Development of dispersive solid phase extraction based on magnetic metal organic framework for the extraction of sunitinib in biological samples and its determination by high performance liquid chromatography-tandem mass spectrometry.

Herein, a simple, sensitive, and reliable dispersive solid phase extraction was reported for the efficient extraction of sunitinib from biological samples. To facilitate the extraction of the desired analyte from urine and plasma samples, magnetic MIL-101Cr (NH2) @SiO2 @ NiFe2O4 was synthesized by a hydrothermal method and applied as an effective sorbent during the extraction process. After adsorption of the drug using 10 mg of MIL-101Cr (NH2) @ SiO2 @ NiFe2O4 nanoparticles through vortexing (1 min), the sorbent was separatedfrom the sample solution using a magnet. To eluate the drug, the sorbent containing the sunitinib was contacted with 100 µL dimethylformamide. The eluent was analyzed by high performance liquid chromatography-tandem mass spectrometry. Reasonable validation data consisting of low limits of detection (0.14, 0.35, and 0.70 ng mL-1 in deionized water, plasma, and urine) and quantification (0.48, 1.2, and 2.4 ng mL-1 in deionized water, plasma, and urine, respectively), a wide linear range of the calibration curve (0.48-200, 1.2-200, and 2.4-100 ng mL-1 in deionized water, plasma, and urine, respectively) good extraction recovery (76 %), and low relative standard deviations for inter- and intra-day precisions (6.9 %) were obtained by the method. Eventually, the proposed procedure was effectively implemented on both plasma and urine samples, yielding successful outcomes.

Development of a Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Sunitinib Unaffected by Light-Induced Isomerization.

Sunitinib is an oral multi-targeted tyrosine kinase inhibitor approved for treating metastatic renal cell carcinoma. This study reports a specific and sensitive competitive enzyme-linked immunosorbent assay (ELISA) for the pharmacokinetic evaluation of sunitinib. Anti-sunitinib serum was obtained from mice by using N-(2-(diethylamino)ethyl)-5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxamide (DFPC) as a hapten, which has the same substructure as sunitinib, in order to avoid the effects of structural changes in the geometrical isomers of sunitinib. Enzyme labeling of sunitinib with horseradish peroxidase was similarly performed using DFPC. Serum sunitinib concentrations below the limit of quantification of 0.52 ng/mL were reproducibly measurable. This ELISA was specific for sunitinib (Z- and E-isomers) and showed very low cross-reactivity (0.094%) with its major metabolite, N-desethyl sunitinib. Its analytical applicability was demonstrated by a kinetic study with human liver microsomes. In addition, the levels of sunitinib measured by ELISA in a kinetic study with human liver microsomes were comparable with those measured by HPLC, and there was a strong correlation between the values determined by both methods (y = 1.065x - 51.2, R2 = 0.9804). The developed ELISA provides for the specific and sensitive quantification of sunitinib without the influence of its major metabolite or light-induced geometric isomers. This ELISA will be a valuable tool in pharmacokinetic studies of sunitinib.

A new high-performance liquid chromatography-tandem mass spectrometry method for the determination of sunitinib and N-desethyl sunitinib in human plasma: Light-induced isomerism overtaking towards therapeutic drug monitoring in clinical routine.

