Novel LC–MS assays impacting CYP and transporter drug–drug interaction evaluations
2018; Future Science Ltd; Volume: 10; Issue: 9 Linguagem: Inglês
10.4155/bio-2018-0086
ISSN1757-6199
Autores Tópico(s)Antibiotics Pharmacokinetics and Efficacy
ResumoBioanalysisVol. 10, No. 9 ForewordFree AccessNovel LC–MS assays impacting CYP and transporter drug–drug interaction evaluationsRagu RamanathanRagu Ramanathan*Author for correspondence: E-mail Address: ragu.ramanathan@pfizer.com PDM-NCE, Pfizer World Wide RD, Groton, CT 06340, USAPublished Online:29 May 2018https://doi.org/10.4155/bio-2018-0086AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Drug–drug interaction (DDI) potentials of new chemical entities (NCEs) are a major concern when patients require treatment with multiple drug regimens at the same time. When drug X is co-administered with drug Y, either drug has the potential to partially or totally alter absorption, distribution, metabolism and excretion (ADME) properties of the other. The alterations in the ADME properties of the drug X could result from inhibition or induction of Cytochrome P450 (CYP) enzymes or transporters and, thus, alter the co-administered drug's efficacy and safety. To ensure safety and efficacy of drugs, regulatory agencies across the globe and the pharmaceutical industries have embraced evaluations of DDI potential of an NCE early in the drug development program and continued postmarketing. The guidance documents, from the US FDA, provide recommendations on drug interaction study design, data analysis, implications for dosing and labeling recommendations [1,2]. In addition, a collaborative group of scientists, from the academia, regulatory agencies, pharmaceutical industry and contract research organizations (CROs), regularly organize conferences and publish white papers based on new research, new clinical studies and findings from recent regulatory reviews. For example, breakthroughs and outcomes, from rapidly growing transporter sciences, are disseminated through outputs from the International Transporter Consortium [3–5].There is a growing interest in exploring novel LC–MS/MS, LC-high resolution accurate mass spectrometry (LC–HRMS) or LC–MS/HRMS assays early in drug development to ensure DDI-related liabilities to establish safety and efficacy of an NCE. This special issue brings together some of the novel LC–MS applications and outlook from leaders in the field.In vitro cytochrome P450 DDI projections before the start of clinical trialsDDI potentials, related to CYP and UDP-glucuronosyltranferase (UGT) enzymes, have been extensively investigated because collectively both enzymes account for more than 90% of the biotransformation of the NCEs used for human treatments [6,7]. Very recently, Yu et al. [8] reviewed a total of 103 drugs, including 14 combination therapies and published the data under a title of 'Risk of clinically relevant pharmacokinetic-based drug–drug interactions with drugs approved by the US Food and Drug Administration between 2013 and 2016'. Investigations of the ADME properties and DDI profiles of the drugs approved between 2013 and 2016, using the University of Washington Drug Interaction Database and each drug's NDA information, showed CYP3A to be involved in >65% of DDIs. Several LC–MS/MS-based high-throughput CYP inhibition and induction assays have been reported over the last 20 years to ensure the safety and efficacy of drugs when required for co-administration [9,10]. The commentary entitled 'Application of in vitro CYP and transporter assays to predict clinical drug–drug interactions' by Volpe and Balimane [11] provides the needed background on enzymes including Phase I, Phase II and others as well as outlines experimental protocols recommended in the regulatory guidance documents. This commentary further details in vitro DDI screening options available including human liver microsomes, recombinant enzymes (rCYP), S9 fractions and human hepatocytes. More specific contributions, from individual CYPs to biotransformation or elimination pathways, are elucidated using isoform-specific chemical inhibitors and/or inhibitory antibodies [12,13]. Drug depletion or metabolite formation or combinations of both are measured using reaction phenotyping experiments. Ideally, all the CYP-based DDI potential information should be available before an NCE is advanced into first-in human trials. However, the FDA recommends, at minimum, the evaluation of the major CYP enzymes such as CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A for the potential metabolism-mediated drug interactions. If these CYPs are not involved in the metabolism of the drug, other CYPs such as, CYP2J2, CYP4F2, CYP2E1 or non-CYP Phase I enzymes should be evaluated [14,15]. In this Special Focus Issue, the article by Ramanathan et al. [16] provides details on the use of HRMS-based in vitro cocktail assay for cassette analysis involving ten CYP enzymes [16]. Within assay positive-to-negative polarity switching, HPLC-HRMS method allowed quantification of eight and