Faculty of applied medical science.

Minocycline and valproic acid are potential adjuvant therapies for the treatment of human immunodeficiency virus (HIV)-associated cognitive impairment. The purpose of this study was to determine whether minocycline alone or in combination with valproic acid affected atazanavir plasma concentrations. Twelve adult HIV-infected subjects whose regimen included atazanavir (300 mg)-ritonavir (100 mg) daily for at least 4 weeks were enrolled. Each subject received atazanavir-ritonavir on day 1, atazanavir-ritonavir plus 100 mg minocycline twice daily on days 2 to 15, and atazanavir-ritonavir plus 100 mg minocycline twice daily and 250 mg valproic acid twice daily on days 16 to 30 with meals. The subjects had 11 plasma samples drawn over a dosing interval on days 1, 15, and 30. The coadministration of minocycline and valproic acid with atazanavirritonavir was well tolerated in all 12 subjects (six male; mean [؎ standard deviation] age was 43.1 [8.2] years). , respectively. Similar decreases in atazanavir exposure were seen after the addition of valproic acid. The GMRs (95% CI) for atazanavir AUC 0-24 , C min , and C max with and without minocycline plus valproic acid were 0.68 (0.43 to 1.06), 0.50 (0.24 to 1.06), and 0.66 (0.41 to 1.06), respectively. Coadministration of neither minocycline nor minocycline plus valproic acid appeared to influence the plasma concentrations of ritonavir (P > 0.2). Minocycline coadministration resulted in decreased atazanavir exposure, and there was no evidence that the addition of valproic acid mediated this effect.

The objective of the study was to measure antiretroviral exposures in four physiological compartments during pregnancy, delivery, and postpartum. This prospective, open-label, longitudinal study collected paired blood plasma (BP) and genital tract (GT) aspirates antepartum, at delivery, and up to 12 weeks postpartum. Antiretroviral cord BP and amniotic fluid concentrations were also measured. Drug concentrations were analyzed by validated high-performance liquid chromatography/UV and liquid chromatography/tandem mass spectrometry methods, with secondary compartment concentrations presented as the percentage of BP. Fourteen women taking lamivudine plus zidovudine and either lopinavir-ritonavir (n ‫؍‬ 7), nelfinavir (n ‫؍‬ 6), or nevirapine (n ‫؍‬ 1) were enrolled; four also received tenofovir. GT penetration relative to BP was highest for the nucleoside reverse transcriptase inhibitors compared to the protease inhibitors and nevirapine. Only antepartum nelfinavir GT penetration was significantly higher than in the second trimester (geometric mean ratio [GMR], 179.3) or third trimester (GMR, 41.9). Compared to nonpregnant historical controls, antepartum GT penetration was significantly lower (P < 0.05) for zidovudine (GMR, 0.25) and lopinavir (GMR, 0.03); postpartum lopinavir GT penetration continued to be significantly lower (GMR, 0.27). Cord BP exposures were highest for lamivudine and tenofovir (>100%), with cord BP levels of the remaining drugs ranging from 49 to 86% of that of the respective BP level. Amniotic exposures for lamivudine, zidovudine, tenofovir, and nelfinavir were >100%, nevirapine exposure was 53%, and lopinavir and ritonavir exposures were <6% that of BP. We conclude that GT, cord BP, and amniotic fluid exposures vary within and between antiretroviral drug classes and biologic sites. Measurement of antiretroviral exposure in maternal genital secretions, cord BP, and amniotic fluid may be needed to identify signals of subtherapeutic or supratherapeutic drug exposure.

