LC–MS/MS Estimation of Rociletinib Levels in Human Liver Microsomes: Application to Metabolic Stability Estimation

Abstract

Background

Rociletinib (CO-1686; RLC) is a new, small molecule that is orally administered to inhibit mutant-selective covalent inhibitor of most epidermal growth factor receptor (EGFR)-mutated forms, including T790M, L858R, and exon 19 deletions, but not exon 20 insertions. Non–small-cell lung cancer (NSCLC) with a gene mutation that encodes EGFR is sensitive to approved EGFR inhibitors, but usually resistance develops, which is frequently mediated by T790M EGFR mutation. RLC is an EGFR inhibitor found to be active in preclinical models of EGFR-mutated NSCLC with or without T790M.

Methods

In silico drug metabolism prediction of RLC was executed with the aid of the WhichP450 module (StarDrop software package) to verify its metabolic liability. Second, a fast, accurate, and competent LC-MS/MS assay was developed for RLC quantification to determine its metabolic stability. RLC and bosutinib (BOS) (internal standard; IS) were separated using an isocratic elution system with a C₁₈ column (reversed stationary phase).

Results

The developed LC-MS/MS analytical method showed linearity of 5–500 ng/mL with r² ≥ 0.9998 in the human liver microsomes (HLMs) matrix. A limit of quantification of 4.6 ng/mL revealed the sensitivity of the analytical method, while the acquired inter- and intra-day accuracy and precision values below 4.63% inferred the method reproducibility. RLC metabolic stability estimation was calculated using intrinsic clearance (20.15 µL/min/mg) and in vitro half-life (34.39 min) values.

Conclusion

RLC exhibited a moderate extraction ratio indicative of good bioavailability. The developed analytical method herein is the first LC-MS/MS assay for RLC metabolic stability.

Keywords:

• rociletinib
• metabolic stability assessment
• in vitro half-life
• validated LC-MS/MS methodology

Acknowledgment

The authors extend their sincere appreciation to the Deanship of Scientific Research at the King Saud University for funding this work through the Research Group Project No. RG-1435-025.

Abbreviations

RLC, rociletinib; ESI, electrospray ionization; HLMs, human liver microsomes; TKIs, tyrosine kinase inhibitors; LC-MS/MS, liquid chromatography tandem mass spectrometry; EGFR, epidermal growth factor receptor; NSCLCs, Non-small cell lung cancers; Clint, intrinsic clearance; t_1/2, half-life; ABL, Abelson murine leukemia; MET, met proto-oncogene; ROS1, v-ros UR2 sarcoma virus oncogene homolog 1; Axl, receptor tyrosine kinase.

Ethics Approval

The study design using in vitro experiments with commercially available human liver microsomes exempts it from the need of the Ethics Committees approval.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest for this work.