Principles and practice of HIV-protease inhibitor pharmacoenhancement

Continually maintaining maximally suppressive drug concentrations represents a key defence against the emergence of resistance. If drug levels fall and replication occurs, the opportunity for mutant virus to be selected occurs. It has been increasingly recognized that variability in the pharmacokinetics of antiretrovirals, particularly protease inhibitors (PIs), means that drug exposure is not always optimal, giving the virus a chance to replicate. A significant number of patients receiving PIs two or three times daily will have trough (C_trough or C_min) plasma concentrations, which are close to, or below, the plasma protein binding‐corrected inhibitory concentration (IC₅₀ or IC₉₅) during the dosing interval. It is primarily in this context that therapeutic drug monitoring of PIs has been proposed as an aid to patient management, to ensure that patients maintain adequate drug concentrations throughout the dosing interval. Ideally, an antiretroviral drug will have a pharmacokinetic (PK) profile that maintains drug levels well above the viral inhibitory concentration throughout the entire dosing interval. Beneficial drug–drug interactions have been shown to improve PI pharmacokinetics. Ritonavir (RTV) inhibits the key enzymes that limit the bioavailability or speed the metabolism of other PIs. It is therefore increasingly used for boosting and maintaining PI plasma concentrations. At low (100 mg twice a day) doses it acts as a pharmacoenhancer of indinavir (IDV), amprenavir, saquinavir, lopinavir and to a more limited degree nelfinavir. Using a pharmacoenhancer with a PI results in increased exposure to the PI, higher C_min levels, and in most cases prolonged elimination half‐lives. The long‐term clinical benefits of PK enhancing are unknown as are the long‐term toxicities, although the incidence of nephrolithiasis with IDV appears increased when IDV is combined with low‐dose RTV in HIV‐infected patients. Head‐to‐head clinical comparisons of boosted PI regimens will help answer some of the questions that remain with regard to PK enhancement.     

Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System

Retroviral protease inhibitors (PIs) are fundamental pillars in the treatment of HIV infection and acquired immunodeficiency syndrome (AIDS). Currently used PIs are designed against HIV-1, and their effect on HIV-2 is understudied. Using a modular HIV-2 protease cassette system, inhibition profiling assays were carried out for protease inhibitors both in enzymatic and cell culture assays. Moreover, the treatment-associated resistance mutations (I54M, L90M) were introduced into the modular system, and comparative inhibition assays were performed to determine their effect on the susceptibility of the protease. Our results indicate that darunavir, saquinavir, indinavir and lopinavir were very effective HIV-2 protease inhibitors, while tipranavir, nelfinavir and amprenavir showed a decreased efficacy. I54M, L90M double mutation resulted in a significant reduction in the susceptibility to most of the inhibitors with the exception of tipranavir. To our knowledge, this modular system constitutes a novel approach in the field of HIV-2 protease characterization and susceptibility testing.     

Effects of ritonavir and amprenavir on insulin sensitivity in healthy volunteers

BACKGROUND: Some HIV protease inhibitors (PIs) have been shown to induce insulin resistance in vitro but the degree to which specific PIs affect insulin sensitivity in humans is less well understood. METHODS: In two separate double-blind, randomized, cross-over studies, we assessed the effects of a single dose of ritonavir (800 mg) and amprenavir (1200 mg) on insulin sensitivity (euglycemic hyperglycemic clamp) in healthy normal volunteers. RESULTS: Ritonavir decreased insulin sensitivity (-15%; P = 0.008 versus placebo) and non-oxidative glucose disposal (-30%; P = 0.0004), whereas neither were affected by amprenavir administration. CONCLUSION: Compared to previously performed studies of identical design using single doses of indinavir and lopinavir/ritonavir, a hierarchy of insulin resistance was observed with the greatest effect seen with indinavir followed by ritonavir and lopinavir/ritonavir, with little effect of amprenavir.     

