Clinical Evaluation Based on a New Approach to Improve the Accuracy of 4β-Hydroxycholesterol Measurement as a Biomarker of CYP3A4 Activity

Abstract

This study examines 4β-Hydroxycholesterol (4β-HC), which is considered to be a potential marker for the CYP3A4 induction of new chemical entities (NCEs) in drug development. To ensure the use of 4β-HC as a practical biomarker, it is necessary to accurately measure 4β-HC and demonstrate that CYP3A4 induction can be appropriately assessed, even for weak inducers. In clinical trials of NCEs, plasma is often collected with various anticoagulants, in some cases, the plasma is acidified, then stored for an extended period. In this study, we examined the effects of these manipulations on the measurement of 4β-HC, and based on the results, we optimized the plasma collection and storage protocols. We also found that a cholesterol oxidation product is formed when plasma is stored, and by monitoring the compound, we were able to identify when plasma was stored inappropriately. After evaluating the above, clinical drug-drug interaction (DDI) studies were conducted using two NCEs (novel retinoid-related orphan receptor γ antagonists). The weak CYP3A4 induction by the NCEs (which were determined based on a slight decline in the systemic exposure of a probe substrate (midazolam)), was detected by the significant increase in 4β-HC levels (more specifically, 4β-HC/total cholesterol ratios). Our new approach, based on monitoring a cholesterol oxidation product to identify plasma that is stored inappropriately, allowed for the accurate measurement of 4β-HC, and thus, it enabled the evaluation of weak CYP3A4 inducers in clinical studies without using a probe substrate.

Keywords: 4β-hydroxycholesterol; biomarkers; cholesterol oxidation; cytochrome P450 CYP3A4 inducers; drug–drug interaction study; mass spectrometry.

Figure 1

Typical MRM chromatograms analyzing plasma that was obtained with K2-EDTA, then analyzed without storage (A), or analyzed after 13 months of storage in the dark at −20 °C (B) or −80 °C (C). Each plasma sample was saponified, extracted with hexane, and analyzed with LC-MS/MS in the MRM mode. Extended MRM chromatograms between the retention times of 6.2–9.0 min are shown. Other details are described in the Methods section. Identification of 4β-HC and 4α-HC was performed by comparing the two markers against commercial standards. The correlation chart between the production of peak X and the relative error (RE) of 4β-HC in the plasma was analyzed during the long-term storage test (D). Peak X was corrected in accordance with the internal standard 4β-HC-d7 (i.e., the ratio of the area of peak X to the area of 4β-HC-d7 was calculated). A correlation diagram was created by plotting the peak area ratio of peak X and 4β-HC-d7 after long-term storage at −20 °C on the Y-axis and the RE shown in Table 1B on the X-axis (D).

Figure 2

Typical Q1 chromatogram and MS spectrum of peak X (A); product ion spectrum of peak X (B). An extended Q1 chromatogram between the retention times of 14.0–20.0 min is shown. K2-EDTA plasma collected from healthy volunteers, stored at −20 °C for 13 months in the dark, was analyzed by LC-HRMS. Details of this process are shown in the Methods section. The sample was saponified, extracted with hexane, and analyzed using LC-HRMS. Refer to the Methods section for details regarding the conditions.

Figure 3

The 4β-HC concentrations and 4β-HC/TC ratios before and after the repeated administration of JTE-451 (A) and JTE-761 (B). The correlation chart between plasma 4β-HC concentrations or 4β-HC/TC ratios and the AUC of midazolam (C). Clinical DDI studies were conducted using weak inducers of CYP3A4 (JTE-451 and JTE-761), and blood collection and plasma sample storage were performed based on the results of 4β-HC stability tests. Midazolam and 4β-HC were measured using plasma, and the total cholesterol was measured using serum. The percentage (%) changes in 4β-HC concentration, 4β-HC/TC ratio, and midazolam AUC demonstrate their differences before and after the administration of NCEs. ^# Two subjects out of eighteen subjects opted out of the clinical study. ^## One subject out of sixteen subjects opted out of the clinical study. * p < 0.01, n.s.: not significant.