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.

Analytical aspects of sunitinib and its geometric isomerism towards therapeutic drug monitoring in clinical routine.

Sunitinib malate, an oral multi-targeted tyrosine kinase inhibitor approved for the treatment of metastatic renal cell carcinoma, gastrointestinal stromal tumor, and well-differentiated pancreatic neuroendocrine tumors, has been identified as a potential candidate for therapeutic drug monitoring approach. Nevertheless, the development of an analytical assay suitable for clinical application for the quantification of the plasma concentration of sunitinib and its active metabolite, N-desethyl sunitinib, is limited by its Z/E isomerization when exposed to light. Several LC-MS/MS methods already published require protection from light during all sample handling procedures to avoid the formation of E-isomer, which makes them not suitable for clinical practice. In order to obtain a simple and fast procedure to reconvert the E-isomer, formed during sample collection and treatment without light protection, and, thus, to have only Z-isomer peak to quantify, we studied the Z/E photodegradation with special attention to the condition allowing the reverse reaction in plasma matrix. After 30 min of light exposure, the E-isomer maximum percentage of both the analytes was reached (44% of E-sunitinib and 20% of E-N-desethyl sunitinib; these percentages were calculated with respect to the sum of E + Z). Moreover, the formation of the E-isomer increased up to 20% after lowering the pH of the solution. Since the reverse reaction takes place when the pre-exposed solution is placed in dark, we followed the E to Z-isomer kinetics into the autosampler. The conversion rate was very slow when the autosampler was set at 4 °C (after 4 h the mean percentages of E-isomer were 50% for sunitinib and 22% for N-desethyl sunitinib). The reconversion rate was considerably accelerated with the increasing of the temperature: incubating the analytical solution in a heated water bath for 5 min at 70 °C we obtained the quantitative (99%) reconversion of the E- to the Z-isomer. No effect of concentration was observed, while the presence of acids inhibited the reconversion. Based on these results, a simple and fast procedure was setup to quantitatively reconvert the E-isomer formed during sample collection and processing without light protection into its Z-form thus leading to a single peak to quantify. The application of this additional step allows to develop a LC-MS/MS method suitable to clinical practice, due to its practicality and speed.

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.

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.

Peptide biosensors for anticancer drugs: Design in silico to work in denaturizing environment.

One of the main targets in current clinical oncology is the development of a cheap device capable of monitoring in real-time the concentration of a drug in the blood of a patient. This would allow fine-tuning the dosage according to the patient's metabolism, a key condition to reduce side effects. By using surface plasmon resonance and fluorescence spectroscopy we here show that short peptides designed in silico by a recently developed algorithm are capable of binding the anticancer drug irinotecan (CPT-11) with micromolar affinity. Importantly, the recognition takes place in the denaturating solution used in standard therapeutic drug monitoring to detach the drug from the proteins that are present in human plasma, and some of the peptides are capable of distinguishing CPT-11 from its metabolite SN-38. These results suggest that the in silico design of small artificial peptides is now a viable route for designing sensing units, opening a wide range of applications in diagnostic and clinical areas.

Nanocarriers in cancer clinical practice: a pharmacokinetic issue.

The advent of nanocarriers for drug delivery has given rise to new intriguing scenarios in the cancer field. Nanocarriers indeed partly overcome the limits of traditional cytotoxic drugs principally changing the pharmacokinetic behavior of the parental drug. The peculiar characteristics of these systems strongly minimize the adverse reactions and ensure a more precise release of the compound to the tumor site. Several nanocarriers have been developed for the delivery of cytotoxic drugs such as paclitaxel and doxorubicin in order to improve both the outcome and the patients' quality of life. The aims of this review are to describe in detail the pharmacokinetics of nanocarriers, already marketed or in advanced clinical phases, for paclitaxel and doxorubicin, to highlight the main differences with the parental drugs, and to underline, in a critical manner, benefits and disadvantages related to the use of these new drug delivery systems.

Candidate germline polymorphisms of genes belonging to the pathways of four drugs used in osteosarcoma standard chemotherapy associated with risk, survival and toxicity in non-metastatic high-grade osteosarcoma.

This study aimed to identify associations between germline polymorphisms and risk of high-grade osteosarcoma (HGOS) development, event-free survival (EFS) and toxicity in HGOS patients treated with neo-adjuvant chemotherapy and surgery.Germline polymorphisms of 31 genes known to be relevant for transport or metabolism of all four drugs used in HGOS chemotherapy (methotrexate, doxorubicin, cisplatin and ifosfamide) were genotyped in 196 patients with HGOS and in 470 healthy age and gender-matched controls. Of these 196 HGOS patients, a homogeneously treated group of 126 patients was considered for survival analyses (survival cohort). For 57 of these, treatment-related toxicity data were available (toxicity cohort).Eleven polymorphisms were associated with increased risk of developing HGOS (p < 0.05). The distribution of polymorphisms in patients was characterized by a higher Shannon entropy. In the survival cohort (n = 126, median follow-up = 126 months), genotypes of ABCC2_1249A/G, GGH_452T/C, TP53_IVS2+38G/C and CYP2B6*6 were associated with EFS (p < 0.05). In the toxicity cohort (n = 57), genotypes of ABCB1_1236T/C, ABCC2_1249A/G, ABCC2_3972A/G, ERCC1_8092T/G, XPD_23591A/G, XRCC3_18067T/C, MTHFR_1298A/C and GGH_16T/C were associated with elevated risk for toxicity development (p < 0.05).The results obtained in this retrospective study indicate that the aforementioned germline polymorphisms significantly impact on the risk of HGOS development, EFS and the occurrence of chemotherapy-related toxicity. These findings should be prospectively validated with the aim of optimizing and tailoring HGOS treatment in the near future.