Differential effect of HIV protease inhibitors on adipogenesis: intracellular ritonavir is not sufficient to inhibit differentiation

OBJECTIVES: Lipodystrophy is a major side effect of HIV protease inhibitor (PI) antiretroviral therapy. It has been shown that protease inhibitors interfere in vitro with adipocyte differentiation. However, there is no evidence that PIs accumulate into preadipocytes and adipocytes and that intra-cellular accumulation is sufficient to alter differentiation. We assessed the effect of six different PIs on the differentiation of cells from four clonal lines. We also studied the capacity of ritonavir to accumulate both into drug-sensitive and drug-resistant cultured adipocytes. METHODS: Adipocyte differentiation of mouse 3T3-F442A, 3T3-L1 and Ob1771 cells as well as embryonic stem cells were investigated at pharmacological concentrations of indinavir, saquinavir, ritonavir, amprenavir, nelfinavir and lopinavir. We used a sensitive ELISA to determine intracellular concentration of ritonavir from 3T3-L1 and Ob1771 preadipocytes. RESULTS: Nelfinavir and lopinavir inhibited adipocyte differentiation whereas amprenavir was ineffective. Indinavir, saquinavir and ritonavir inhibited differentiation of 3T3-L1 and 3T3-F442A cells but did not alter differentiation of either Ob1771 or embryonic stem cells. We showed that ritonavir accumulated in preadipocytes and fully differentiated 3T3-L1 adipocytes as a function of its extracellular concentration. Although Ob1771 cells were resistant and 3T3-L1 cells were sensitive to ritonavir, the drug accumulated to similar levels in both cases. CONCLUSIONS: Protease inhibitors inhibit adipocyte differentiation depending on the cell model used. We showed for the first time that ritonavir accumulates into preadipocytes and adipocytes, suggesting a direct effect on intracellular targets. However, intracellular accumulation was clearly not sufficient as Ob1771 cells remained resistant to the inhibitory effect of ritonavir.     

High rate of proV47A selection in HIV-2 patients failing lopinavir-based HAART

We describe the emergence of the proV47A mutation in three out of five HIV-2-infected individuals failing lopinavir/ritonavir-based HAART. The appearance of such mutated variants resulted in high levels of phenotypic resistance to lopinavir, cross-resistance to indinavir, amprenavir, and hypersusceptibility to saquinavir. A search in HIV-2 databases revealed that proV47A is present in 8.6% of protease inhibitor (PI)-experienced patients but absent in all PI-naive patients. Its selection may be a common mutational pathway for developing resistance to lopinavir/ritonavir in HIV-2.     

The effect of increasing alpha1-acid glycoprotein concentration on the antiviral efficacy of human immunodeficiency virus protease inhibitors

The effect of a 4-fold increase in alpha1-acid glycoprotein (AGP) on the antiviral efficacy of 5 human immunodeficiency virus (HIV) protease inhibitors (PIs) was examined by the effect of HIV PIs on p24 production in peripheral blood mononuclear cells infected with protease wild-type and PI-resistant HIV isolates. For wild-type virus, the efficacy of the PIs at trough concentrations was unaffected by a 4-fold increase in AGP. With the partially HIV PI-resistant isolate, a 4-fold increase in AGP resulted in 2%, 30%, 37%, 37%, and 42% loss of activity for indinavir, saquinavir, nelfinavir, ritonavir, and amprenavir, respectively. The high-level HIV PI-resistant isolate had a greater loss in activity. The change in IC50 secondary to the addition of AGP was the greatest for ritonavir, nelfinavir, and amprenavir and lowest for indinavir. These data suggest that the target plasma concentration for the highly bound HIV PIs may need to be raised in subjects with elevated AGP who harbor partially PI-resistant isolates.     

Saquinavir induces stable and functional expression of the multidrug transporter P-glycoprotein in human CD4 T-lymphoblastoid CEMrev cells

Background

The multidrug transporter P‐glycoprotein (P‐gp) is expressed in HIV‐1 target cells, in a range of pharmacological barriers and in AIDS‐associated tumours. P‐gp substrates include HIV‐1 protease inhibitors (PIs) and anticancer drugs, which are efficiently effluxed from multidrug‐resistant (MDR) cells.

