Cognitive ageing is premature among a community sample of optimally treated people living with HIV.

OBJECTIVES: Evidence of premature cognitive ageing amongst people living with HIV (PLHIV) remains controversial due to previous research limitations including underpowered studies, samples with suboptimal antiretroviral access, varying rate of virological control, high rate of AIDS, over-representation of non-community samples, and inclusion of inappropriate controls. The current study addresses these limitations, while also considering mental health and non-HIV comorbidity burden to determine whether PLHIV showed premature cognitive ageing compared with closely comparable HIV-negative controls. METHODS: This study enrolled 254 PLHIV [92% on antiretroviral therapy; 84% with HIV RNA < 50 copies/mL; 15% with AIDS) and 72 HIV-negative gay and bisexual men [mean (SD) age = 49 (10.2) years] from a single primary care clinic in Sydney, Australia. Neurocognitive function was evaluated with the Cogstate Computerized Battery (CCB) at baseline and 6 months after. Linear mixed-effects (LME) models examined main and interaction effects of HIV status and chronological age on the CCB demographically uncorrected global neurocognitive z-score (GZS), adjusting for repeated testing, and then adjusting sequentially for HIV disease markers, mental health and comorbidities. RESULTS: HIV status and age interacted with a lower GZS (ß = -0.43, P < 0.05). Higher level of anxiety symptoms (ß = -0.11, P < 0.01), historical AIDS (ß = -0.12, P < 0.05) and historical HIV brain involvement (ß = -0.12, P < 0.05) were associated with lower GZS. CONCLUSIONS: We found a robust medium-sized premature ageing effect on cognition in a community sample with optimal HIV care. Our study supports routine screening of cognitive and mental health among PLHIV aged ≥ 50 years.

Associations between CD36 gene polymorphisms, fat tolerance and oral fat preference in a young-adult population.

BACKGROUND/OBJECTIVES: CD36 is known to be an orosensory receptor for dietary long-chain fatty acids, as well as being involved in the chemosensory mechanisms within the human gut. Recent data have demonstrated an association between CD36 single-nucleotide polymorphisms (SNPs) and lipid consumption behaviours in humans. This study aimed to test for associations between CD36 SNPs and response to a high-fat meal in a young healthy Australian cohort. Secondary associations were tested between CD36 gene variants and fasting lipid parameters, body composition, cardiovascular disease (CVD) risk factors and measures of oral fat preference. SUBJECTS/METHODS: Two SNPs (rs1527479 and rs1984112) were assessed for associations with response to a 75 g saturated fat oral fat tolerance test (OFTT), whole-body substrate oxidation, fasting plasma lipids, CVD risk factors and self-reported habitual diet questionnaires. Genotyping was performed using real-time polymerase chain reaction. RESULTS: Cross-sectional data were collected on 56 individuals (28 m, 28 f; 24.9±3.3 years), with 42 completing participation in a high-fat OFTT. No genotypic associations were evident in anthropometric data or self-reported fat preference measures. AA SNP carriers at rs1984112 exhibited significantly elevated fasting triglyceride when compared with non-carriers (P=0.024). This group also tended to have an elevated response to a high-fat meal (P=0.078). CONCLUSIONS: Although these data show the potential pleiotropic influence of CD36 SNP rs1984112 on lipoprotein accumulation in a young healthy cohort, further assessment in a larger cohort is warranted.

Characterization of nelfinavir binding to plasma proteins and the lack of drug displacement interactions.

OBJECTIVES: To determine the characteristics of the binding of nelfinavir and active M8 to alpha1-acid glycoprotein (AAG) and human serum albumin (HSA), and to examine the displacement effects of drugs binding extensively to AAG (ritonavir and saquinavir) or to HSA (salicylic acid and valproic acid). METHODS: Free drugs were separated by equilibrium dialysis after incubation with human plasma or purified plasma proteins and after co-incubation with potential displacers. Association constants were estimated from double-reciprocal plots of the data. RESULTS: Nelfinavir and M8 free fractions [fractions of unbound drug (fus)] were 0.42+/-0.08% (mean+/-standard deviation) and 0.64+/-0.07%, respectively. For the two analytes, respectively, association constants were 7.25 x 10(7)/m and 3.33 x 10(7)/m for AAG and 1.11 x 10(6)/m and 7.92 x 10(5)/m for HSA. Nelfinavir fu in an AAG solution was significantly (P < 0.01) increased by the addition of ritonavir or saquinavir, whereas it was unaltered by addition of these drugs to whole plasma. Similarly, fu in an HSA solution was significantly increased (P < 0.01) by the addition of salicylic acid or valproic acid, whereas there was no difference in the free fraction in plasma. CONCLUSIONS: The affinity of nelfinavir for human plasma proteins was higher than that of M8, and both nelfinavir and M8 showed higher affinity to AAG than to HSA. The free fraction of nelfinavir was not affected by drugs that bind extensively to AAG or albumin when these drugs were added to whole plasma in combination, suggesting a compensatory effect of alternate binding proteins.

