Antiretroviral medication adherence and class- specific resistance in a large prospective clinical trial.

OBJECTIVE: To assess the association between adherence to antiretroviral therapy and the presence of class-specific antiretroviral medication resistance. DESIGN: Secondary analysis of prospective clinical trial data. METHODS: Participants randomized to the protease inhibitor or nonnucleoside reverse transcriptase inhibitor (NNRTI) strategies of the Community Programs for Clinical Research on AIDS (CPCRA) Flexible Initial Retrovirus Suppressive Therapies (FIRST) Study were included. Adherence was measured by 7-day self-report. Virological failure was defined as an HIV-RNA more than 1000 at or after 4 months. The association between cumulative adherence and the development of class-specific genotypic resistance was assessed by Cox regression analysis. RESULTS: Included were 457 and 446 antiretroviral-naive participants on the protease inhibitor and NNRTI strategies, respectively. The median time to initial virological failure in the protease inhibitor strategy was 1.2 years; 135 (30%) individuals failed with resistance. The median time to initial virological failure in the NNRTI strategy was 3.0 years; 127 (28%) failed with resistance. No association was found between cumulative adherence and protease inhibitor resistance [hazard ratio 1.1, 95% confidence interval (CI) 0.9-1.4 per 10% lower adherence]. However, lower cumulative adherence was associated with an increased risk of NNRTI resistance at initial virological failure (hazard ratio 1.2, 95% CI 1.1-1.3 per 10% lower adherence). In both strategies, lower cumulative adherence was associated with an increased risk of nucleoside reverse transcriptase inhibitor (NRTI) resistance at initial virological failure. CONCLUSION: Adherence-resistance relationships are class-specific. For NRTIs and NNRTIs, initial virological failure with resistance is more likely at lower levels of cumulative adherence.

Antiretroviral rounds. Too many options?

We asked three experts which antiretroviral regimen they would recommend next for a patient with triple-class resistance and low viremia--and got three different answers.

Effectiveness of commercial inhibitors against subtype F HIV-1 protease.

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.

Polymorphisms in HIV-1 subtype C proteases and the potential impact on protease inhibitors.

OBJECTIVES: (i) To review data on the genetic profile of the protease (PR) gene of human immunodeficiency virus (HIV)-1-C primary isolates relative to HIV-1-B; (ii) to examine data on the susceptibility of HIV-1-C isolates harbouring polymorphisms to PR inhibitors (PI) and the development of resistance; and (iii) to identify gaps required for an improved understanding of the role of polymorphisms in resistance development of HIV-1-C to PI. METHOD: Literature review. RESULTS: Significant differences exist between the baseline nucleotide and amino acid sequences of PR of HIV-1-B and HIV-1-C. Some of the amino acid substitutions seen in HIV-1-B when exposed to PI occur naturally in HIV-1-C isolates. Studies used different methodologies and interpretation systems to evaluate the phenotypic significance of polymorphisms seen in subtype C viruses, with conflicting outcomes. The evolutionary path to the resistance of HIV-1-C to PI may be different from that of HIV-1-B. CONCLUSIONS: Infection with HIV-1-C is driving the AIDS epidemic in regions of the world with the most urgent needs for the management of the disease. More and more individuals will require PR inhibitors in second-line therapies, as access to antiretrovirals progresses. It is proposed that a standardized protocol be adopted to evaluate the phenotypic significance of the highly polymorphic HIV-1-C PR to PR inhibitors with the aim of better informing the tailoring of treatment regimens for optimal clinical benefit.

Greater tenofovir-associated renal function decline with protease inhibitor-based versus nonnucleoside reverse-transcriptase inhibitor-based therapy.