Objectives

The aim of this study was to investigate the effect on human CD4 T‐lymphoblastoid CEM^rev cells of saquinavir and other PIs in terms of P‐gp expression and to characterize the functional and biochemical patterns of PI‐induced P‐gp molecules.

Methods

CEM^rev cells no longer expressing detectable amounts of P‐gp were cultured for a prolonged period in the presence of 10 μg/mL saquinavir (CEM^saq10) and tested for P‐gp expression and function. Subsequently, CEM^saq10 cells were transferred into medium containing 15 μg/mL saquinavir (CEM^saq15) and cultured for several months. These cell lines were continuously monitored for P‐gp expression, function and immunochemical patterns. A similar strategy was adopted to determine whether other PIs, such as ritonavir and indinavir, were able to induce P‐gp expression in CEM^rev cells.

Results

Compared with the drug‐diluent control, the exposure of CEM^rev cells to 10μg/mL saquinavir induced, in a consistent fraction of cells (45–50%), de novo expression of functioning P‐gp molecules. The transfer of CEM^saq10 cells to 15μg/mL saquinavir was associated with a dramatic increase in P‐gp expression and function (85–90% of CEM^saq15 cells expressed P‐gp and effluxed P‐gp dye substrates). These saquinavir‐induced P‐gp molecules included 75‐kDa molecules as well as the classical 170‐kDa form of P‐gp, suggesting induction of a particular isoform of P‐gp termed mini‐P‐glycoprotein. Conversely, ritonavir and indinavir induced transient P‐gp expression in a small percentage of the CEM^rev cells.

Conclusions

Treatment of human CD4 T‐lymphoblastoid CEM^rev cells with saquinavir caused over‐expression of functioning P‐gp molecules. This de novo acquired MDR phenotype, which differed from that induced by other PIs, was stable, as expression and activity of P‐gp were observed in CEM^saq10 and CEM^saq15 cells during prolonged in vitro culturing, even in drug‐free conditions.

     

Comparison of HIV type 1 protease inhibitor susceptibility results in viral samples analyzed by phenotypic drug resistance assays and by six resistance algorithms: an analysis of a subpopulation of the CHORUS cohort

Drug resistance testing is increasingly used to guide treatment decisions in patients infected with HIV-1. A number of rules-based algorithms have been designed to predict drug resistance profiles based on the HIV-1 genotypic data. Drug-resistance mutations in 206 viral samples from protease inhibitor (PI)-experienced subjects with HIV-1 infection were assessed, and the level of susceptibility of the samples predicted using seven unique algorithms. kappa scores were used to compare agreement of results obtained using each of the predictive algorithms with the phenotypic assay results. Good overall agreement between the different algorithms and the phenotypic results was observed. Good or excellent agreement was observed between the results obtained by the predictive algorithms and the phenotypic assay results for ritonavir, indinavir, saquinavir, and nelfinavir. For amprenavir and lopinavir, there were marked differences between the different algorithms, with poor agreement (kappa < 0.40) obtained with four of the seven algorithms for amprenavir. For lopinavir, poor agreement was obtained with three of seven algorithms using the 2.5-fold biological cut-off and four of seven with the clinical cut-off of 10. Atazanavir susceptibility was evaluated for concordance among six algorithms, with a range of 23-50% of the samples maintaining susceptibility. Although this cohort of patients included many who were highly antiretroviral experienced, predictive algorithms demonstrated good agreement with phenotype for several Pls. For those where discordance among algorithms existed, further improvement will likely occur as drug resistance pathways for the more recently approved PIs are elucidated.     

