Development and implementation of urinary transporter biomarkers to facilitate assessment of drug-drug interaction.

Aim: Novel urinary biomarker evaluation approaches to support inhibition assessment for renal transporters (e.g., OCT2, multidrug and toxin extrusion proteins [MATEs]). Methods: Highly sensitive and robust hydrophilic interaction chromatography-MS/high-resolution MS assays, for urine and plasma, were developed and characterized to evaluate transporter biomarkers including N1-methyladenosine and N1-methylnicotinamide. Results: The assays were simple and reliable with good selectivity and sensitivity, and successfully supported a clinical drug-drug interaction study with a drug candidate that presented in vitro inhibition of OCT2 and MATEs. Conclusion: The multiplexed assays enable a performance comparison, including biomarker specificity and sensitivity, that should increase the confidence in early clinical OCT2/MATEs drug-drug interaction risk assessment.

Cytochrome P450 Enzyme Inhibition and Herb-Drug Interaction Potential of Medicinal Plant Extracts Used for Management of Diabetes in Nigeria.

BACKGROUND AND OBJECTIVE: The use of herbal medicines is common in Africa, and patients often use a combination of herbs and drugs. Concurrent herbal and pharmaceuticals treatments can cause adverse effects through herb-drug interactions (HDI). This study evaluated the potential risk of HDI for five medicinal plants, Vernonia amygdalina, Ocimum gratissimum, Moringa oleifera, Azadirachta indica, and Picralima nitida, using in vitro assays. Patients with diabetes and some other disease conditions commonly use these medicinal plants in Nigeria, and little is known regarding their potential for drug interaction, despite their enormous use. METHODS: Crude extracts of the medicinal plants were evaluated for reversible and time-dependent inhibition (TDI) activity of six cytochrome P450 (CYP) enzymes using pooled human liver microsomes and cocktail probe-based assays. Enzyme activity was determined by quantifying marker metabolites' formation using liquid chromatography-mass spectrometry/mass spectrometry. The drug interaction potential was predicted for each herbal extract using the in vitro half-maximal inhibitory concentration (IC50) values and the percentage yield. RESULTS: O. gratissimum methanol extracts reversibly inhibited CYP 1A2, 2C8, 2C9 and 2C19 enzymes (IC50: 6.21 µg/ml, 2.96 µg/ml, 3.33 µg/ml and 1.37 µg/ml, respectively). Additionally, V. amygdalina methanol extract inhibited CYP2C8 activity (IC50: 5.71 µg/ml); P. nitida methanol and aqueous extracts inhibited CYP2D6 activity (IC50: 1.99 µg/ml and 2.36 µg/ml, respectively) while A. indica methanol extract inhibited CYP 3A4/5, 2C8 and 2C9 activity (IC50: 7.31 µg/ml, 9.97 µg/ml and 9.20 µg/ml, respectively). The extracts showed a potential for TDI of the enzymes when incubated at 200 µg/ml; V. amygdalina and A. indica methanol extracts exhibited TDI potential for all the major CYPs. CONCLUSIONS: The medicinal plants inhibited CYP activity in vitro, with the potential to cause in vivo HDI. Clinical risk assessment and proactive monitoring are recommended for patients who use these medicinal plants concurrently with drugs that are cleared through CYP metabolism.

Identification of Appropriate Endogenous Biomarker for Risk Assessment of Multidrug and Toxin Extrusion Protein-Mediated Drug-Drug Interactions in Healthy Volunteers.

Endogenous biomarkers are emerging to advance clinical drug-drug interaction (DDI) risk assessment in drug development. Twelve healthy subjects received a multidrug and toxin exclusion protein (MATE) inhibitor (pyrimethamine, 10, 25, and 75 mg) in a crossover fashion to identify an appropriate endogenous biomarker to assess MATE1/2-K-mediated DDI in the kidneys. Metformin (500 mg) was also given as reference probe drug for MATE1/2-K. In addition to the previously reported endogenous biomarker candidates (creatinine and N1 -methylnicotinamide (1-NMN)), N1 -methyladenosine (m1 A) was included as novel biomarkers. 1-NMN and m1 A presented as superior MATE1/2-K biomarkers since changes in their renal clearance (CLr ) along with pyrimethamine dose were well-correlated with metformin CLr changes. The CLr of creatinine was reduced by pyrimethamine, however, its changes poorly correlated with metformin CLr changes. Nonlinear regression analysis (CLr vs. mean total concentration of pyrimethamine in plasma) yielded an estimate of the inhibition constant (Ki ) of pyrimethamine and the fraction of the clearance pathway sensitive to pyrimethamine. The in vivo Ki value thus obtained was further converted to unbound Ki using plasma unbound fraction of pyrimethamine, which was comparable to the in vitro Ki for MATE1 (1-NMN) and MATE2-K (1-NMN and m1 A). It is concluded that 1-NMN and m1 A CLr can be leveraged as quantitative MATE1/2-K biomarkers for DDI risk assessment in healthy volunteers.

A Multiplexed HILIC-MS/HRMS Assay for the Assessment of Transporter Inhibition Biomarkers in Phase I Clinical Trials: Isobutyryl-Carnitine as an Organic Cation Transporter (OCT1) Biomarker.

There is a growing interest in using endogenous compounds as drug transporter biomarkers to facilitate drug-drug interaction (DDI) risk assessment in early phase I clinical trials. Compared to other drug transporters, however, no valid biomarker for hepatic organic cation transporter (OCT) 1 has been described to date. The present work represents the first report of an endogenous compound, isobutyryl-l-carnitine (IBC), as a potential clinical OCT1 biomarker for DDI assessment. A hydrophilic interaction chromatography (HILIC)-mass spectrometry/high resolution mass spectrometry (MS/HRMS) assay with a simple sample preparation method was developed. The assay is capable of simultaneously quantifying multiple endogenous compounds, including IBC, thiamine, N1-methylnicotinamide (1-NMN), creatinine, carnitine, and metformin, which is a probe for OCT1 and OCT2 and MATE1 and MATE2K (multidrug and toxin extrusion proteins) in clinical studies. The HRMS assay was fit-for-purpose validated in human plasma and demonstrated good linearity, accuracy, and precision for all analytes. It was further applied to two phase I clinical trials to evaluate potential biomarkers for OCT1 and additional cation transporters (renal OCT2, MATE1, and MATE2K). The clinical data demonstrated that plasma IBC changes correlated well with in vitro data and supported its use as a liver OCT1 biomarker. The described HILIC-MS/HRMS assay can be used as a "biomarker cocktail" to simultaneously assess clinical DDI risk for the inhibition of OCT1/2 and MATEs in clinical studies with new drug candidates.