BACKGROUND: Plasma concentrations of tenofovir increase when the drug is coadministered with some ritonavir-boosted protease inhibitors (PI/r). We hypothesized that tenofovir disoproxil fumarate (TDF)-treated patients taking PI/r-based regimens would have a greater decline in renal function than patients receiving nonnucleoside reverse-transcriptase inhibitor (NNRTI)-based therapy. METHODS: We compared the estimated decline in renal function among 146 human immunodeficiency virus type 1 (HIV-1)-infected patients receiving a TDF+PI/r- (n = 51), TDF+NNRTI- (n = 29), or non-TDF-containing (n = 66) regimen. Plasma tenofovir concentrations were measured at study week 2, and rates of creatinine clearance (CrCl) were estimated using the Cockcroft-Gault (C-G) and Modification of Diet in Renal Disease (MDRD) equations. Mixed-effects models were used to analyze regimen type and tenofovir concentration as predictors of change in CrCl from baseline to weeks 24 and 48. RESULTS: Decreases in C-G estimates of CrCl were not significantly different among the 3 groups during the first 24 weeks of therapy. However, in adjusted analyses, patients receiving TDF+PI/r had a greater rate of decline in CrCl than did the TDF+NNRTI group (for C-G, -13.9 vs. -6.2 mL/min/year [P = .03]; for MDRD, -14.7 vs. -4.5 mL/min/1.73 m(2)/year [P = .02]). Among TDF-treated patients, tenofovir plasma concentration was not associated with CrCl over time. CONCLUSIONS: Treatment with TDF and PI/r was associated with greater declines in renal function over 48 weeks compared with TDF+NNRTI-based regimens.

Overview of boosted protease inhibitors in treatment-experienced HIV-infected patients.

Antiretroviral drug combinations that include two nucleoside reverse transcriptase inhibitors and a protease inhibitor (PI) can suppress HIV replication to undetectable levels, improving the prognosis of HIV-infected individuals. The aim of therapy is complete virological suppression, with a current goal of <50 copies/mL HIV-1 RNA, in order to minimize the occurrence of drug resistance. Improved understanding of the pharmacology of PIs, primarily the importance of adequate drug exposure, has led to the widespread administration of PIs combined with a low 'boosting' dose of ritonavir. The combination of PIs with ritonavir can improve treatment responses in both treatment-naive and -experienced patients. Boosted PIs are an important therapeutic option for HIV and extensive data exist supporting their use. Use of individual agents should be guided by a resistance test at all stages of treatment from naive through to highly treatment-experienced patients. Currently, seven boosted PIs have both US and European licensing approval: indinavir, saquinavir, lopinavir, fosamprenavir, atazanavir, tipranavir and darunavir (formerly TMC114). The preferred first-line option in the USA is lopinavir. Many of the older PIs are less effective and/or have less favourable tolerability profiles. Emergent PI resistance is a major challenge in treatment, and it can be accelerated by partial suppression of viral load through inappropriate therapy combinations. Using the newer boosted PIs, which have more robust resistance profiles, with an optimized background regimen may increase the likelihood of complete viral suppression. This review discusses the relative strengths and weaknesses of boosted PIs in current practice.

Direct and fast determination of antiretroviral drugs by automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry in human plasma.

BACKGROUND: In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis. METHODS: The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-microL sample volume, which was only diluted with 200 microL of H2O (containing 500 ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up. RESULTS: The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6 min, subsequent analyses 3.3 min between injections) and has been validated in terms of the limit of detection (LOD, 2-70 ng/mL), lower limit of quantification (LLOQ, 78-156 ng/mL), linearity (R2, 0.9971-0.9989), linear concentration range (from LLOQ to 10,000 ng/mL), intra- and inter-day precision (< 13.5% at LLOQ, < 7.5% at high concentrations), proficiency testing accuracy (78-127%), laboratory internal accuracy (86-113%), recovery (60-110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability. CONCLUSION: Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.

Simultaneous determination of HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in human plasma by high-performance liquid chromatography-tandem mass spectrometry.

We report a precise and accurate method for simultaneous quantification of protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in plasma. An internal standard was added to samples prior to protein precipitation with acetonitrile followed by addition of ammonium formate buffer. Analysis was by HPLC-MS/MS. Calibration curves were validated over concentration ranges encompassing both subtherapeutic and potentially 'toxic' drug concentrations. Inter- and intra-assay variation were below 11% and PI recovery was above 87%. The bioanalytical method described is successfully applied to measure PI concentrations obtained from clinical pharmacokinetic studies and routine therapeutic drug monitoring (TDM).

