Abstract 333: Development and validation of a LC-MS/MS method for the quantification of the tropomyosin receptor kinase (Trk) inhibitor pegcantratinib in human skin tumors
Authors: Zangarini Monique , Rajan Neil , Danilenko Marina , Berry Philip , Veal Gareth J. 
Published: July 2016

Background: Pegcantratinib is the first topically applied tropomyosin receptor kinase inhibitor allowing retention in the skin to exert its effects at nanomolar concentrations.

Pegcantratinib is now under early clinical evaluation as an anti-tumour agent in patients with germline mutations in the tumour suppressor gene CYLD, which include multiple clinical presentations of rare, skin appendage tumours. No pharmacological treatment is currently available and patients face repeated and disfiguring surgery to control tumour burden. We report on the development and validation of an analytical method to quantify pegcantratinib in skin tumour biopsies of patients to determine tumour penetration.

Methods: A sensitive and specific HPLC-MS/MS (API 4000) assay coupled with in-source CID was developed and validated according to EMA guidelines, for direct quantitative measurement of pegcantratinib in biopsies of human skin tumours. The sample preparation procedure required a 10 mg sample volume and involved protein precipitation with acetonitrile following tissue homogenization and addition of internal standard. Reversed-phase chromatography under gradient conditions (MP A - 1% formic acid; MP B - 1% formic acid in acetonitrile) was applied with separation on a Kinetex 2.6 μm C18 column (100 Å, 50 × 4.6 mm). Detection was obtained by SRM, following the transitions m/z 419.0 → 376.4. The method is rapid and selective, allowing good resolution of peaks in 2.4 min.

Results: The assay was fully validated and the method exhibited good sensitivity, precision, and accuracy, with overall precision expressed as CV ≤8.5% and accuracy in the range 96-99%. Recovery was high (≥85%) and consistent with CV ≤13%. No matrix effect was observed in four independent matrix sources including 3 different tumours and normal skin, the calculated CV was 5%. The limit of quantitation was 2.5 ng/ml, with precision and accuracy of 6% and 99%, respectively. The assay developed was linear in the range 1.0-500 ng/ml, with mean precision &lt;8% and accuracy within the range 96-105%. Inter/intra-day precision and accuracy were always &lt;8.5% and within the range 95-99%, respectively. Pegcantratinib is stable in tumour homogenate for at least 4 hours at room temperature, for 10 days at 4°C, before and after extraction, and over 3 freeze-thaw cycles. Preliminary data of an ex-vivo incubation of skin tumours biopsies, from two patients, at 5μM pegcantratinib for 1, 3 and 6h, showed that drug achieved concentrations of 1.5-2.5 μM in the tumours analysed.

Conclusion: We have successfully developed a LC-MS/MS method to measure the anti-tumour agent pegcantratinib in human skin tumour samples. Analysis of samples obtained from the Phase 1b/2a clinical trial is now underway.

Acknowledgements: Work supported by the Department of Health and Wellcome Trust through the Health Innovation Challenge Fund and Cancer Research UK.

Citation Format: Monique Zangarini, Neil Rajan, Marina Danilenko, Philip Berry, Gareth J. Veal. Development and validation of a LC-MS/MS method for the quantification of the tropomyosin receptor kinase (Trk) inhibitor pegcantratinib in human skin tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 333.