debrisoquine], 250 mg chlorzoxazone, and 100 mg dapsone) separately and in combination on 3 occasions over a period of 5 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 each of the metabolizing enzymes. The geometric mean ratio and 90% confidence interval of the phenotypic indices were included within the 80% to 125% bioequivalence range for each of the probe drugs. There were no statistically significant differences between the phenotypic indices determined after administration of the 5-drug and 6-drug cocktails. However, there was a small but statistically significant increase (7.5%, P = .03) in the 8-hour urinary flurbiprofen recovery ratio after administration of the 6-drug cocktail compared with that after administration of flurbiprofen alone. The 6-drug cocktail was well tolerated. The results of this study show that caffeine (CYP1A2), chlorzoxazone (CYP2E1), dapsone (N-acetyltransferase 2), debrisoquin (CYP2D6), flurbiprofen (CYP2C9), and mephenytoin (CYP2C19) can be simultaneously administered in low doses without metabolic interaction.

Appropriate Phenotyping Procedures for Drug Metabolizing Enzymes and Transporters in Humans and Their Simultaneous Use in the “Cocktail” Approach

Article

Mar 2007

Phenotyping for drug metabolizing enzymes and transporters is used to assess quantitatively the effect of an intervention (e.g., drug therapy, diet) or a condition (e.g., genetic polymorphism, disease) on their activity. Appropriate selection of test drug and metric is essential to obtain results applicable for other substrates of the respective enzyme/transporter. The following phenotyping metrics are recommended based on the level of validation and on practicability: CYP1A2, paraxanthine/caffeine in plasma 6 h after 150 mg caffeine; CYP2C9, tolbutamide plasma concentration 24 h after 125 mg tolbutamide; CYP2C19, urinary excretion of 4'-OH-mephenytoin 0-12 h after 50 mg mephenytoin; CYP2D6, urinary molar ratio debrisoquine/4-OH-debrisoquine 0-8 h after 10 mg debrisoquine; and CYP3A4, plasma clearance of midazolam after 2 mg midazolam (all drugs given orally).

Development of the “Inje Cocktail” for High-throughput Evaluation of Five Human Cytochrome P450 Isoforms in vivo

Article

Dec 2007
CLIN PHARMACOL THER

To develop and validate an in vivo cocktail method for high-throughput phenotyping of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A, 12 healthy subjects received five probe drugs alone or simultaneously. The in vivo phenotyping index of CYP2C9, the ratio of 8 h urine concentration of losartan to its metabolite after a single administration of losartan, was not significantly different from that obtained using the five-drug cocktail. Similarly, the ratios of [omeprazole]/[5-hydroxyomeprazole] (CYP2C19) and [paraxanthine]/[caffeine] (CYP1A2) in 4 h plasma samples and the log ratio of [dextromethorphan]/[dextrorphan] (CYP2D6) in 8 h urine samples and the 4 h plasma concentrations of midazolam (CYP3A) after single administration or well-established three-drug cocktail of caffeine, omeprazole, and dextromethorphan were not significantly different from those after the new five-drug cocktail. In conclusion, the new five-drug cocktail regimen, named the "Inje cocktail," can be used as a tool to phenotype in vivo enzyme activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A with only 4 h blood sampling and 8 h urine collection following simultaneous administration of the five probe drugs.

A High-Throughput Assay Using Liquid Chromatography-Tandem Mass Spectrometry for Simultaneous In Vivo Phenotyping of 5 Major Cytochrome P450 Enzymes in Patients

Abstract

No full-text available

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