Simple and simultaneous determination of the hiv-protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir plus M8 nelfinavir metabolite and the nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma by reversed-phase liquid chromatography.

Several studies suggest that therapeutic drug monitoring of protease inhibitors and nonnucleoside reverse transcriptase inhibitors may contribute to the clinical outcome of HIV-infected patients. Because of the growing number of antiretroviral drugs and of drug combinations than can be administered to these patients, an accurate high-performance liquid chromatographic (HPLC) method allowing the simultaneous determination of these drugs may be useful. To date, the authors present the first simultaneous HPLC determination of the new protease inhibitor atazanavir with all the others currently in use (M8 nelfinavir metabolite included) and the 2 widely used nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine. This simple HPLC method allows the analysis all these drugs at a single ultraviolet wavelength following a 1-step liquid-liquid extraction procedure. A 500-muL plasma sample was spiked with internal standard and subjected to liquid-liquid extraction using by diethyl ether at pH 10. HPLC was performed using a Symmetry Shield RP18 and gradient elution. All the drugs of interest and internal standard were detected with ultraviolet detection at 210 nm. Calibration curves were linear in the range 50-10,000 ng/mL. The observed concentrations of the quality controls at plasma concentrations ranging from 50 to 5000 ng/mL for these drugs showed that the overall accuracy varied from 92% to 104% and 92% to 106% for intraday and day-to-day analysis, respectively. No metabolites of the assayed compounds or other drugs commonly coadministered to HIV-positive patients were found to coelute with the drugs of interest or with the internal standard. This assay was developed for the purpose of therapeutic monitoring (TDM) in HIV-infected patients.

Topological models for the prediction of HIV-protease inhibitory activity of tetrahydropyrimidin-2-ones.

Relationship between the topological indices and HIV-protease inhibitory activity of tetrahydropyrimidine-2-ones has been investigated. Three topological indices, Wiener's index--a distance based topological descriptor, Zagreb group parameter--an adjacency based topological descriptor and eccentric connectivity index--an adjacency-cum-distance based topological descriptor were used for the present investigations. A dataset comprising of 80 substituted tetrahydropyrimidine-2-one analogues was selected for the present studies. The values of the Wiener's index, Zagreb group parameter and eccentric connectivity index for each of the 80 compounds comprising the dataset were computed using an in-house computer program. The dataset was divided randomly into training and test sets. Resultant data was analyzed and suitable models were developed after identifying the active ranges in the training set. Subsequently, a biological activity was assigned to each of the compound involved in the test set using these models, which was then compared with the reported HIV-protease inhibitory activity. Accuracy of prediction using these models was found to vary from a minimum of approximately 86% to a maximum of approximately 88%.

Improved structure-activity relationship analysis of HIV-1 protease inhibitors using interaction kinetic data.

Despite the availability of large amounts of data for HIV-protease inhibitors and their effectiveness with wild type and resistant enzyme, there is limited knowledge about how this and other information can be systematically applied to the development of new antiviral compounds. To identify in vitro parameters that correlate with the efficacy of HIV inhibitors in cell culture, the relationships between inhibition, interaction kinetic, and cell culture parameters for HIV-1 protease inhibitors were analyzed. Correlation, cluster, and principal component analysis of data for 37 cyclic and linear compounds revealed that the affinities (K(D)) determined from SPR-biosensor binding studies correlated better to cell culture efficacy (ED(50)) than that of the inhibition constants (K(i)), indicating that the conventional use of K(i) values for structure-activity relationship analysis of HIV-1 inhibitors should be seriously reconsidered. The association and dissociation kinetic rate constants (k(on) and k(off)) alone showed weak correlations with ED(50) values. However, ED(50) values were most related to the free enzyme concentration in the viral particle ([E]), calculated from the rate constants and the total enzyme concentration in a viral particle. A structure-activity relationship analysis of the current data set was found to be valid for all classes of compounds analyzed. In summary, use of affinity, based on interaction kinetic rate constants, rather than inhibition constants, and theoretical consideration of the physiological conditions in the virus particle provide improved structure-activity relationship analysis of HIV-1 protease inhibitors.

