Residual activity of two HIV antiretroviral regimens prescribed without virological monitoring.

Virological residual activity (VRA) denotes the degree of HIV RNA suppression achieved by antiretroviral therapy in the presence of resistant virus. This concept is particularly important in resource-limited settings, where rapid switching after detection of virological failure may not be feasible. Using data from the NORA trial, we estimated VRA for two regimens-zidovudine-lamivudine-abacavir (ZDV-3TC-ABC) and zidovudine-lamivudine-nevirapine (ZDV-3TC-NVP)-and related this to the phenotypic drug sensitivity of the component drugs in the two regimens. Plasma samples at weeks 0, 48, and 96 were retrospectively assayed for HIV-1 RNA, and genotypic/phenotypic resistance testing was performed if HIV-1 RNA exceeded 1,000 copies/ml. Virological residual activity (VRA) was defined as the difference between log(10)(HIV RNA) at week 48 or 96 and week 0 and related to 50% inhibitory concentration (IC(50)) relative to wild-type virus for ZDV and ABC (fold change [FC]). Twenty-seven samples in the ZDV-3TC-NVP group and 56 in the ZDV-3TC-ABC group contributed to the analysis. Mean VRA was significantly higher in the ZDV-3TC-ABC group than in the ZDV-3TC-NVP at week 48 (1.62 versus 0.90) and week 96 (1.29 versus 0.78). There was a weak and nonsignificant relationship between VRA and ZDV FC, with VRA decreasing by 0.1 log(10) copies/ml per 2-fold increase in ZDV. The association with ABC FC was much stronger, with a marked reduction in VRA occurring at ABC FC values greater than approximately 2. This information should be considered in future treatment guidelines relevant to resource-poor settings.

The non-nucleoside reverse transcriptase inhibitor efavirenz stimulates replication of human immunodeficiency virus type 1 harboring certain non-nucleoside resistance mutations.

We measured the effects of non-nucleoside reverse transcriptase (RT) inhibitor-resistant mutations K101E+G190S, on replication fitness and EFV-resistance of HIV(NL4-3). K101E+G190S reduced fitness in the absence of EFV and increased EFV resistance, compared to either single mutant. Unexpectedly, K101E+G190S also replicated more efficiently in the presence of EFV than in its absence. Addition of the nucleoside resistance mutations L74V or M41L+T215Y to K101E+G190S improved fitness and abolished EFV-dependent stimulation of replication. D10, a clinical RT backbone containing M41L+T215Y and K101E+G190S, also demonstrated EFV-dependent stimulation that was dependent on the presence of K101E. These studies demonstrate that non-nucleoside reverse transcriptase inhibitors can stimulate replication of NNRTI-resistant HIV-1 and that nucleoside-resistant mutants can abolish this stimulation. The ability of EFV to stimulate NNRTI-resistant mutants may contribute to the selection of HIV-1 mutants in vivo. These studies have important implications regarding the treatment of HIV-1 with combination nucleoside and non-nucleoside therapies.

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries.

BACKGROUND: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. METHODS: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. RESULTS: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs. CONCLUSION: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Nine-year trends in clinically relevant reduced susceptibility of HIV-1 to antiretrovirals.

BACKGROUND: Temporal changes in HIV-1 resistance both reflect and influence the clinical use of antiretrovirals (ARVs). OBJECTIVE: To determine temporal trends in reduced susceptibility to ARVs and resistance mutations in routine clinical samples (RCS) from HIV infected patients. STUDY DESIGN: Calculated fold-changes (FC) for ARVs were determined for viral genotypes from RCS received between July 1998 and June 2007 using vircoTYPE HIV-1 (Version 4.2.01). The prevalence of isolates with clinically relevant reduced susceptibility and of resistance mutations were determined for consecutive 6-month periods. RESULTS: 242,003 RCS were identified. The prevalence of RCS exhibiting reduced susceptibility to > or =1 drug among any of three ARV classes decreased from 82% to 66.5% and of these, to > or =1 drug in each of the three ARV classes and from 30.5% to 15.7% from December 98 to June 07 (p< or =0.0001). The prevalence of mutations associated with NRTI, NNRTI and PI resistance generally reflected evolving use of these drug classes. Among fully susceptible RCS, isolates that exhibited resistance mutations were rare. CONCLUSIONS: Clinically relevant reduced susceptibility to ARVs declined over the 9-year period. There was a general higher prevalence of reduced susceptibility to RT inhibitors than to PIs.

The HIV-1 protease resistance mutation I50L is associated with resistance to atazanavir and susceptibility to other protease inhibitors in multiple mutational contexts.

