Bioanalysis in clinical development of tasquinimod using liquid chromatography/tandem mass spectrometry.

Tasquinimod (ABR-215050) is an oral drug in clinical development for treatment of patients with castrate resistant prostate cancer. This paper describes a method for the determination of tasquinimod in human plasma. The method is based on liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) using stable isotope labeled tasquinimod as internal standard (IS). The plasma samples were processed by protein precipitation using acidic acetonitrile containing the IS. The precipitated samples were centrifuged and the supernatant was injected directly into the LC-MS/MS system. Chromatographic separation was performed on a reversed phase column using fast gradient elution, with a total run cycle time of 4 min. The method was validated with respect to accuracy, precision, dynamic range, lower limit of quantification, selectivity and robustness. Furthermore, the stability of tasquinimod in spiked plasma, in processed extracts and in incurred samples was thoroughly studied. The method was validated in the range of 1.0-2400 nmol/L, defining the lower and upper limits of quantification. The repeatability, reproducibility and overall bias were 1.5-7.1%, 3.5-7.4%, and 1.3-4.7%, respectively, in the range of 1-2000 nmol/L. Excellent selectivity was demonstrated in the validation, as well as in study samples from both healthy volunteers and cancer patients. Robustness was demonstrated by the calculated carry-over as low as 0.06%, and by an incurred sample reproducibility (ISR) experiment where 97% of the reanalyzed samples fulfilled the acceptance criteria of 20% deviation from initial analysis result. Also, tasquinimod was found to be stable in all investigated matrices, including in incurred samples. In an incurred sample stability (ISS) investigation, tasquinimod was demonstrated to be stable for 24 months, and 97% of the reanalyzed samples were within 20% from the initial analysis result. In conclusion, the method was demonstrated to be accurate, precise, robust and reliable for the determination of tasquinimod. The method was successfully used in several clinical studies for the support of pharmacokinetic and pharmacodynamic evaluations.

Aniline in hydrolyzed urine and plasma--possible biomarkers for phenylisocyanate exposure.

There are few studies on phenylisocyanate (PhI) exposure, although there are studies indicating that PhI is a very potent chemical sensitizer. The aim of this study was to evaluate aniline in urine and plasma as possible biomarkers of exposure to PhI. Occupational airborne exposure to PhI was measured during one day for 11 workers exposed to thermal degradation products from polyurethane with filters impregnated with 2-methoxyphenyl piperazine. A urine sample was collected from each worker on measurement day, and plasma samples were collected within the following 2 weeks. Urine and plasma samples also were collected from four unexposed subjects. The biological samples were hydrolyzed and analyzed with gas chromatography mass spectrometry. The time-weighted averages (TWA) for the workers were between 0.1 and 1.6 microg/m3. Aniline levels in urine were in the same range for the exposed and unexposed workers, but there was a significant correlation between air and urinary levels (Pearson's correlation coefficient r = 0.518; p = 0.05). All exposed workers had higher levels in the plasma samples than the highest control, and there was a significant correlation between the plasma levels and measured air levels (r = 0.675; p = 0.008). The conclusion is that aniline in hydrolyzed urine and plasma are possible biomarkers of exposure to PhI, and that the plasma biomarker is more sensitive, at least at this rather low exposure.

Biological monitoring of exposure to 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate.

OBJECTIVES: Biological monitoring of occupational sensitizers, such as 1,5-naphthalene diisocyanate (NDI) and 4,4'-methylenediphenyl diisocyanate (MDI) is of high importance. In this study, 1,5-naphthalenediamine (NDA) and 4,4'-methylenedianiline (MDA) in hydrolysed urine and plasma were evaluated as biomarkers of exposure to NDI and MDI, respectively. METHODS: The air exposure to NDI and MDI was monitored for 30 exposed workers at four different plants. In parallel, urinary as well as blood plasma samples were collected. Biomarker levels were determined in hydrolysed urine and plasma by means of gas chromatography-mass spectrometry. RESULTS: Air exposure to both MDI and NDI was correlated to their corresponding urinary and plasma biomarkers. The correlation coefficients for the associations between air and biomarker levels were in the range of 0.51-0.65 and 0.53-0.96 for MDI and NDI, respectively. For NDI, but not for MDI, the significance and correlation coefficients were increased by adjusting the urinary biomarker levels for creatinine content or density. CONCLUSIONS: Biomarker and air levels of MDI and NDI were correlated, but there was a large individual variation.

Identification and characterisation of adducts between serum albumin and 4,4'-methylenediphenyl diisocyanate (MDI) in human plasma.

Diisocyanates are potent inducers of airways disease. Methylenediphenyl diisocyanate (MDI) is a widely used diisocyanate in the chemical industry. The aim of this study was to identify major and also immunologically relevant protein conjugates of MDI in plasma. Plasma was obtained from an MDI-exposed worker. The plasma was dialysed and then fractionated using ion exchange chromatography (IEC) and gel filtration. These fractions and also aliquots of unfractioned plasma were hydrolysed, derivatised and analysed for isocyanate adduct content using gas chromatography-mass spectrometry. In addition, immunologically relevant proteins were identified through specific IgG immunoblotting using pooled sera from two exposed workers. It was shown by dialysis that 96% of the hydrolysed MDI derivatives were protein bound and that 95% of the MDI adducts co-eluted with serum albumin in plasma using IEC. All MDI-protein adducts co-eluted with serum albumin using gel filtration. IgG immunoblotting showed a major 66 kDa protein and also some intermolecular reactions in serum albumin. This study shows serum albumin to be the major protein in plasma that forms adducts in vivo with MDI. Thus, a quick and simple quantitative method for biological monitoring may be developed for MDI exposure. The results also showed that MDI-specific IgG antibodies preferentially bind to the serum albumin in in-vitro-synthesised MDI-plasma protein conjugates.