two probe compounds in the positive and negative ionization modes respectively, while monitoring for loratadine and its metabolites. Although modern triple quadrupole mass spectrometer can be utilized in a similar manner to the HRMS assay detailed in this manuscript, the triple quadrupole-based assays lack the selectivity of an HRMS method and fail to provide the additional qualitative data or the options to postacquisition interrogation of the data for metabolites or other components.Translating in vitro transporter DDI projections to the clinicA recent FDA publication reports that in vitro-based transporter DDI results in a high number of false positive rates (∼30%) and categorizes NCEs as potential candidates for dedicated probe drug-based clinical DDI evaluations [17]. Rapidly advancing drug transporter sciences suggest that selected compounds, endogenous to human plasma and urine, may serve as biomarkers for specific transporters [18–21]. The evolution of such endogenous biomarkers to the frontlines of drug discovery and development would allow drug researchers to confirm or refute in vitro-based transporter DDI projections in the first-in-human clinical trial of an NCE and ensure safety and efficacy. There are two commentary articles in this Special Focus Issue on transporters; the commentary, by Rodrigues [22] entitled 'A pharmaceutical industry perspective on transporter and CYP mediated DDI – organic anion transporting polypeptides (OATPs)', focuses on liver transporters, while kidney transporters are covered in the commentary entitled 'Pharmaceutical industry perspective on transporter and CYP mediated DDI – kidney transporter biomarkers' by Shen [23]. Both of these commentaries provide information about the endogenous biomarkers available to investigate transporter-related DDIs in early clinical studies to add or eliminate dedicated clinical DDI trials involving the dosing of probe drugs. Additionally, an early understanding of the transporters, with respect to an NCE, would allow the clinical teams to design safer studies by establishing patient inclusion/exclusion criteria, genetic polymorphism and NCE exposure related to diseased state.Potential OATP clinical biomarkersPotential OATP clinical biomarker: coproporphyrin-I and coproporphyrin-IIICoproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) historically, endogenously present coproporphyrin isomers have been used as biomarker for occupational and environmental exposure to lead, arsenic or mercury as well as for diagnosing Dubin–Johnson's syndrome [24–30]. Only very recently, coproporphyrin isomers, CP-I and CP-III, have emerged as potential biomarkers of organic anion transporting polypeptide (OATP)-mediated DDIs [31–36]. Under typical LC–MS conditions, CP-I and CP-III form both singly ([M+H]+) and doubly charged ([M+2H]2+) precursor ions at m/z 655.3 and 328.14, respectively. In human plasma and urine, the doubly charged precursor-ions are present at five- to ten-times the intensity of the corresponding singly charged precursor ions. For LC–MS quantification, it is important to utilize the higher abundant ions because endogenous or background plasma levels of CP-I and CP-III range 500–900 and 50–500 pg/ml, respectively. In the article by King-Ahmed et al. [37], a novel, validated, automated LC–MS assays for the quantitation of CP-I and CP-III in human plasma and demonstrate for the first time that first-in-human clinical study samples can be used to evaluate DDI liabilities. In this study, sensitivity and extraction recoveries were improved respectively by using the doubly charged ([M+2H]2+) precursor ions at m/z 328.1 and the incorporation of a supported liquid extraction. Additionally, the throughput of the assay was improved by the incorporation of automation and three-times charcoal stripped plasma was used as surrogate matrix to minimize quantitative interferences from endogenously present proporphyrins. Another important outcome from the research published by King-Ahmed et al. [37] is a similar AUC-fold changes in CP-I and Atorvastatin. Atorvastatin is used as a probe drug for evaluating OATP-mediated DDIs. Similarly, the article by Kandoussi et al. [38] demonstrates a robust UHPLC–MS/MS assay using the singly charged ([M+H]+) precursor ions of CP-I/CP-III at m/z 655.3 and a supported liquid extraction method for sample clean-up and automation. The assay was validated and applied to clinical studies to confirm suitability of CPs as a potential substitute for DDI study.Potential OATP clinical biomarker: selected bile acidsBile acids (BAs) have been used as disease biomarkers for decades, recently, researchers have developed and validated BA biomarkers to screen, diagnose and monitor the progression of Niemann–Pick disease [39]. Only recently, selected BAs in plasma have been proposed as endogenous biomarkers for DDIs involving hepatic drug transporters such as OATP1B1 and OATP1B3. Selected BAs, if established, as biomarkers of OATPs, would likely facilitate DDI evaluations better than using CP-I/CP-III