Abstract
Irinotecan is currently used in several cancer regimens mainly in colorectal cancer (CRC). This drug has a narrow therapeutic range and treatment can lead to side effects, mainly neutropenia and diarrhea, frequently requiring discontinuing or lowering the drug dose. A wide inter-individual variability in irinotecan pharmacokinetic parameters and pharmacodynamics has been reported and associated to patients’ genetic background. In particular, a polymorphism in the UGT1A1 gene (UGT1A1*28) has been linked to an impaired detoxification of SN-38 (irinotecan active metabolite) to SN-38 glucuronide (SN-38G) leading to increased toxicities. Therefore, therapeutic drug monitoring of irinotecan, SN-38 and SN-38G is recommended to personalize therapy. In order to quantify simultaneously irinotecan and its main metabolites in patients’ plasma, we developed and validated a new, sensitive and specific HPLC–MS/MS method applicable to all irinotecan dosages used in clinic. This method required a small plasma volume, addition of camptothecin as internal standard and simple protein precipitation. Chromatographic separation was done on a Gemini C18 column (3 μM, 100 mm x 2.0 mm) using 0.1% acetic acid/bidistilled water and 0.1% acetic acid/acetonitrile as mobile phases. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring. The standard curves were linear (R2 ≥0.9962) over the concentration ranges (10–10000 ng/mL for irinotecan, 1–500 ng/mL for SN-38 and SN-38G and 1–5000 ng/mL for APC) and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy, determined on three quality control levels for all the analytes, were always <12.3% and between 89.4% and 113.0%, respectively. Moreover, we evaluated this bioanalytical method by re-analysis of incurred samples as an additional measure of assay reproducibility. This method was successfully applied to a pharmacokinetic study in metastatic CRC patients enrolled in a genotype-guided phase Ib study of irinotecan administered in combination with 5-fluorouracil/leucovorin and bevacizumab.
Highlights
Irinotecan (CPT-11) (Fig. 1) is a semisynthetic analogue of the natural alkaloid camptothecin (CPT)
In order to obtain a sensitive, specific and rapid method to quantify irinotecan and its main metabolites in human plasma, we developed and validated, according to the Food Drug and Administration (FDA) and the European Medicines Agency (EMA) guidance on bioanalytical method validation [11,12–13], a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method that requires a small volume of plasma sample (100 μL), only simple treatment with methanol added with acetic acid (0.1% v/v) and a reasonable time of analysis
The daughter ion with the highest signal was used as quantifier, as follows: 587.4 >124.2 for CPT11, 393.3>349.3 for SN-38, 569.3>393.2 for SN-38 glucuronide (SN-38G), 619.2>393.3 for APC and 349.2>305.1 for Internal Standard (IS), all expressed in m/z
Summary
Irinotecan (CPT-11) (Fig. 1) is a semisynthetic analogue of the natural alkaloid camptothecin (CPT). It is a prodrug topoisomerase I inhibitor and it is activated by the enzyme carboxylesterase to 7-ethyl-10-hydroxycamptothecin (SN-38) (Fig. 1). Compared with the parent drug, SN-38 is 100- to 1000-times more cytotoxic [1]. CPT-11 is mainly used in colorectal cancer (CRC) diagnosed patients with metastases, with recorded relapse or progression after application of standard 5-fluorouracil (5-FU)-based therapy [2]. CPT-11 is used as single agent or in combination with other chemotherapeutic (i.e. FOLFIRI: infusional 5-FU, leucovorin, and irinotecan) or biological (i.e. cetuximab and bevacizumab) agents. These regimens include several dosages of CPT-11
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