Abstract 4648: Development and validation of an HPLC-UV method for Sirolimus (sir) quantification in human blood and application to cancer patients in routine clinical practice
Authors: Escudero-Ortiz Vanesa , Valenzuela Belen , Rebollo Joseba , Sureda Manuel , Brugarolas Antonio 
Published: May 2015

Abstract
Background and objective: After oral administration, SIR (mTOR inhibitor) is rapid absorbed with a bioavailability around 14%. SIR is distributed mostly into red blood cells, 92% is bound to proteins and its apparent steady state volume of distribution is higher than 650 L. The apparent clearance has been determined to be 13 L/h. SIR undergoes extensive hepatic and intestinal metabolism via CYP3A4 and CYP3A5, as well as excretion by P-glycoprotein. The purpose of this study was to develop a simple and sensitive HPLC method with UV-Visible detection for SIR blood quantification to allow therapeutic drug monitoring (TDM) in cancer patients. A pilot phase I study in advanced cancer patients was initiated to evaluate the effect of grapefruit juice in SIR pharmacokinetics.
Methods: After a liquid-liquid extraction with 1-chlorobutane, SIR and paclitaxel (internal standard, IS) are separated in Ultrabase C18 column using a mobile phase consisting of a mixture of methanol and water in a proportion 77:23 pumped at a constant flow rate of 1 mL/min. The column was maintained at 50°C and the eluent was monitored at a wavelength of 277 nm. Validation experiments were carried out after the guidelines for Bioanalytical Method Validation published by the FDA and the EMA. SIR was administered once weekly as an oral solution in three dose leves (10, 20 or 30 mg). Natural grapefruit juice (250 cc) was administered half an hour before SIR administration, once a week.
Results: The calibration curve was linear in the range of 10-700 ng/mL. The limit of quantification was determined to be 10 ng/mL. SIR and IS retention times were 12.2 and 3.4 min, respectively. Inter- and intra-day coefficients of variation were less than 8%. The mean extraction recovery from plasma was higher than 67%. In the first six patients evaluated, SIR blood concentrations were well quantified and showed that the analytical method was capable of measuring the blood concentrations of SIR in advanced cancer patients undergoing SIR therapy. In all patients, SIR concentrations were significantly increased by grapefruit juice at all dose levels because its clearance was reduce around 50% confirming previous results published by Cohen E et al. (Clin Cancer Res, 2012).
Conclusion: The simple, rapid and sensitive HPLC-UV method developed and validated for the measurement of SIR in human blood using paclitaxel as IS can be applied easily in routine clinical practice for the TDM of SIR. Co-administration of grapefruit juice and SIR must be evaluated in the context of Phase II and III clinical trials in order to explore alternative and safer treatments.
Note: This abstract was not presented at the meeting.
Citation Format: Vanesa Escudero-Ortiz, Belen Valenzuela, Joseba Rebollo, Manuel Sureda, Antonio Brugarolas. Development and validation of an HPLC-UV method for Sirolimus (sir) quantification in human blood and application to cancer patients in routine clinical practice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4648. doi:10.1158/1538-7445.AM2014-4648