Bas Thijssen

A novel self-microemulsifying formulation of paclitaxel for oral administration to patients with advanced cancer

To explore the parmacokinetics, safety and tolerability of paclitaxel after oral administration of SMEOF#3, a novel Self-Microemulsifying Oily Formulation, in combination with cyclosporin A (CsA) in patients with advanced cancer. Seven patients were enrolled and randomly assigned to receive oral paclitaxel (SMEOF#3) 160 mg+CsA 700 mg on day 1, followed by oral paclitaxel (Taxol®) 160 mg+CsA 700 mg on day 8 (group I) or vice versa (group II). Patients received paclitaxel (Taxol®) 160 mg as 3-h infusion on day 15. The median (range) area under the plasma concentration–time curve of paclitaxel was 2.06 (1.15–3.47) μg h ml−1 and 1.97 (0.58–3.22) μg h ml−1 after oral administration of SMEOF#3 and Taxol®, respectively, and 4.69 (3.90–6.09) μg h ml−1 after intravenous Taxol®. Oral SMEOF#3 resulted in a lower median Tmax of 2.0 (0.5–2.0) h than orally applied Taxol® (Tmax=4.0 (0.8–6.1) h, P=0.02). The median apparent bioavailability of paclitaxel was 40 (19–83)% and 55 (9–70)% for the oral SMEOF#3 and oral Taxol® formulation, respectively. Oral paclitaxel administered as SMEOF#3 or Taxol® was safe and well tolerated by the patients. Remarkably, the SMEOF#3 formulation resulted in a significantly lower Tmax than orally applied Taxol®, probably due to the excipients in the SMEOF#3 formulation resulting in a higher absorption rate of paclitaxel.

Incorporation of concentration data below the limit of quantification in population pharmacokinetic analyses

Handling of data below the lower limit of quantification (LLOQ), below the limit of quantification (BLOQ) in population pharmacokinetic (PopPK) analyses is important for reducing bias and imprecision in parameter estimation. We aimed to evaluate whether using the concentration data below the LLOQ has superior performance over several established methods. The performance of this approach (“All data”) was evaluated and compared to other methods: “Discard,” “LLOQ/2,” and “LIKE” (likelihood‐based). An analytical and residual error model was constructed on the basis of in‐house analytical method validations and analyses from literature, with additional included variability to account for model misspecification. Simulation analyses were performed for various levels of BLOQ, several structural PopPK models, and additional influences. Performance was evaluated by relative root mean squared error (RMSE), and run success for the various BLOQ approaches. Performance was also evaluated for a real PopPK data set. For all PopPK models and levels of censoring, RMSE values were lowest using “All data.” Performance of the “LIKE” method was better than the “LLOQ/2” or “Discard” method. Differences between all methods were small at the lowest level of BLOQ censoring. “LIKE” method resulted in low successful minimization (<50%) and covariance step success (<30%), although estimates were obtained in most runs (~90%). For the real PK data set (7.4% BLOQ), similar parameter estimates were obtained using all methods. Incorporation of BLOQ concentrations showed superior performance in terms of bias and precision over established BLOQ methods, and shown to be feasible in a real PopPK analysis.

A sensitive combined assay for the quantification of paclitaxel, docetaxel and ritonavir in human plasma using liquid chromatography coupled with tandem mass spectrometry