The new potent combination of antiretrovirals etravirine, darunavir, and ritonavir requires a new bioanalytical method for clinical pharmacology investigations and potential therapeutic drug monitoring. The development and validation of a novel LC-MS method for the simultaneous quantification of the most recently FDA-approved protease inhibitor and non-nucleoside reverse transcriptase inhibitor is described. This novel method was developed and validated using a sub-2 μm particle column, and provides excellent chromatographic separation and peak shape for all three analytes and internal standard. The method was validated over the range of 0.002-2.0 μg/mL. Intraand inter-day accuracy of all analytes ranged from 88 to 106%, and intra-and inter-day precision was <7%. Dilution of samples 2-, 5-, and 10-fold maintained accuracy and precision, using a sample volume as low as 10 μL. Finally, the applicability of the method was investigated with clinical samples and external quality assurance proficiency testing samples.

Studying the pharmacokinetics of antiretroviral drugs in breast milk has important implications for the health of both the mother and the infant, particularly in resource-poor countries. Breast milk is a highly complex biological matrix, yet it is necessary to develop and validate methods in this matrix, which simultaneously measure multiple analytes, as women may be taking any number of drug combinations to combat human immunodeficiency virus infection. Here, we report a novel extraction method coupled to high-performance liquid chromatography and tandem mass spectrometry for the accurate, precise, and specific measurement of 7 antiretroviral drugs currently prescribed to infected mothers. Using 200 microL of human breast milk, simultaneous quantification of lamivudine (3TC), stavudine (d4T), zidovudine (ZDV), nevirapine (NVP), nelfinavir (NFV), ritonavir, and lopinavir was validated over the range of 10-10,000 ng/mL. Intraday accuracy and precision for all analytes were 99.3% and ...

To evaluate whether patients with human immunodeficiency virus (HIV) infection who were receiving protease inhibitor therapy had altered bile acid concentrations compared with noninfected control subjects, and whether bile acid concentrations could predict the onset of hepatotoxicity caused by protease inhibitors. Retrospective sample analysis from a prospectively conducted clinical trial. Academic research center. Eleven adults with advanced HIV disease who were taking protease inhibitor-based antiretroviral therapy, one of whom had developed protease inhibitor-induced hepatotoxicity. Plasma concentrations of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and taurocholic acid (TC) were analyzed by using a novel high-performance liquid chromatography with tandem mass spectrometry detection method. Comparisons of the relative contribution of each bile acid to the total bile acid pool were made with previous...

The effects of tipranavir/ritonavir (TPV/r) on hepatic and intestinal P-glycoprotein (P-gp) and cytochrome P450 (CYP) enzyme activity were evaluated in 23 volunteers. The subjects received oral (p.o.) caffeine, warfarin + vitamin K, omeprazole, dextromethorphan, and midazolam and digoxin (p.o. and intravenous (i.v.)) at baseline, during the first three doses of TPV/r (500 mg/200 mg b.i.d.), and at steady state. Plasma area under the curve (AUC)(0-infinity) and urinary metabolite ratios were used for quantification of protein activities. A single dose of TPV/r had no effect on the activity of CYP1A2 and CYP2C9; it weakly inhibited CYP2C19 and P-gp; and it potently inhibited CYP2D6 and CYP3A. Multiple dosing produced weak induction of CYP1A2, moderate induction of CYP2C19, potent induction of intestinal P-gp, and potent inhibition of CYP2D6 and CYP3A, with no significant effects on CYP2C9 and hepatic P-gp. Several P450/transporter single-nucleotide polymorphisms correlated with the ba...

A sensitive, accurate, and precise liquid chromatography-mass spectrometry assay for the determination of tenofovir (TNF) in human vaginal tissue was developed and validated. After homogenization of the tissue, solid-phase extraction on Varian Bond Elut-C(18) column was used for sample clean up. Chromatographic separation of TNF and the internal standard (tolbutamide) was achieved with a Varian Polaris 3C(18)-A reversed-phase analytical column (150 mm x 2 mm). A gradient method using 0.1% formic acid in water and 0.1% formic acid in acetonitrile was employed. Detection of TNF and tolbutamide was achieved by electrospray ionization mass spectrometry in the positive ion mode using 288.05 and 271.00 m/z, respectively. Linear TNF calibration curves were obtained between 1-1,000 ng/mL with a correlation coefficient (r(2)) greater than 0.999. Intra- and inter-day accuracy for TNF ranged from 89.7% and 109.4% and from 97.3% and 104.9%, and precision ranged from 1.3% and 10.9% and 2.6% and ...