The efficacy of calcium carbonate in the treatment of protease inhibitor-induced persistent diarrhea in HIV-infected patients.

BACKGROUND: Although some evidence exists to support the practice of using calcium carbonate to treat nelfinavir-induced diarrhea, there is a lack of data supporting the role of calcium in diarrhea induced by other protease inhibitors (PIs). PURPOSE: The objective of this prospective open-label study is to evaluate the efficacy of calcium carbonate in the treatment of PI-induced persistent diarrhea in HIV-infected patients. METHOD: Along with dietary advice, patients were asked to take oral calcium carbonate 500 mg twice daily for 2 weeks. Visual Analog Scale (VAS) and the National Cancer Institute of Canada (NCIC) scale were used to assess the severity of diarrhea. Data were analyzed using paired t tests to test for differences in VAS and NCIC scores between baseline and 14 days. Pearson correlation was used to explore the relationships between change in diarrhea and patient baseline factors. RESULTS: At day 0, the mean VAS +/- standard deviation was 6.6 +/- 2.1 and decreased to 5.3 +/- 1.9 (p=.01) after 14 days. At day 0, the mean NCIC score was 1.9 +/- 0.8 and decreased to 1.2 +/- 0.9 (p=.005) after 14 days. No baseline patient factors predicted change in NCIC or VAS grade. CONCLUSION: Calcium carbonate is associated with a reduction of diarrhea in individuals with diarrhea induced by PI.

Classification of HIV protease inhibitors on the basis of their antiviral potency using radial basis function neural networks.

HIV protease inhibitors are being used as frontline therapy in the treatment of HIV patients. Multi-drug-resistant HIV mutant strains are emerging with the initial aggressive multi-drug treatment of HIV patients. This necessitates continued search for novel inhibitors of viral replication. These protease inhibitors may further be useful as pharmacological agents for inhibition of other viral replication. Classification models of HIV Protease inhibitors are developed using a data set of 123 compounds containing several heterocycles. Their inhibitory concentrations expressed as log (IC50) ranged from -1.52 to 2.12 log units. The dataset was divided into active and inactive classes on the basis of their antiviral potency. Initially a two-class problem (active, inactive) is explored using k-nearest neighbor approach. In order to introduce non-linearity in the classifier different approaches were investigated. This led to the goal of a fast, simple, minimum user input, radial basis function neural network (RBFNN) classifier development. Then the same two-class problem was resolved using the (RBFNN) classifier. A genetic algorithm with RBFNN fitness evaluator was used to search for the optimum descriptor subsets. The application of majority rules was also tested for the RBFNN classification. The best six descriptor model found by the new cost function showed predictive ability in the high 80% range for an external prediction set.

Atazanavir: improving the HIV protease inhibitor class.

Protease inhibitors are potent agents against HIV but their use is constrained by poor pharmacokinetics, cross-resistance and metabolic toxicities. Atazanavir [Reyataz] is a new protease inhibitors with once-daily dosing and minimal lipid and glycemic effects. Resistance studies of clinical isolates reveal a mutational pattern distinctive from that of other protease inhibitors. Atazanavir selects for the I50L mutation in HIV protease that confers increased susceptibility to other protease inhibitors in vitro. Clinical trials have shown comparable efficacy to nelfinavir (Viracept) and efavirenz (Sustiva) in treatment-naive patients, and in preliminary studies, ritonavir-boosted atazanavir is effective in patients failing previous protease inhibitor-containing regimens. Reversible elevations in bilirubin occur in some patients but are not associated with hepatic injury. Atazanavir improves upon aspects of currently-available protease inhibitors and appears useful for initial and possibly subsequent HIV therapy.