The present study assessed the relationship between central nervous system (CNS) side effects and plasma concentrations of efavirenz (EFV) in Japanese HIV-1-infected patients. Subjects consisted of 69 HIV-1-infected patients (57 therapy-naive and 12 therapy-experienced patients) being treated using EFV in combination with other antiretroviral agents at the outpatient HIV clinic. Successful virological treatment was achieved in 61 patients. Eight patients discontinued EFV containing therapy because CNS symptoms did not resolve (four patients), EFV-specific mutations were detected (two patients), or skin rash was observed (two patients). Mean EFV plasma concentration for 61 effectively treated patients, measured at 15 h postdosing, was 2.42 microg/ml (range: 0.78-6.82 microg/ml). This EFV concentration range contributed to suppressed viral load in these Japanese patients. Adverse CNS effects were observed in 19 patients soon after therapy onset. These effects disappeared within 1 month except for four patients who suffered severe CNS side effects. Mean EFV plasma concentrations were not significantly different between subjects with (2.45 +/- 1.08 microg/ml) and without (2.42 +/- 1.40 microg/ml) CNS side effects. We concluded no correlation existed between the plasma EFV concentration and the emergence of CNS side effects in Japanese HIV-1-infected patients. Further investigations, enforced with the drug concentration measurement at earlier time points and more appropriate assessment of CNS symptoms, are required.

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Darunavir (DRV), a new protease inhibitor (PI), is used to treat human immunodeficiency virus (HIV) type-1. According to in vitro experiments, DRV was active against HIV-1 with PI resistance mutations and against PI resistant clinical isolates. [1][2][3][4] This drug is expected to be effective in antiretroviral treatment-experienced patients, such as those possessing HIV-1 strains which are resistant to more than one PI. [5][6][7][8] Bouche et al. recently determined plasma DRV concentrations using liquid chromatography-tandem mass spectrometry (LC/MS/MS). 9) However, as LC/MS/MS equipment is very expensive and unavailable in conventional hospital laboratories, development of alternate methods is necessary.We have already developed a simple HPLC method for simultaneous quantitative determination of seven HIV protease inhibitors and efavirenz. 10) We expect DRV can be measured using this method because amprenavir, whose chemical structure is quite similar to DRV, was successfully measured.In this study we aimed to validate the measurement of plasma DRV concentrations using the HPLC method. This is the first report where plasma DRV concentration has been measured using this HPLC method. MATERIALS AND METHODSStandard Solutions and Chemicals DRV was supplied by Tibotec Pharmaceuticals Ltd. (Eastgate Village, Eastgate, Little Island, Co Cork, Ireland). The internal standard (IS), 6,7-dimethyl-2,3-di(2-pyridyl)-quinoxaline, was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Stock solutions of DRV and IS were prepared by dissolving accurately weighed amounts of each reference compound in water/ ethanol (50 : 50, v/v) to yield concentrations of 259 mg/ml for DRV, and 588 mg/ml for IS. These stock solutions were stored at Ϫ80°C and thawed until the day of analysis. The stock solution was diluted in drug-free plasma to yield concentrations of 0.13, 1.30, 2.59, 5.18 and 10.36 mg/ml for DRV. All other chemicals and solvents were of analytical grade and have been described in our previous report. 10)Chromatography The HPLC system consisted of a Waters pump (model 515), a 717 plus autosampler, and a 2487 dual l absorbance detector coupled to the Empower TM software (Waters, Milford MA, U.S.A.). The analytical column was a Radial-Pak Nova-Pak C 18 column (4 mm, 8ϫ100 mm, Waters) protected by Guard-Pak Inserts Nova-Pak C 18 precolumn. Absorbance was measured at 205 nm and separations were performed at 30°C. The mobile phase consisted of 39% 50 mM phosphate buffer (pH 5.9), 22% methanol and 39% acetonitrile. The assay run time was 30 min with a flow rate of 1.8 ml/min. Drugs were quantified by measuring the peak areas under the chromatograms. The other equipment and methodology used in this study have been described in our previous report. 10)Sample Preparation Two milliliters of ethyl acetate/nhexane (50 : 50, v/v) containing the IS (3.55 mg/ml) and 1 ml of 0.5 M sodium carbonate were added to a 500 ml plasma sample. The mixture was vortexed and then centrifuged at 3500ϫg for 5 min. The organic layer was separated and e...