Metabolic pathway and structures of CPT-11 and its main metabolites: SN-38, SN-38G, APC and NPC.

<p>Cyt P450: cytochrome P450; UGT1A1: UDP-glucoronosyltransferase 1A1.</p

Development and Validation of a High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for the Simultaneous Determination of Irinotecan and Its Main Metabolites in Human Plasma and Its Application in a Clinical Pharmacokinetic Study

<div><p>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 <i>UGT1A1</i> gene (<i>UGT1A1*28</i>) 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.</p></div

Representative SRM chromatograms.

<p>Panel A: SRM chromatograms of a human blank plasma sample; Panel B: SRM chromatograms of a human blank plasma sample with IS added; Panel C: S/N of SN-38, SN-38G, CPT-11 and APC at the LLOQ (10 ng/mL for CPT-11 and 1 ng/mL for SN-38, SN-38G and APC); Panel D: SRM chromatograms of an extracted plasma sample of a treated patient showing IS, SN-38, SN-38G, CPT-11 and APC. The concentrations measured were 7.42, 16.57, 80.27 and 12.91 ng/mL for SN-38, SN-38G, CPT-11 and APC, respectively.</p

Calibration curve of CPT-11 and its main metabolites SN-38, SN-38G and APC in human plasma.

<p>Calibration curve of CPT-11 and its main metabolites SN-38, SN-38G and APC in human plasma.</p

Plasma concentration-versus-time profiles of CPT-11 and its main metabolites: SN-38, SN-38G and APC.

<p>Panel A: Plasma concentration-<i>versus</i>-time profiles of CPT-11, SN-38, SN-38G and APC in one patient receiving 310 mg/m² of irinotecan during the I and the II administration of the first therapy cycle; Panel B: Plasma concentration-<i>versus</i>-time profiles of CPT-11, SN-38, SN-38G and APC in one patient receiving 370 mg/m² of irinotecan during the I administration of the first therapy cycle. In order to define the pharmacokinetic interactions between CPT-11 and bevacizumab (BV), the pharmacokinetic profile of CPT-11 was evaluated in absence and presence of BV in the same patient. The pharmacokinetic profile of CPT-11 alone was assessed at the first chemotherapy treatment in which BV was administered on day 3 (50 h after the start of CPT-11 infusion). Whereas, irinotecan pharmacokinetics in combination with BV was performed during the second treatment of the first cycle, when BV was administered before CPT-11 dosage.</p

MS/MS mass spectra of CPT-11, SN-38, SN-38G and APC with chemical structures and identification of the main fragment ions.

<p>The fragment ion at <i>m/z</i> *248 of SN-38G is not shown in the MS/MS mass spectrum because it requires, for its formation, a higher collision energy than the other fragments.</p

A new high-performance liquid chromatography-tandem mass spectrometry method for the determination of paclitaxel and 6α-hydroxy-paclitaxel in human plasma: Development, validation and application in a clinical pharmacokinetic study

<div><p>Paclitaxel belongs to the taxanes family and it is used, alone or in multidrug regimens, for the therapy of several solid tumours, such as breast-, lung-, head and neck-, and ovarian cancer. Standard dosing of chemotherapy does not take into account the many inter-patient differences that make drug exposure highly variable, thus leading to the insurgence of severe toxicity. This is particularly true for paclitaxel considering that a relationship between haematological toxicity and plasma exposure was found. Therefore, in order to treat patients with the correct dose of paclitaxel, improving the overall benefit–risk ratio, Therapeutic Drug Monitoring is necessary. In order to quantify paclitaxel and its main metabolite, 6α-hydroxy-paclitaxel, in patients’ plasma, we developed a new, sensitive and specific HPLC–MS/MS method applicable to all paclitaxel dosages used in clinical routine. The developed method used a small volume of plasma sample and is based on quick protein precipitation. The chromatographic separation of the analytes was achieved with a SunFire^™ C18 column (3.5 μM, 92 Å, 2,1 x 150 mm); the mobile phases were 0.1% formic acid/bidistilled water and 0.1% formic acid/acetonitrile. The electrospray ionization source worked in positive ion mode and the mass spectrometer operated in selected reaction monitoring mode. Our bioanalytical method was successfully validated according to the FDA-EMA guidelines on bioanalytical method validation. The calibration curves resulted linear (R² ≥0.9948) over the concentration ranges (1–10000 ng/mL for paclitaxel and 1–1000 ng/mL for 6α-hydroxy-paclitaxel) and were characterized by a good accuracy and precision. The intra- and inter-day precision and accuracy were determined on three quality control concentrations for paclitaxel and 6α-hydroxy-paclitaxel and resulted respectively <9.9% and within 91.1–114.8%. In addition, to further verify the assay reproducibility, we tested this method by re-analysing the incurred samples. This bioanalytical method was employed with success to a genotype-guided phase Ib study of weekly paclitaxel in ovarian cancer patients treated with a wide range of drug’s dosages.</p></div

MS/MS mass spectra of PTX and 6α-OH-PTX with chemical structures and identification of the main fragment ions.

<p>MS/MS mass spectra of PTX and 6α-OH-PTX with chemical structures and identification of the main fragment ions.</p

List of publications related to LC-MS/MS methods for the quantification of PTX in human plasma samples.

<p>List of publications related to LC-MS/MS methods for the quantification of PTX in human plasma samples.</p

Plasma concentration-<i>vs</i>-time profiles of PTX and its main metabolite 6α-OH-PTX in three patients with advanced ovarian cancer.

<p>Patient 1, 2, and 3 received 110 mg/m² of PTX as 1-h intravenous infusion during the first chemotherapy cycle.</p