Disulfiram has been used extensively for alcohol abuse and may have a role in treatment for cocaine addiction. Recent data suggest that disulfiram may also reactivate latent HIV in reservoirs. Disulfiram has complex pharmacokinetics with rapid metabolism to active metabolites, including S-Methyl-N, N-Diethylthiocarbamate (DET-Me) which is formed from cytochrome P450 (CYP450). Assessing disulfiram in HIV-infected individuals with a CYP450 inducing drug (e.g., efavirenz) or a CYP450 inhibiting drug (e.g., HIV-1 protease inhibitors) requires an assay that can measure a metabolite that is formed directly via CYP450 oxidation. Therefore, an assay to measure concentrations of DET-Me in human plasma was validated. DET-Me and the internal standard, S-ethyldipropylthiocarbamate (EPTC) were separated by isocratic ultra performance liquid chromatography using a Waters Acquity HSS T3 column (2.1×100mm, 1.8μm) and detection via electrospray coupled to a triple quadrupole mass spectrometer. Multiple reaction monitoring in positive mode was used with DET-Me at 148/100 and the internal standard at 190/128 with a linear range of 0.500 to 50.0 ng/mL with a five minute run time. Human plasma (500 μL) was extracted using a solid phase procedure. The interassay variation ranged from 1.86 to 7.74% while the intra assay variation ranged from 3.38 to 5.94% over three days. Representative results are provided from samples collected from subjects receiving daily doses of disulfiram 62.5 mg or 250 mg.
The quantification of the integrase inhibitor, elvitegravir, in cerebrospinal fluid will significantly help understand its effects in the central nervous system. An ultra high performance liquid chromatography with tandem mass spectrometry method was successfully validated to measure elvitegravir in human plasma and cerebrospinal fluid. The biological fluid was diluted with methanol and the clear supernatant was injected into the chromatography system. The calibration was linear from 10.0 to 5000 ng/mL in plasma and 1.00 to 25 ng/mL in cerebrospinal fluid. Inter‐day and intra‐day variability ranged from 1.46 to 8.34% and accuracy ranged from –8.36 to 7.50% for quality controls in either plasma or cerebrospinal fluid analysis. The method was then applied to the measurement of elvitegravir concentrations in plasma and cerebrospinal fluid samples from an HIV+ patient enrolled in a clinical study who switched medications of Stribild® to Genvoya®. The validated method is suitable for the quantification of elvitegravir in plasma and cerebrospinal fluid.
The ITX 5061 assay is accurate and reproducible with a wide linear range and will be used for pharmacokinetic analysis and dose-finding studies in HCV-monoinfected patients.
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