A direct determination of thymidine triphosphate concentrations without dephosphorylation in peripheral blood mononuclear cells by LC/MS/MS.

A rapid, sensitive and specific analytical method with minimal sample preparation for the measurement of thymidine triphosphate (TTP) in peripheral blood mononuclear cells (PBMC) by LC/MS/MS has been developed. PBMC were separated from whole blood or buffy coat. The analyte and internal standard were extracted from PBMC with 70% methanol (pH 7.2). These extracts after centrifugation were directly injected onto LC/MS/MS without need for any further sample preparation. The calibration curve was linear over the range 0.8-800 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the range of the standard curve were less than 10%. The overall recovery of TTP was 103.5%.

Pharmacokinetics and pharmacodynamics of thalidomide in HIV patients treated for oral aphthous ulcers: ACTG protocol 251. AIDS Clinical Trials Group.

Thalidomide has increasing clinical benefits, including the healing of aphthous ulcers in patients with HIV. Unfortunately, pharmacological information addressing the pharmacokinetics (PK) of this compound in HIV patients is limited. Concern exists as to whether thalidomide may alter its own metabolism owing to in vitro data previously reported. Furthermore, no information is available defining the relationship between drug exposure and clinical response. This study evaluated the PK and pharmacodynamics (PD) of thalidomide in patients enrolled in AIDS Clinical Trials Group Protocol 251. Study patients had HIV infection and oral aphthous ulcers of at least 2 weeks'duration. Pharmacologic studies were completed in those subjects randomized to receive active thalidomide at a dose of 200 mg daily for the 4-week study period. PK studies involving serial sampling were carried out in 7 subjects following multiple dosing during study weeks 1 and 4. In addition, trough measurements were done in 20 subjects during each of the 4 study weeks to explore the relationship between time-averaged trough values and extent of clinical response. All samples were analyzed using a validated HPLC method, and parameters were determined using noncompartmental PK analysis. Thalidomide oral clearance averaged 0.14 +/- 0.08 and 0.12 +/- 0.05 l/h/kg on weeks 1 and 4 (p = 0.72), while the terminal elimination half-life averaged 5.7 +/- 1.5 and 7.3 +/- 1.7 hours (p = 0.12). The median time-averaged trough value for subjects deemed complete responders was 0.60, while the median value for noncomplete responders was 0.54. Adjusting for baseline CD4 count and initial index ulcer area, no significant effects were observed of increased thalidomide levels on response. In summary, this study provides steady-state PK data in HIV patients managed with thalidomide and suggests negligible effect of chronic dosing on drug clearance (comparing results from weeks 1 and 4). Furthermore, variable trough measurements between patients do not directly influence the effectiveness of thalidomide for oral aphthous ulcers.

An LC-MS-MS method for the determination of indinavir, an HIV-1 protease inhibitor, in human plasma.

A method for the determination of indinavir (IDV) (L-735 524) in human plasma by LC-MS-MS is discussed, and the validation data is presented. The analyte and internal standard are isolated from plasma by a simple acetonitrile precipitation of plasma proteins followed by centrifugation. LC-tandem mass spectrometry in positive ion, multiple reaction monitoring mode used pairs of ions at m/z of 614/421 for indinavir and 628/421 for internal standard, respectively. The calibration curve had a linear range from 3.0 to 12320 ng/ml when linear least square regression weighing 1/x was applied to the concentration versus peak area plot. The advantages of this method are the fast sample preparation, wide dynamic assay range and quick analysis taking only 5 min for each sample run. The robust nature of this assay has been further verified during routine use over several months involving multiple analysts.

Liquid chromatographic determination of urinary 6beta-hydroxycortisol to assess cytochrome p-450 3A activity in HIV positive pregnant women.