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human plasma is described. The drugs were extracted from 200 μL human plasma using liquid-liquid extraction with tertiar-butylmethylether, followed by high performance liquid chromatography analysis using 10 mM ammonium hydroxide pH 10:methanol (3:7, v/v) as mobile phase. Chromatographic separation was obtained using a Zorbax Extend C(18) column. Labelled analogues of the analytes are used as internal standards. For detection, positive ionization electrospray tandem mass spectrometry was used. Method development including optimisation of the mass transitions and response, mobile phase optimisation and column selection are discussed. The method was validated according to FDA guidelines and the principles of Good Laboratory Practice (GLP). The validated range was 0.5-500 ng/mL for paclitaxel and docetaxel and 2-2000 ng/mL for ritonavir. For quantification, quadratic calibration curves were used (r(2)>0.99). The total runtime of the method is 9 min and the assay combines analytes with differences in ionisation and desired concentration range. Inter-assay accuracy and precision were tested at four concentration levels and were within 10% and less than 10%, respectively, for all analytes. Carry-over was less than 6% and endogenous interferences or interferences between analytes and internal standards were less than 20% of the response at the lower limit of quantification level. The matrix factor and recovery were determined at low, mid and high concentration levels. The matrix factor was around 1 for all analytes and total recovery between 77.5 and 104%. Stability was investigated in stock solutions, human plasma, dry extracts, final extracts and during 3 freeze/thaw cycles. The described method was successfully applied in clinical studies with oral administration of docetaxel or paclitaxel in combination with ritonavir.

Development and validation of LC–MS/MS assays for the quantification of E7080 and metabolites in various human biological matrices

To support clinical pharmacokinetic studies with the anticancer agent E7080 (lenvatinib), liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed for the quantification of E7080 and four of its metabolites in human plasma, urine and faeces and of E7080 in whole blood. Cross-analyte interferences between metabolites and parent compound were expected and therefore accounted for early in the method development. Plasma, urine and faeces samples were extracted with acetonitrile. Chromatographic separation was achieved on a 50 mm × 2.1 mm I.D. XTerra MS C18 column, with a 0.2 mL/min flow and gradient elution starting with 100% formic acid in water, followed by an increasing percentage of acetonitrile. Whole blood samples were extracted with diethyl ether and extracts were injected on a 150 mm × 2.1mm I.D. Symmetry Shield RP8 column. Detection was performed using an API3000 triple quadrupole mass spectrometer, with a turbo ion spray interface, operating in positive ion mode. Using 250 μL of plasma, E7080 and its metabolites could be quantified between 0.25 and 50.0ng/mL. The quantifiable ranges of E7080 in whole blood, urine and faeces were 0.25-500 ng/mL, 1.00-500 ng/mL and 0.1-25μg/g, using sample volumes of 250 μL, 200 μL and 250 mg, respectively. Calibration curves in all matrices were linear with a correlation coefficient (r(2)) of 0.994 or better. At the lower limit of quantification, accuracies were within ±20% of the nominal concentration with CV values less than 20%. At the other concentrations the accuracies were within ±15% of the nominal concentration with CV values below 15%. The developed methods have successfully been applied in a mass balance study of E7080.

Validation of high-performance liquid chromatography–tandem mass spectrometry assays for the quantification of eribulin (E7389) in various biological matrices

This paper presents specific and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assays for the quantification of the novel anticancer agent eribulin in human plasma, whole blood, urine and faeces. These assays, developed to support clinical pharmacological studies with the drug, quantify eribulin concentration ranges of 0.2-100ng/mL for plasma, 0.5-100 ng/mL for whole blood and urine and 0.1-25 μg/g for faeces, using sample volumes of 500 μL or 250 μg (faeces). Samples were prepared with liquid-liquid extraction, separated on a C18 column with gradient elution and analysed with a triple quadrupole MS, in positive ion mode. A structural analogue of eribulin was used as internal standard for the quantification. The assays were linear with correlation coefficients (r(2)) of 0.99 and better, whereby the deviation from nominal concentrations ranged from -8.2 to 8.9% with CV values of maximally 14.2%. Stability assessments demonstrated that eribulin is stable at -20°C in plasma, whole blood, urine and faeces for at least 38, 4, 10.5 and 5 months, respectively. In conclusion, the validation results show that the assays are specific and accurate and can therefore adequately be applied to support clinical studies of eribulin.