because BAs are present at higher concentrations in plasma than CP-I/CP-III. However, relatively complex pool of circulating BAs, require liquid chromatographic separation of multiple isomers before MS quantification. Over the last 5 years, bioanalytical quantification using UHPLC–HRMS has evolved as an alternative to the conventional methods using LC coupled with triple quadrupole mass spectrometry (LC–MS/MS) to quantify analytes present in complex systems. Rago et al. [40] quantified six BAs in human plasma using a multiplexed HRMS method and demonstrated, for the first time in a first-in-human clinical study, GDCA-3S and GDCA-3G as potential biomarkers based on observed four- to fivefold increase in plasma AUC (vs placebo) following administration of a compound known to be present as an OATP1B1/3 inhibitor in vitro.Potential OCT2 & MATE clinical biomarker: 1-NMNN1-methylnicotinamide (1-NMN) has been proposed as a potential transporter of clinical biomarker for the evaluation of DDIs involving organic cation transporters (OCT2) and multidrug and toxin extrusion protein transporters (MATEs). 1-NMN, formed by the methylation of nicotinamide, is a polar molecule with an [M+H]+ at m/z of 137 and requires hydrophilic interaction chromatography for separation from numerous endogenous interferences. The article by Luo et al. [41] demonstrates the development, qualification and application of a highly selective and sensitive assay for 1-NMN in human plasma and urine. For the first time, 1-NMN was evaluated as a potential DDI biomarker following administration of a compound known to present as an OCT-2 inhibitor in vitro. The detailed assay provides improved selectivity through chromatographic separation of isobaric precursors and MS separation of isobaric product ions.Options to increasing bioanalytical throughput to ensure safety & efficacy of NCEsThe articles in this special issue bring together some novel strategies to improve bioanalytical throughput in early discovery through clinical studies. The HPLC-HRMS full scan with polarity switching assay detailed in the article by Ramanathan et al. [16] provides options for increasing throughput of human in vitro cocktail DDI assay. An HPLC-HRMS full-scan polarity switching assay was developed for simultaneously assessing the activities of drug metabolizing enzymes CYP3A4/5, CYP2D6, CYP2C19, CYP2C9, CYP1A2, CYP2B6 and CYP2C8. The assay also provides the options to simultaneously evaluate an NCE and its major metabolites involved in impacting the major drug metabolizing enzymes.The research published by King-Ahmed et al. [37] and Kandoussi et al. [38] demonstrates options for increasing bioanalytical throughput by the incorporation of sample processing automation. Rago et al. [40] demonstrate options for multiplexed detection and quantification of six BAs using UHPLC-HRMS and UHPLC-MS/HRMS. Availability of the HRMS raw data with each sample provides the options for postacquisition scouting for new biomarkers, NCEs and metabolites. For the first-time, Luo et al. [41] demonstrate the application of pooling method, called 'AUC pooling' or 'time-proportional pooling' or 'Hamilton pooling' for clinical biomarker application [42]. AUC pooling method has been extensively applied to assure metabolite exposures as part of metabolite in safety testing evaluations [43]. Luo et al. [41] evaluated NCE dose-dependent changes in 1-NMN using equal volume pooled-, AUC pooled and individual samples and demonstrate that higher-throughput AUC pooling method provide the same directional biomarker changes as data acquired following analysis of individual samples. Overall, AUC-pooling approach conserved clinical samples and conserved sample analysis resources while providing data to support or refute in vitro DDI projections.Overall, in this Special Focus Issue, articles demonstrate the rapid expansion of interest in using sensitive and selective LC–MS assays for evaluations of DDI potentials early in the development of an NCE to assure safety and efficacy to patients. Endogenous transporter biomarkers, coproporphyrin isomers, CP-I and CP-III and BAs have been evaluated as possible candidate biomarkers for organic anion-transporting polypeptides (OATP1B1 and OATP1B3). 1-NMN has been proposed as a potential transporter clinical biomarker for the evaluation of DDIs involving OCT2 and MATEs. In the near future, similar to biomarkers reported in this Special Focus Issue, several DDI biomarkers will be proposed as potential biomarkers for confirming or refuting in vitro DDI projection. 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Lett. 11(1), 21–28 (2017).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 10, No. 9 Follow us on social media for the latest updates Metrics Downloaded 1,186 times History Received 26 March 2018 Accepted 28 March 2018 Published online 29 May 2018 Published in print May 2018 Information© 2018 Newlands PressFinancial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download
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