MDR1 G1199A polymorphism alters permeability of HIV protease inhibitors across P-glycoprotein-expressing epithelial cells

OBJECTIVE: To evaluate the impact of the human multidrug resistance gene (MDR1) G1199A polymorphism (amino acid change Ser400Asn) on P-glycoprotein (P-gp)-dependent transepithelial permeability and uptake kinetics of HIV protease inhibitors (PI), by using recombinant epithelial cells expressing wild-type MDR1 (MDR1wt) or the G1199A variant (MDR1(1199A)). METHODS: Using a recombinant expression system developed previously, the transepithelial permeability and uptake kinetic parameters of five PI, amprenavir, indinavir, lopinavir, ritonavir, and saquinavir were estimated across polarized epithelial cells. RESULTS: For all PI, the transepithelial permeability ratio (basolateral-to-apical transport divided by apical-to-basolateral transport) was significantly greater in MDR1(1199A) cells than MDR1wt cells: amprenavir (1.7-fold), indinavir (1.8-fold), lopinavir (1.5-fold), ritonavir (2.8-fold), and saquinavir (2.1-fold). However, the impact of G1199A on P-gp activity appeared to primarily influence drug permeability in the apical-to-basolateral direction. Kinetic analysis of ritonavir and saquinavir uptake by MDR1wt- and MDR1(1199A)-expressing cells showed that Vmax was similar, while uptake Km was significantly higher in cells expressing the G1199A variant suggesting that alterations in P-gp-dependent efflux mediated by G1199A were due to changes in transporter affinity. CONCLUSIONS: Alterations in transepithelial permeability of HIV PI due to the G1199A polymorphism may impact oral bioavailability of PI and penetration into cells and tissues of the lymphoid and central nervous systems.     

Analysis of HIV cross-resistance to protease inhibitors using a rapid single-cycle recombinant virus assay for patients failing on combination therapies.

OBJECTIVE: To assess the patterns of HIV phenotypic cross-resistance to protease inhibitors (PI) in patients experiencing viral load rebound on combination therapy including a PI. METHODS: Phenotypic analysis of sensitivity to indinavir, nelfinavir, saquinavir, ritonavir and amprenavir was carried out using a single-cycle recombinant virus assay. Viral protease was sequenced by automated dideoxynucleotide chain termination. RESULTS: Of the 108 patients studied, 68 had received indinavir, 50 ritonavir, 25 saquinavir and eight nelfinavir. The majority (71%) had received only one PI. The incidence of cross-resistance between indinavir, nelfinavir, ritonavir and saquinavir was high (60-90%). Cross-resistance to amprenavir was less frequent (37-40%). However there was some correlation between levels of sensitivity to amprenavir and indinavir (r2 = 0.34; P < 0.01). Conversely, the correlation between levels of sensitivity to indinavir and saquinavir was poor (r2 = 0.25), particularly for patients who had not received saquinavir. The degree of cross-resistance correlated with the level of resistance and with the total number of mutations in the protease gene (P < 0.05, chi square test) but could not be significantly correlated to any one particular mutation or combination of mutations. Mutation 184V was significantly associated with cross-resistance to amprenavir, with no mutations at codon 50 observed, while mutations associated with cross-resistance to saquinavir differed according to the treatment received. CONCLUSIONS: These results suggest that, although the total number of protease mutations correlates with the degree of cross-resistance, the specific mechanisms accounting for primary resistance and for cross-resistance may be different.     

Penetration of enfuvirtide, tenofovir, efavirenz, and protease inhibitors in the genital tract of HIV-1-infected men

One likely mechanism of virological failure is poor antiretroviral drug diffusion in sites of viral replication such as the genital tract. We measured antiretroviral drug concentrations in blood and semen in 13 HIV-infected men failing treatment. Enfuvirtide did not cross the blood-testis barrier, whereas tenofovir accumulated in semen. Unlike indinavir, semen concentrations of lopinavir, amprenavir, saquinavir and efavirenz were ineffective. These are worrying findings, because suboptimal semen drug concentrations may enhance the risk of sexually transmitted drug-resistant HIV variants.     