Assessing the activity of CYP3A4 is important for predicting the pharmacokinetic behavior of protease inhibitors in HIV positive patients, especially in pregnant women. The endogenous hormonal ratio of 6beta-hydroxycortisol (beta-OHF) to cortisol (F) in the urine is an index for metabolic enzyme activity of cytochrome p-450 (CYP) 3A4. Because the ratio is a unique way to assess the enzyme activity without using any exogenous probes for this isozyme, it is practical for use in pregnant women. In this paper, we describe a method using high performance liquid chromatography (HPLC) for 6beta-OHF in urine from pregnant women to estimate the ratio of 6beta-OHF/F. Urinary 6beta-OHF was measured by using C18-cartridge solid phase extraction and isocratic HPLC. Aliquots (1 ml) of urine samples spiked with internal standard, 6beta-hydroxyprednisolone (6beta-OHPSL), were alkalinized with NaOH, then applied to C18-cartridges, which were washed with water and hexane and eluted with ethyl acetate. After the effluents were dried and reconstituted in 10% acetonitrile, the samples were analyzed by HPLC using an isocratic mobile phase (acetic acid/acetonitrile/50 mM potassium dihydrogenphosphate: 0.2/9/90.8; v/v) and ultraviolet detection at 245 nm. The recoveries of 6beta-OHF from C18 cartridges were 93.2 and 93.9% when the authentic 6beta-OHF was added to the urine sample at the concentration of 50 and 300 ng/ml, respectively. Intra- and inter-day variations estimated at concentrations of 113-674 ng/ml were 2.9-5.6 and 4.9-8.1%, respectively. The method was applied to morning urine samples collected from HIV-positive pregnant women managed with protease inhibitor containing anti-retroviral regimens.

Altered pharmacokinetics of indinavir by a novel nonnucleoside reverse transcriptase inhibitor (HBY-097): a pharmacokinetic evaluation in HIV-positive patients.

HBY-097 (HBY), an investigational nonnucleoside reverse transcriptase inhibitor (NNRTI), and indinavir (IDV) share a common metabolic pathway, cytochrome P4503A4 (CYP3A4), and may clinically be used together as well as with zidovudine (ZDV). Thus, the potential pharmacokinetic (PK) interaction between these drugs was evaluated. HBY (500 mg Q8H), IDV (800 mg Q8H), and ZDV (200 mg Q8H) were given to 8 HIV-infected subjects. Serial plasma samples were collected at baseline (ZDV and IDV alone) and day 11 (all 3 drugs) to determine PK parameters using noncompartmental analysis. PK parameters for ZDV in the presence and absence of HBY were not appreciably different. However, both the maximum (Cmax) and minimum (Cmin) concentrations of IDV were significantly reduced, from a mean of 7514 +/- 1636 and 146 +/- 81 mcg/L to 4725 +/- 2494 mcg/L and 54 +/- 24 mcg/L (p < .05) after addition of HBY. Furthermore, apparent clearance (CL/F) of IDV before and after 11 days of concomitant HBY administration was significantly higher, from 0.69 +/- 0.14 to 1.94 +/- 0.63 L/h/kg (p < .05) with an associated reduction in area under the curve (AUC0-8) from 16,034 +/- 4903 to 6134 +/- 2701 mg/L/h (p < .05). The increase in IDV CL/F is consistent with the observed metabolic induction effects of other NNRTIs. The results of this trial showed that HBY significantly alters the pharmacokinetic parameters of IDV at the dose studied.

High-performance liquid chromatographic determination of ribavirin in whole blood to assess disposition in erythrocytes.

Ribavirin is an antiviral agent used in the treatment of chronic hepatitis C virus infection. One of the limitations associated with the use of ribavirin is a reversible anemia caused by its accumulation in erythrocytes. Therefore, it is of interest to determine ribavirin levels in erythrocytes, as well as in plasma, as these measurements may be predictive of hematotoxicity. In the present study, we describe a high-performance liquid chromatographic (HPLC) assay for ribavirin in whole blood to estimate concentrations of free ribavirin and phosphorylated anabolites in erythrocytes. Since ribavirin exists primarily as phosphorylated anabolites (mono-, di-, and triphosphates) in erythrocytes, whole-blood extracts were initially dephosphorylated with acid phosphatase. The enzyme-treated samples were subjected to phenyl boronic acid column extraction for cleanup. The purified fraction was analyzed by reversed-phase HPLC, which was optimized for determination of ribavirin levels in whole blood. The recoveries of ribavirin from whole blood ranged from 63.1 to 90.7% at concentrations ranging from 1.67 to 40.0 microM. Intra- and interassay variations estimated at these concentrations were 3.2 to 10.4 and 4.7 to 11.7%, respectively. This method was used to quantitate ribavirin in samples both treated and untreated with acid phosphatase to estimate the extent of intracellular phosphorylation in erythrocytes. The method was also used to evaluate the effects of dipyridamole, a nucleoside transporter inhibitor, on ribavirin disposition in erythrocytes in in vitro experiments.