Development and clinical validation of an LC–MS/MS method for the quantification of pazopanib in DBS

BACKGROUND Pazopanib is approved for the treatment of renal cell carcinoma and soft tissue sarcoma. Analyses show increased benefit in patients with plasma trough concentrations ≥20.5 μg/ml compared with patients with lower concentrations. METHODS & RESULTS We developed a DBS assay as a patient friendly approach to guide treatment. The method was validated according to US FDA and EMA guidelines and European Bioanalysis Forum recommendations. Influence of spot homogeneity, spot volume and hematocrit were shown to be within acceptable limits. Analysis of paired clinical samples showed a good correlation between the measured plasma and DBS concentrations (R(2) of 0.872). CONCLUSION The method was successfully validated, applied to paired clinical samples and is suitable for application to therapeutic drug monitoring of pazopanib.

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of Dexamphetamine in human plasma

HighlightsA LC–MS/MS assay is described for the quantification of dexamphetamine in plasma.The method is a new combination of simplicity, high sensitivity and full validation.The developed method is fast, easy‐to‐use and economic.Low m/z measuring problems in the clinical setting problems are briefly addressed.Applicability of the method for clinical studies was shown. ABSTRACT Dexamphetamine is registered for the treatment of attention deficit hyperactivity disorder and narcolepsy. Current research has highlighted the possible application of dexamphetamine in the treatment of cocaine addiction. To support clinical pharmacologic trials a new simple, fast, and sensitive assay for the quantification of dexamphetamine in human plasma using liquid chromatography tandem mass spectrometry (LC–MS/MS) was developed. Additionally, it is the first reported LC–MS assay with these advantages to be fully validated according to current US FDA and EMA guidelines. Human plasma samples were collected on an outpatient basis and stored at nominally −20 °C. The analyte and the internal standard (stable isotopically labeled dexamphetamine) were extracted using double liquid–liquid extraction (plasma‐organic and organic‐water) combined with snap‐freezing. The aqueous extract was filtered and 2 &mgr;L was injected on a C18‐column with isocratic elution and analyzed with triple quadrupole mass spectrometry in positive ion mode. The validated concentration range was from 2.5–250 ng/mL and the calibration model was linear. A weighting factor of 1 over the squared concentration was applied and correlation coefficients of 0.997 or better were obtained. At all concentrations the bias was within ±15% of the nominal concentrations and imprecision was ≤15%. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines on method validation. In conclusion, the developed method to quantify dexamphetamine in human plasma was fit to support a clinical study with slow‐release dexamphetamine.

Fast and straightforward method for the quantification of pazopanib in human plasma using LC-MS/MS

Background: Pazopanib is an angiogenesis inhibitor approved for renal cell carcinoma and soft-tissue sarcoma. Studies indicate that treatment with pazopanib could be optimized by adapting the dose based on measured pazopanib plasma concentrations. Methods: We describe the validation and clinical application of a fast and straightforward method for the quantification of pazopanib in human plasma for the purpose of therapeutic drug monitoring and bioanalytical support of clinical trials. Stable isotopically labeled 13C,2H3-pazopanib was used as internal standard. Plasma samples were prepared for analysis by protein precipitation using methanol and diluted with 10 mmol/L ammonium hydroxide buffer. Chromatographic separation was performed on a C18 column using isocratic elution with ammonium hydroxide in water and methanol. For detection, a tandem mass spectrometer, equipped with a turbo ion spray interface was used in positive ion mode at m/z 438 → m/z 357 for pazopanib and m/z 442 → m/z 361 for the internal standard. Results: Final runtime was 2.5 minutes. All validated parameters were within pre-established limits and fulfilled the FDA and EMA requirements for bioanalytical method validation. After completion of the validation, the routine application of the method was tested by analyzing clinical study samples that were collected for the purpose of therapeutic drug monitoring. Conclusions: In conclusion, the described method was successfully validated and was found to be robust for routine application to analyze samples from cancer patients treated with pazopanib.