The objective of the study was to measure antiretroviral exposures in four physiological compartments during pregnancy, delivery, and postpartum. This prospective, open-label, longitudinal study collected paired blood plasma (BP) and genital tract (GT) aspirates antepartum, at delivery, and up to 12 weeks postpartum. Antiretroviral cord BP and amniotic fluid concentrations were also measured. Drug concentrations were analyzed by validated high-performance liquid chromatography/UV and liquid chromatography/tandem mass spectrometry methods, with secondary compartment concentrations presented as the percentage of BP. Fourteen women taking lamivudine plus zidovudine and either lopinavir-ritonavir (n ‫؍‬ 7), nelfinavir (n ‫؍‬ 6), or nevirapine (n ‫؍‬ 1) were enrolled; four also received tenofovir. GT penetration relative to BP was highest for the nucleoside reverse transcriptase inhibitors compared to the protease inhibitors and nevirapine. Only antepartum nelfinavir GT penetration was significantly higher than in the second trimester (geometric mean ratio [GMR], 179.3) or third trimester (GMR, 41.9). Compared to nonpregnant historical controls, antepartum GT penetration was significantly lower (P < 0.05) for zidovudine (GMR, 0.25) and lopinavir (GMR, 0.03); postpartum lopinavir GT penetration continued to be significantly lower (GMR, 0.27). Cord BP exposures were highest for lamivudine and tenofovir (>100%), with cord BP levels of the remaining drugs ranging from 49 to 86% of that of the respective BP level. Amniotic exposures for lamivudine, zidovudine, tenofovir, and nelfinavir were >100%, nevirapine exposure was 53%, and lopinavir and ritonavir exposures were <6% that of BP. We conclude that GT, cord BP, and amniotic fluid exposures vary within and between antiretroviral drug classes and biologic sites. Measurement of antiretroviral exposure in maternal genital secretions, cord BP, and amniotic fluid may be needed to identify signals of subtherapeutic or supratherapeutic drug exposure.

Objective-Evaluate the pharmacokinetics (PK) of lamivudine (3TC), stavudine (d4T), and nevirapine (NVP) in HIV-infected Malawian children receiving quartered tablet multiples of Triomune 40™ (GT) compared to individual generic (GL) and trade (TL) liquids Design-prospective, randomized, 3-way crossover study [8-<12kg;18-<22kg; or 28-<32kg] taking Triomune 40™ were recruited and randomized to receive GT twice daily (¼, ½, or ¾ tablets using Malawi treatment guidelines); GL twice daily (in the equivalent dose of GT), or TL twice daily (dosed using weight and age from DHHS Pediatric treatment guidelines). After 10 days of one formulation, 6-hour PK sampling was performed, and subjects crossed over to subsequent formulations. C 0h , AUC 0-6h , C max, and T max were generated for each antiretroviral (ARV).

A simple reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of three HIV non-nucleoside reverse transcriptase inhibitors (nevirapine, delavirdine, and efavirenz) in human blood plasma is described. The method was validated over the range of 10 ng / ml to 50 mg / ml for nevirapine, 25 ng / ml to 25 mg / ml for delavirdine, and 10 ng / ml to 10 mg / ml for efavirenz. The method is accurate (average accuracies over eight concentrations ranging from 87.3 to 113%), and precise (within-day and between-day precision measures ranging from 0.12 to 7.9% and 0.26 to 5.9%, respectively). All three non-nucleoside reverse transcriptase inhibitors proved to be stable under various conditions. Due to its simplicity, this assay can readily be used for investigational or clinical monitoring of plasma concentrations.