Sunitinib is approved for advanced renal cell cancer, imatinib-resistant or -intolerant gastrointestinal stromal tumors and pancreatic neuroendocrine cancers. It is prescribed at a fixed dose but its plasma exposure shows large inter-individual variations. Taking into account the narrow therapeutic window and the positive exposure-efficacy relationship, there is a robust rationale for its therapeutic drug monitoring. In fact, a target plasma concentration of sunitinib plus its active metabolite, N-desethyl sunitinib, ≥50 ng/mL was suggested. In order to quantify sunitinib and N-desethyl sunitinib in patients' plasma, we developed and validated a new LC-MS/MS method applicable to clinical routine. In solution, sunitinib and N-desethyl sunitinib undergo to photo-isomerization and many published methods overcome this problem by conducting the entire procedures of samples collection and handling under strictly light-protection. Our method is based on a simple and fast procedure that quantitatively reconverts the E-isomer of both analytes, obtained during sample draw and processing without light-protection, into their Z-forms. Moreover, our method uses a small plasma volume (30 µL) and the analytes are extracted by a rapid protein precipitation. It was validated according to EMA-FDA guidelines. The calibration curves resulted linear (R2 always >0.993) over the concentration ranges (0.1-500 ng/mL for sunitinib, 0.1-250 ng/mL for N-desethyl sunitinib) with a good precision (within 7.7 % for sunitinib and 10.8% for N- desethyl sunitinib) and accuracy (range 95.8-102.9% for sunitinib and 92.3-106.2% for N-desethyl sunitinib). This method was applied to a pharmacokinetic study in one patient treated with sunitinib. Moreover, as incurred samples reanalysis is an established part of the bioanalytical process to support clinical studies, its assessment was performed early in order to assure that any reproducibility issues was detected as soon as possible. The percentage difference between the two runs resulted within ±20% in all the re-analysed samples for both sunitinib and N- desethyl sunitinib.

Development of a validated LC-MS/MS method for the in vitro and in vivo quantitation of sunitinib in glioblastoma cells and cancer patients.

Sunitinib is a multi-targeted tyrosine kinase inhibitor approved for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumor and is currently being investigated against other forms of malignant tumors. Recently great interest has emerged for the application of sunitinib to glioblastoma treatment. In order to have a method with broad applicability it will be of importance to have access to a method that could be applied both in human plasma and cell uptake studies. No method has been reported thus far for the estimation of sunitinib uptake in glioma cells. We therefore set out to develop a method that could be applied for quantifying sunitinib in human plasma and in cell uptake studies. The method was validated and accredited according to ISO 17025:2005 guideline in human plasma and successfully applied to cancer patient plasma. Also, the method was effectively recruited to establish a protocol for the evaluation of sunitinib accumulation into M095K glioma cells. This method could significantly contribute to developmental phases in repurposing this drug in different cancer types.

Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib.

Sunitinib is a multitargeted tyrosine kinase inhibitor associated with idiosyncratic hepatotoxicity. The mechanisms of this toxicity are unknown. We hypothesized that sunitinib undergoes metabolic activation to form chemically reactive, potentially toxic metabolites which may contribute to development of sunitinib-induced hepatotoxicity. The purpose of this study was to define the role of cytochrome P450 (P450) enzymes in sunitinib bioactivation. Metabolic incubations were performed using individual recombinant P450s, human liver microsomal fractions, and P450-selective chemical inhibitors. Glutathione (GSH) and dansylated GSH were used as trapping agents to detect reactive metabolite formation. Sunitinib metabolites were analyzed by liquid chromatography-tandem mass spectrometry. A putative quinoneimine-GSH conjugate (M5) of sunitinib was detected from trapping studies with GSH and dansyl-GSH in human liver microsomal incubations, and M5 was formed in an NADPH-dependent manner. Recombinant P450 1A2 generated the highest levels of defluorinated sunitinib (M3) and M5, with less formation by P450 3A4 and 2D6. P450 3A4 was the major enzyme forming the primary active metabolite N-desethylsunitinib (M1). In human liver microsomal incubations, P450 3A inhibitor ketoconazole reduced formation of M1 by 88%, while P450 1A2 inhibitor furafylline decreased generation of M5 by 62% compared to control levels. P450 2D6 and P450 3A inhibition also decreased M5 by 54 and 52%, respectively, compared to control. In kinetic assays, recombinant P450 1A2 showed greater efficiency for generation of M3 and M5 compared to that of P450 3A4 and 2D6. Moreover, M5 formation was 2.7-fold more efficient in human liver microsomal preparations from an individual donor with high P450 1A2 activity compared to a donor with low P450 1A2 activity. Collectively, these data suggest that P450 1A2 and 3A4 contribute to oxidative defluorination of sunitinib to generate a reactive, potentially toxic quinoneimine. Factors that alter P450 1A2 and 3A activity may affect patient risk for sunitinib toxicity.