Failure to detect nelfinavir in the cerebrospinal fluid of HIV-1--infected patients with and without AIDS dementia complex.

OBJECTIVE: To assess the penetration of the HIV-1 protease inhibitor, nelfinavir, into cerebrospinal fluid (CSF). DESIGN: Nelfinavir, a commonly used HIV-1 protease inhibitor (PI), is highly effective for reducing plasma viral load. It is deployed clinically in combination with other antiretroviral agents, including nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). Despite its potency based on plasma HIV-1 RNA results, its effectiveness in reducing HIV-1 RNA levels (i.e., viral load) in the central nervous system (CNS) is less certain. We sampled the CSF as a surrogate for brain because this fluid also is separated from the blood by a barrier to free diffusion, the blood-CSF barrier (BCB), which shares properties with the blood-brain barrier (BBB). These studies of nelfinavir CSF pharmacokinetics exploited the multiple CSF samples derived from individual study subjects who were enrolled in studies the primary objective of which was to compare viral kinetics in CSF and blood in response to antiviral therapy. METHODS: Six study subjects, four with and two without AIDS dementia complex, underwent multiple lumbar punctures (LP). Intervals of CSF sampling after drug dosing were varied (from 0.48 hours to 10.3 hours after nelfinavir administration) to quantitate nelfinavir concentrations throughout the steady-state dosing interval. In four study subjects, CSF sampling was accompanied by assessment of nelfinavir levels in plasma before and after LP, whereas in the other two subjects, a single plasma sample was obtained before or after the LP. In total, 25 CSF samples were analyzed. Nelfinavir concentrations in CSF and plasma were determined using an high-performance liquid chromatography (HPLC) method with a limit of quantitation of 25 and 50 ng/ml, respectively. RESULTS: Plasma concentrations before and after LP averaged 2420+/-1365 ng/ml and 2528+/-1132 ng/ml, respectively. Nelfinavir was not detected in any of the CSF samples and levels >25 ng/ml were not present in the CSF. Thus, standard therapy with nelfinavir does not result in CSF drug concentrations at or exceeding the IC95 level for most HIV-1 isolates. However, study subjects with high CSF viral loads experienced a marked reduction in the context of the combination-drug regimen including nelfinavir with two subjects showing a comparable CSF response with that in plasma. CONCLUSIONS: Nelfinavir does not appreciably penetrate into the CSF. The clinical importance of this observation is not certain, in that in four study subjects who initiated nelfinavir in combination with other antiretroviral therapy, a comparable degree of viral suppression was obtained in both the CSF and the blood when sampled 4 weeks or later after initiating therapy.

Simple high-performance liquid chromatographic determination of the protease inhibitor indinavir in human plasma.

Indinavir is a member of a class of protease inhibitors that actively prevent the acquired immunodeficiency syndrome virion from maturing. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of indinavir in human plasma. Indinavir and the internal standard were isolated from the plasma by ether extraction. The residue after evaporation of ether was reconstituted with buffer and injected onto a C4 reversed-phase column eluted isocratically with a mobile phase consisting of 35:65 (v/v) of acetonitrile and buffer. A wavelength of 210 nm was found to be optimum for detection. The calibration range of this assay was from 10 to 5000 ng/ml and coefficients of variation for the assay ranged from 4.6% to 11.0% for three different drug concentrations and the limit of quantitation was 10 ng/ml. During the validation, short-term stability of the drug in plasma, stability during heat deactivation and on repeated freezing and thawing of plasma was evaluated. The overall recovery of indinavir by the ether extraction method was 91.4%. This HPLC assay was found to be a simple and reproducible method for monitoring indinavir levels in human plasma obtained during clinical trials of the drug.

High-performance liquid chromatographic determination of pyrazoloacridine, a nitro-9-methoxyacridine anticancer agent, in human plasma.

Pyrazoloacridine (PZA) is a 9-methoxy substituted acridine with a reducible nitro group. PZA has shown selective solid tumor cytotoxicity with activity against hypoxic cells, non-cycling cells and cells expressing the multidrug resistant phenotype. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of PZA in human plasma to support phase II clinical trials. PZA and ethyl orange, the internal standard, were isolated from human plasma by precipitating plasma proteins with methanol, and centrifuging to pellet the proteins. The resulting supernatant was injected onto a cyanopropyl HPLC column eluted isocratically with a mobile phase consisting of 125 mM ammonium acetate buffer pH 4.75-acetonitrile (76:24, v/v). A single wavelength at 460 nm was used for detection. Relative standard deviations for the assay ranged from 5.0% to 12.2% for four different drug concentrations and the limit of quantitation was 100 ng/ml. During the validation short term stability of the drug in plasma and stability of PZA on repeated freezing and thawing of plasma was evaluated. Overall recovery of PZA was 88%. This simple assay was found suitable for studying the clinical pharmacokinetics of PZA.