Understanding the pharmacokinetics of drugs in peripheral body compartments, such as the genital tract, is particularly important in the infectious diseases arena. However, extracting drugs from small volumes of viscous, proteinacious substances like cervicovaginal fluid is particularly challenging. The goal of this study was to develop a method to quantify raltegravir, an HIV-1 integrase inhibitor, in the female genital tract. The method included sample preparation with perchloric acid followed by solid-phase extraction, separation with reverse-phase high-performance liquid chromatography, and detection with an ultraviolet wavelength of 218 nm. The method was linear from 0.05 to 10.0 mg/L, with minimal endogenous interference. The method was accurate (1.2-11.0% deviation) and precise (1.1-12.6% CV) for both within and between-day analyses. The ability to detect raltegravir in the female genital tract is essential for future investigations of raltegravir as an agent for prevention of HIV acquisition, and this method will be used for clinical studies further evaluating pharmacokinetic-pharmacodynamic relationships in this body compartment.

An accurate, sensitive and specific reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors zalcitabine, lamivudine, didanosine, stavudine, zidovudine, and abacavir with the non-nucleoside reverse transcriptase inhibitor nevirapine in human blood plasma is described. The new Polarity dC C silica column used in this method provides better resolution and peak shape than all 18 other columns tested. Also, four different ultraviolet wavelengths were used for accurate and specific quantitation of the analytes. The method was validated over the range of 10-10 000 ng / ml for all analytes except zalcitabine (10-5000 ng / ml). This method is accurate (average accuracies of three different concentrations ranged from 97.2 to 105%), and precise (within-and between-day precision measures ranged from 0.5 to 5.1% and 0.5 to 5.6%, respectively), and is currently being used for determination of plasma drug concentrations in our laboratory. 

The highly potent non-nucleoside reverse transcriptase inhibitor UC-781 is under development as a potential microbicide to prevent sexual transmission of human immunodeficiency virus type 1 (HIV-1). A sensitive and reproducible liquid chromatography-mass spectrometric method has been developed and validated for the quantification of the drug in New Zealand white rabbit plasma after liquid-liquid extraction procedure. The method was validated over the range of 1-500 ng mL(-1). Average recoveries of the extraction method were high and consistent: 72%. The method is accurate with average accuracies over three QC (n=30) concentrations ranging from 99.9% to 106.1%, and precise (within-day and between-day precision measures ranging from 2.2% to 9.9% and 6.50% to 9.0%, respectively). Plasma from other three species proved that extraction method did no affect analyte and internal standard stability. Due to its critical and consequential use, this assay could be readily used for investigatio...

Liquid chromatography plays the important and critical role in the field of clinical pharmacology of evaluating drugs in biological matrices. Studying antiretroviral drugs in the female genital tract has important implications for using drugs as a vaginal microbicide for prevention of HIV-1 sexual transmission. Accurate measurement of drug levels is extremely important in optimizing drug concentration in gel formulation. Extracting drugs from small volumes of viscous, proteinaceous substances like cervicovaginal fluid (CVF) is a practically challenging process. The proposed method was designed to introduce procedure for sample collection and drug extraction procedure for CVF matrix before the chromatographic separation. Based on this extraction method, we validated a reverse phase high performance liquid chromatography with electrospray ionization mass spectrometry assay in order to quantify UC781 in female genital tract compartment. The LC-MS method was validated based on a novel extraction technique which proved to be efficient in reducing analyte degradation with an average extraction efficiency of 72%. This method is accurate, demonstrating an average accuracy over three QC (n = 30) concentrations ranging from 99.9% to 106.1%. Average precision within-day and between-day ranged from 3.1% to 10.2% and 5.1% to 6.4%, respectively. We demonstrated that the analyte was able to maintain its stability under various conditions using this extraction method. The sample preparation, extraction, and the powerful liquid chromatography and mass spectrometry can readily be applied for accurate quantification of similar drugs in CVF.