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

Abstract

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.

Influence of YES1 Kinase and Tyrosine Phosphorylation on the Activity of OCT1

“…This analysis was performed using plasma and liver specimens from wild-type mice and OCT1/2-deficient mice, and ultimately resulted in the identification of various structurally named molecules of possible significance, including isobutyryl-l-carnitine (IBC). Interestingly, while we were completing the current study, Luo et al reported that IBC is also a potentially useful endogenous biomarker to predict OCT1-mediated drug-drug interactions in humans (Luo et al, 2020). These collective findings are largely congruent with prior studies by Kim et al on the transport of carnitines in liver-specific OCT1-knockout mice (Kim et al, 2017).…”

“…We also observed that reduced hepatic levels of IBC in wild-type mice were accompanied by significantly elevated levels in plasma ( Figure 4B), that IBC levels in the kidney were negligible ( Figure 4B) regardless of mouse genotype, and that additional deficiency of MATE1 ( Figure 4C), which forms a functional unit with OCT1 in the liver and with OCT2 in the kidney, did not influence the results. These findings suggest that IBC, a natural four-carbon acylcarnitine involved in fatty acid oxidation and organic acid metabolism, serves as a bona fide biomarker for hepatic OCT1 function, a conclusion, that is in line with a recent clinical report (Luo et al, 2020). We next evaluated the impact of dasatinib on concentrations of IBC and found that administration of the TKI resulted in a transient, statistically significant increase in the plasma levels of IBC in wild-type mice, but not in OCT1/2-deficient mice or OCT1/ 2/MATE1-deficient mice ( Figure 4D).…”

“…Although this observation seems counterintuitive, it should be noted that OCT1 can serve as a bi-directional hepatic transporter to either mediate the electrogenic cellular influx or alternatively to mediate efflux of organic cations under trans-zero conditions, depending on the substrate concentration gradient. Regardless of the mechanistic basis, the recorded discrepancy with the recently published human study (Luo et al, 2020) suggests that further investigation into the use of IBC as an OCT1 biomarker in the context of transport inhibitors is warranted.…”

2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome ( Part 1 –Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos)

“…The improved performance of HRMS systems has triggered more bioanalysts to switch from triple quadrupole MS to HRMS for quantitative analysis when additional selectivity was required [56][57][58]. There was consensus that the conclusions reached in the 2015 White Paper in Bioanalysis, i.e., HRMS technology is fit for use in regulated bioanalysis for PK and biomarker assays [10] are still valid, and there have been regulatory submissions already.…”

Isobutyrylcarnitine as a Biomarker of OCT1 Activity and Interspecies Differences in its Membrane Transport

“…In genome-wide association studies, isobutyrylcarnitine (IBC) was strongly associated with OCT1 genetic polymorphisms (Suhre et al, 2011) and may thus be a suitable endogenous biomarker of OCT1 activity (Luo et al, 2020). IBC is a metabolite of valine, when its acyl residue is transferred from isobutyryl-CoA to carnitine (Ramsay et al, 2001) (Figure 1).…”