Foscarnet and ganciclovir pharmacokinetics during concomitant or alternating maintenance therapy for AIDS-related cytomegalovirus retinitis.

INTRODUCTION: The use of foscarnet and ganciclovir as a combination treatment for cytomegalovirus retinitis is increasing because of limitations associated with single agent therapy. METHODS: The pharmacokinetics of foscarnet and ganciclovir were determined in 13 patients receiving either concomitant therapy (regimen A) or daily alternating therapy (regimen B) for maintenance of cytomegalovirus disease. For regimen A, 60 mg/kg intravenous foscarnet and 3.75 mg/kg ganciclovir were sequentially administered daily; for regimen B, 120 mg/kg foscarnet and 6 mg/kg ganciclovir were administered on alternating days. For both regimens, serial blood sampling for pharmacokinetic analysis was performed for each drug alone (day 1 or 2) and after 2 weeks of combination therapy. Plasma samples for foscarnet and ganciclovir analysis were performed by means of high-performance liquid chromatography. Pharmacokinetic analysis was performed with noncompartmental methods. RESULTS: For regimen A, the plasma clearance (CL) of foscarnet did not change in the presence of ganciclovir, averaging 0.12 +/- 0.08 and 0.11 +/- 0.02 L/hr/kg on study days 2 and 14, respectively (p = 0.34). The volume of distribution (VSS) and mean residence time (MRT) also did not change significantly. CL and MRT of foscarnet did not change for regimen B, although a slight increase in VSS was observed before (0.38 +/- 0.05 L/kg) and after (0.46 +/- 0.07 L/kg) alternating therapy (p = 0.03). Ganciclovir CL did not change for either regimen, with mean values of 0.21 +/- 0.10 and 0.25 +/- 0.10 L/hr/kg (regimen A, p = 0.17) and 0.32 +/- 0.10 and 0.34 +/- 0.11 L/hr/kg (regimen B, p = 0.24). MRT and VSS were also not significantly different. CONCLUSION: These plasma data suggest that further dosage adjustments are unnecessary for or alternating maintenance therapy.

Determination of ticarcillin in human plasma and in urine by reversed-phase LC.

A liquid chromatographic assay for ticarcillin (ticar.) in plasma and urine is described. For analysis, the internal standard cefoperazone (cfp) is dissolved in acetonitrile, which is used for precipitating the protein. The supernatant is evaporated, reconstituted in running mobile phase and injected directly onto the reversed-phase C18 column, with detection at 205 nm. The mobile phase is composed of water-acetonitrile-o-phosphoric acid-tetramethylammonium chloride (TMA). Coefficients of variation for reproducibility were in the range of 2.2-15.5% for extra-low, low, medium and high controls. Limits of detection were 0.5 microgram ml-1 for plasma and 1 microgram ml-1 for urine. No interference from other cephalosporins or other antibiotics was noted. This liquid chromatographic assay is simple, accurate, requires no extraction and overcomes previous problems related to the drug's peak splitting due to isomerization.

Paired ion reversed-phase HPLC assay for the determination of iothalamic acid and para aminohippuric acid in urine.

A paired ion reversed-phase high performance liquid chromatographic method for simultaneous determination of iothalamic acid (Io) and para aminohippuric acid (PAH) in urine is described. The method uses a single internal standard for both drugs. The only sample preparation required is dilution of urine (1:100 or 1:500) with deionized water. The internal standard is added to a small aliquot of the diluted specimen and injected. For HPLC, a C8 column and a mobile phase consisting of potassium phosphate buffer with dodecyl triethylammonium phosphate IP reagent, 25% organic modifier with UV detection at 254 nm was used. Within day and between day variation for the assay were in the range of 1.48-9.46% for iothalamic acid and 1.84-10.36% for para aminohippuric acid for four levels of concentration. Limits of quantitation were 50.0 micrograms ml-1 for iothalamic acid and 75.0 micrograms ml-1 for para aminohippuric acid. Mean recovery was 98.55% for Io and 97.79% for PAH. This isocratic HPLC assay is simple, rapid and relatively inexpensive.