Drug-drug interactions and the pharmacotherapy of HIV infection

Knowledge of drug-drug interactions is crucial to HIV therapeutics. Recent reports in this area include reduced atazanavir exposure with coadministration of omeprazole or rifampin; increased hepatic toxicity with coadministration of saquinavir and rifampin; reduced buprenorphine exposure with concurrent efavirenz administration; absence of clinically significant interactions of depomedroxyprogesterone with nevirapine, efavirenz, or nelfinavir; increased atazanavir and saquinavir exposure with the double-boosted regimen of atazanavir/saquinavir/ritonavir; reduced amprenavir, lopinavir, and saquinavir exposure with the addition of tipranavir/ritonavir therapy; and reduced lopinavir and amprenavir exposure with the addition of fosamprenavir or fosamprenavir/ritonavir to lopinavir/ritonavir. This article summarizes a presentation on drug-drug interactions in HIV therapeutics by Angela D. M. Kashuba, PharmD, at the International AIDS Society-USA course in Los Angeles in April 2005.     

Factors associated with accelerated atherosclerosis in HIV-1-infected persons treated with protease inhibitors

Recent evidence suggests that as a group protease inhibitors (PIs) may accelerate certain factors associated with atherosclerosis. The objective of this study was to evaluate the effect of individual PIs (indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) on certain factors associated with atherosclerosis. Persons who took saquinavir and/or ritonavir were compared with those on other PIs. Between May 2000 and July 2001, the lipid profiles, C-reactive protein (CRP) levels, coronary artery calcium (CAC) scores, and blood cell morphologic parameters were measured in 98 black adult participants aged 25 to 45 years with HIV-1 infection in Baltimore, Maryland. Among these 98, there were 55 (56.1%) taking PIs. Students' t-test and chi2 test were used to detect the between-group differences. Study participants in both the PI and non-PI groups were similar in age, sex, body mass index, blood pressure, red and white blood cell counts, time since HIV diagnosis, and duration on anti-retroviral therapy. Compared with those who took non-PI regimens, those who took indinavir, nelfinavir, or saquinavir had significantly higher levels of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). Those taking any PI had significantly higher total cholesterol and low-density lipoprotein. Those taking nelfinavir, ritonavir, or saquinavir were more likely to have a higher CAC score (>5) than those on non-PI regimens. There were no differences in the lipid profiles, MCV, MCH, CRP, and CAC between those taking saquinavir and/or ritonavir and those taking other PIs. Overall, the changes noted might lead to anticipation of clinical changes linked to accelerated atherosclerosis in patients on PIs.     

Hepatotoxicity associated with protease inhibitor-based antiretroviral regimens with or without concurrent ritonavir

OBJECTIVE: To determine the incidence of significant liver enzyme elevations following the initiation of protease inhibitor (PI)-based antiretroviral therapy (ART) with or without pharmacokinetic boosting with ritonavir (RTV), and to define the role of chronic viral hepatitis in its development. DESIGN: Prospective, cohort analysis of 1161 PI-naive, HIV-infected patients receiving RTV-boosted (lopinavir, indinavir and saquinavir) and unboosted PI-based ART (indinavir, nelfinavir) that had at least one liver enzyme measurement before and during therapy. METHODS: The incidence of grade 3 and 4 liver enzyme elevations among persons with and without hepatitis B and/or C co-infection treated with PI-based ART were compared. Severe hepatotoxicity was defined as an increase in serum liver enzyme >/= 5-times the upper limit of the normal range or 3.5-times an elevated baseline level. RESULTS: The incidence of grade 3 or 4 elevations among PI-naive patients was: nelfinavir, 11%; lopinavir/RTV (200 mg/day), 9%; indinavir, 13%; indinavir/RTV (200-400 mg/day), 12.8%; and saquinavir/RTV (800 mg/day), 17.2%. The risk was significantly greater among persons with chronic viral hepatitis (63% of cases); however, the majority of hepatitis C virus (HCV)-infected patients treated with nelfinavir (84%), saquinavir/RTV (74%), indinavir, 86%, indinavir/RTV (90%) or lopinavir/RTV (87%) did not develop hepatotoxicity. CONCLUSIONS: Our data suggest that the lopinavir/RTV is not associated with a significantly increased risk of hepatotoxity among HCV-infected and uninfected patients compared with an alternative PI-based regimen, nelfinavir. Accordingly, other medication-related factors (e.g, efficacy and non-hepatic toxicity) should guide individual treatment decisions.     

