ABSTRACT
Tolvaptan is a competitive vasopressin V2-receptor antagonist that inhibits water reabsorption in the renal collecting ducts. A selective and sensitive liquid chromatography-tandem mass spectrometry method for determining tolvaptan and its nine metabolites in rat serum was developed and validated. An analogue of tolvaptan was used as an internal standard. Sample preparation involved protein precipitation following solid-phase extraction. Chromatographic separation was performed on a C18 reversed-phase column with a linear gradient elution. The flow rate was 0.25 mL/min, and total run time was 30 min. The analytes were detected by tandem mass spectrometry using an electrospray ionization interface in positive ion mode and multiple reaction monitoring. The calibration curve showed linearity over the concentration range from 5 to 1,000 ng/mL for each analyte. The lower limit of quantification using 0.1 mL of rat serum was 5 ng/mL for each analyte. Precision did not exceed 5.7 %, and accuracy as relative error were within ± 7.5 % for all analytes. The validated method was successfully applied to evaluate the pharmacokinetics of oral tolvaptan in rats, indicating the systemic exposure to tolvaptan in females eight times larger than that in males.
Subject(s)
Antidiuretic Hormone Receptor Antagonists/blood , Benzazepines/blood , Animals , Antidiuretic Hormone Receptor Antagonists/metabolism , Antidiuretic Hormone Receptor Antagonists/pharmacokinetics , Benzazepines/metabolism , Benzazepines/pharmacokinetics , Biotransformation , Calibration , Chromatography, High Pressure Liquid/methods , In Vitro Techniques , Limit of Detection , Molecular Structure , Rats, Sprague-Dawley , Reference Standards , Reproducibility of Results , Solid Phase Extraction , Tandem Mass Spectrometry/methods , TolvaptanABSTRACT
Hepatitis C virus (HCV)-specific CD8+ cytotoxic T lymphocytes (CTL) contribute to viral clearance in acute, self-limited hepatitis C as well as to liver cell injury in the more frequent cases with chronic hepatitis C. Although HLA class I-peptide tetramers have been used to detect circulating HCV epitope-specific CTL with a high sensitivity and specificity, this technique has been targeted exclusively to the most frequent HLA haplotypes in the Caucasian population and the large number of HCV-infected Asian patients, most of whom are HLA-A24 positive, have not been studied. The current study determines the frequency, phenotype, and clinical significance of HCV-specific CD8(+) T lymphocytes with five different HLA-A*2402 tetramers in 43 HCV infected Japanese patients and 32 controls. Overall, tetramer(+) cells were detected in the blood of 33 of 43 patients at frequencies of 0.064-0.75% CD8(+)CD4(-)CD14(-)CD19(-) T lymphocytes. Interestingly, although the T cell response was always targeted multispecifically against epitopes in different HCV proteins, the relative frequency of cells stained with individual tetramers differed between patients. Furthermore, tetramer(+)CD8(+) T lymphocytes were highly activated, but the phenotypes of different tetramer(+) cells varied in each patient. In conclusion, HLA-A24 restricted, HCV-specific CD8(+) T lymphocytes are found at similar frequencies in Asian patients as HLA-A2 restricted, HCV-specific CD8(+) T lymphocytes in Caucasian patients. Differences in the frequency and activation status of individual tetramer(+) cell populations suggest that CD8(+) T lymphocytes with different HCV epitope specificity may mediate differential pathogenetic effects in chronic hepatitis C.