Cross-validation of a mass spectrometric-based method for the therapeutic drug monitoring of irinotecan: implementation of matrix-assisted laser desorption/ionization mass spectrometry in pharmacokinetic measurements.

Irinotecan is a widely used antineoplastic drug, mostly employed for the treatment of colorectal cancer. This drug is a feasible candidate for therapeutic drug monitoring due to the presence of a wide inter-individual variability in the pharmacokinetic and pharmacodynamic parameters. In order to determine the drug concentration during the administration protocol, we developed a quantitative MALDI-MS method using CHCA as MALDI matrix. Here, we demonstrate that MALDI-TOF can be applied in a routine setting for therapeutic drug monitoring in humans offering quick and accurate results. To reach this aim, we cross validated, according to FDA and EMA guidelines, the MALDI-TOF method in comparison with a standard LC-MS/MS method, applying it for the quantification of 108 patients' plasma samples from a clinical trial. Standard curves for irinotecan were linear (R (2) ≥ 0.9842) over the concentration ranges between 300 and 10,000 ng/mL and showed good back-calculated accuracy and precision. Intra- and inter-day precision and accuracy, determined on three quality control levels were always <12.8 % and between 90.1 and 106.9 %, respectively. The cross-validation procedure showed a good reproducibility between the two methods, the percentage differences within 20 % in more than 70 % of the total amount of clinical samples analysed.

Clinical validity of a DPYD-based pharmacogenetic test to predict severe toxicity to fluoropyrimidines.

Pre-therapeutic DPYD pharmacogenetic test to prevent fluoropyrimidines (FL)-related toxicities is not yet common practice in medical oncology. We aimed at investigating the clinical validity of DPYD genetic analysis in a large series of oncological patients. Six hundred three cancer patients, treated with FL, have been retrospectively tested for eight DPYD polymorphisms (DPYD-rs3918290, DPYD-rs55886062, DPYD-rs67376798, DPYD-rs2297595, DPYD-rs1801160, DPYD-rs1801158, DPYD-rs1801159, DPYD-rs17376848) for association with Grade ≥3 toxicity, developed within the first three cycles of therapy. DPYD-rs3918290 and DPYD-rs67376798 were associated to Grade ≥3 toxicity after bootstrap validation and Bonferroni correction (p = 0.003, p = 0.048). DPYD-rs55886062 was not significant likely due to its low allelic frequency, nonetheless one out of two heterozygous patients (compound heterozygous with DPYD-rs3918290) died from toxicity after one cycle. Test specificity for the analysis of DPYD-rs3918290, DPYD-rs55886062 and DPYD-rs67376798 was assessed to 99%. Among the seven patients carrying one variant DPYD-rs3918290, DPYD-rs55886062 or DPYD-rs67376798 allele, not developing Grade ≥3 toxicity, 57% needed a FL dose or schedule modification for moderate chronic toxicity. No other DPYD polymorphism was associated with Grade ≥3 toxicity. Our data demonstrate the clinical validity and specificity of the DPYD-rs3918290, DPYD-rs55886062, DPYD-rs67376798 genotyping test to prevent FL-related Grade ≥3 toxicity and to preserve treatment compliance, and support its introduction in the clinical practice.

Development and validation of a HPLC-UV method for the quantification of antiepileptic drugs in dried plasma spots.

BACKGROUND: Therapeutic drug monitoring (TDM) of antiepileptic drugs is widely used in clinical practice to optimise therapy, but it is limited by technical problems and cost considerations. The aim of the present study was: 1) to validate a chromatographic method for the concomitant determination of levetiracetam, lamotrigine, ethosuximide, felbamate, rufinamide, zonisamide and monohydroxycarbamazepine; 2) to develop it for dried plasma spot (DPS) assessing its reliability against the classical determination from plasma; and 3) test its clinical application. METHODS: Extraction of plasma samples and DPS was done by simple precipitation. Chromatographic analysis was performed using high performance liquid chromatography with ultraviolet detection. After validation, both methods were applied for the quantification of plasma samples from patients on antiepileptic therapy. RESULTS: Mean inter- and intra-day accuracy and precision were <15% for all compounds both in plasma and in DPS samples. DPS samples were considered stable under tested conditions. Measurements between plasma and DPS samples appeared related (p<0.0001). Bland-Altman analysis revealed accordance in lamotrigine values with mean overestimation of concentration for DPS sample of 2.8%. Also for monohydroxycarbamazepine data the agreement was acceptable (mean overestimation of 9.2%). For levetiracetam mean difference was 7.6%, while for ethosuximide mean percentage difference was 20.6%. CONCLUSIONS: The developed methods simplify TDM of antiepileptic drugs. This is particularly relevant for the method on dried spot sample devices because it facilitates further sample handling, stability and shipments making the management of therapies in epileptic patients easier also in hospitals devoid of a dedicated laboratory.