Inhibition of autoprocessing of natural variants and multidrug resistant mutant precursors of HIV-1 protease by clinical inhibitors

Self-cleavage at the N terminus of HIV-1 protease from the Gag-Pol precursor (autoprocessing) is crucial for stabilizing the protease dimer required for onset of mature-like catalytic activity, viral maturation, and propagation. Among nine clinical protease inhibitors (PIs), darunavir and saquinavir were the most effective in inhibiting wild-type HIV-1 group M precursor autoprocessing, with an IC(50) value of 1-2 μM, 3-5 orders of magnitude higher than their binding affinities to the corresponding mature protease. Accordingly, both group M and N precursor-PI complexes exhibit T(m)s 17-21 °C lower than those of the corresponding mature protease-PI complexes suggestive of markedly reduced stabilities of the precursor dimer-PI ensembles. Autoprocessing of group N (natural variant) and three group M precursors bearing 11-20 mutations associated with multidrug resistance was either weakly responsive or fully unresponsive to inhibitors at concentrations up to a practical limit of approximately 150 μM PI. This observation parallels decreases of up to 8 × 10(3)-fold (e.g., 5 pM to 40 nM) in the binding affinity of darunavir and saquinavir to mature multidrug resistant proteases relative to wild type, suggesting that inhibition of some of these mutant precursors will occur only in the high μM to mM range in extreme PI-resistance, which is an effect arising from coordinated multiple mutations. An extremely darunavir-resistant mutant precursor is more responsive to inhibition by saquinavir. These findings raise the questions whether clinical failure of PI therapy is related to lack of inhibition of autoprocessing and whether specific inhibitors can be designed with low-nM affinity to target autoprocessing.     

Low body weight and type of protease inhibitor predict discontinuation and treatment-limiting adverse drug reactions among HIV-infected patients starting a protease inhibitor regimen: consistent results from a randomized trial and an observational cohort

Departments of Infectious Diseases, ¹ Hvidovre Hospital, University of Copenhagen, Hvidovre, ² Rigshospitalet, University of Copenhagen, Copenhagen, ³ Odense University Hospital, Odense, ⁴ Aalborg Hospital, Aalborg and ⁵ Marselisborg University Hospital, Aarhus, Denmark Objectives To assess predictors for discontinuation and treatment‐limiting adverse drug reactions (TLADR) among patients starting their first protease inhibitor (PI). Methods Data on patients starting a PI regimen (indinavir, ritonavir, ritonavir/saquinavir and saquinavir hard gel) in a randomized trial (RAS, n = 318) and an observational cohort (OBC, n = 505) were used to document reasons for discontinuation and TLADR. Risk factors for discontinuation of the initial PI/developing TLADR were assessed in Cox models. Results A total of 43 (RAS) and 48% (OBC) discontinued the initial PI therapy within less than 2 years. In both populations TLADR were the most common reason for discontinuation. The incidence of TLADR in RAS was: 8.5 (indinavir), 66.0 (ritonavir), 15.6 (saquinavir hard gel) per 100 person‐years of follow‐up (P < 0.001). Body weight and type of PI initiated were independent risk factors for treatment discontinuation and TLADR in both groups. In OBC, the risk of developing TLADR increased by 12% per 5 kg lower body weight when starting the PI regimen [the relative hazard (RH) was 1.12 (95% confidence interval: 1.05–1.19) per 5 kg lighter], and starting ritonavir was associated with a three‐ to sixfold higher risk of TLADR relative to other PI regimens. Very similar results were documented in RAS [RH for body weight was 1.18 (1.07–1.29)]. Conclusions Nearly half of the patients stopped treatment with the initial PI, most commonly as a result of adverse drug reactions. Low body weight and initiation of ritonavir relative to other PIs were associated with an increased risk of TLADRs. Very consistent results were found in a randomized trial and an observational cohort.