BACKGROUND: The HIV-1 protease mutation I50 L causes atazanavir resistance but increases susceptibility to other PIs. Predicted phenotypic FC values were obtained from viral genotypes, using the virtual Phenotype-LM bioinformatics tool (powering vircoTYPE). OBJECTIVE: To evaluate I50 L's effect on susceptibility to 8 PIs, in a large genotype database. STUDY DESIGN: I50 L containing routine clinical isolate samples in Virco's genotype database were paired with samples having like patterns (or profiles) of IAS-USA-defined primary PI mutations, but lacking I50 L. Using vircoTYPE (version 4.1), the median predicted FC for each mutational profile was determined. I50 L-associated shifts in FC were evaluated using drug-specific CCOs. RESULTS: We selected 307 and 37098 samples with and without I50 L. These corresponded to 31 mutation patterns of > or =3 samples each. I50 L caused resistance to atazanavir in all 31 mutation contexts, but was associated with higher susceptibility for other PIs. The largest I50 L-associated shifts in median predicted FC were: 1.2 to 42.4 (atazanavir), 10.2 to 3.2 (amprenavir), 3.3 to 0.5 (darunavir), 13 to 0.5 (indinavir), 34.9 to 1.3 (lopinavir), 22.3 to 1.3 (nelfinavir), 5.2 to 0.3 (saquinavir) and 29.9 to 5.2 (tipranavir). CONCLUSIONS: The PI mutation I50 L causes clinically relevant resistance and increased susceptibility to atazanavir and other PIs respectively.

Prediction of HIV-1 drug susceptibility phenotype from the viral genotype using linear regression modeling.

Linear regression modeling on a database of HIV-1 genotypes and phenotypes was applied to predict the HIV-1 resistance phenotype from the viral genotype. In this approach, the phenotypic measurement is estimated as the weighted sum of the effects of individual mutations. Higher order interaction terms (mutation pairs) were included to account for synergistic and antagonistic effects between mutations. The most significant mutations and interactions identified by the linear regression models for 17 approved antiretroviral drugs are reported. Although linear regression modeling is a statistical data-driven technique focused on obtaining the best possible prediction, many of these mutations are also known resistance-associated mutations, indicating that the statistical models largely reflect well characterized biological phenomena. The performance of the models in predicting in vitro susceptibility phenotype and virologic response in treated patients is described. In addition to a high concordance with in vitro measured fold change, which was the primary aim of model design, the models per drug show good predictivity of therapy response for regimens including that drug, even in the absence of other clinically relevant factors such as background regimen.

Updated European recommendations for the clinical use of HIV drug resistance testing.

In most European countries, HIV drug resistance testing has become a routine clinical tool. However, its practical implementation in a clinical context is demanding. The European HIV Drug Resistance Panel was established to make recommendations to clinicians and virologists on this topic and to propose quality control measures. The panel recommends resistance testing for the following indications: i) drug-naive patients with acute or recent infection; ii) therapy failure, including suboptimal treatment response, when treatment change is considered; iii) pregnant HIV-1-infected women and paediatric patients with detectable viral load when treatment initiation or change is considered; and iv) genotype source patient when post-exposure prophylaxis is considered. In addition, for drug-naive patients with chronic infection in whom treatment is to be started, the panel suggests that resistance testing should be strongly considered and recommends testing the earliest sample for drug resistance if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%. The panel does not favour genotyping over phenotype, however it is anticipated that genotyping will be used more often because of its greater accessibility, lower cost and faster turnaround time. For the interpretation of resistance data, clinically validated systems should be used to the greatest extent possible. It is mandatory that laboratories performing HIV resistance tests take regular part in quality assurance programs. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and meet regularly to discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response. The panel also encourages the timely collection of epidemiological information to estimate the impact of transmission of resistant HIV and the prevalence of HIV-1 non-B subtypes in the different European countries.

Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: intrinsic stability predicts lifelong persistence.

Viral replication and latently infected cellular reservoirs persist in HIV-infected patients achieving undetectable plasma virus levels with potent antiretroviral therapy. We exploited a predictable drug resistance mutation in the HIV reverse transcriptase to label and track cells infected during defined intervals of treatment and to identify cells replenished by ongoing replication. Decay rates of subsets of latently HIV-infected cells paradoxically decreased with time since establishment, reflecting heterogeneous lymphocyte activation and clearance. Residual low-level replication can replenish cellular reservoirs; however, it does not account for prolonged clearance rates in patients without detectable viremia. In patients receiving potent antiretroviral therapy, the latent pool has a heterogeneous and dynamic composition that comprises a progressively increasing proportion of stable lymphocytes. Eradication will not be achieved with complete inhibition of viral replication alone.

DPC 681 and DPC 684: potent, selective inhibitors of human immunodeficiency virus protease active against clinically relevant mutant variants.

Human immunodeficiency virus (HIV) protease inhibitors (PIs) are important components of many highly active antiretroviral therapy regimens. However, development of phenotypic and/or genotypic resistance can occur, including cross-resistance to other PIs. Development of resistance takes place because trough levels of free drug are inadequate to suppress preexisting resistant mutant variants and/or to inhibit de novo-generated resistant mutant variants. There is thus a need for new PIs, which are more potent against mutant variants of HIV and show higher levels of free drug at the trough. We have optimized a series of substituted sulfonamides and evaluated the inhibitors against laboratory strains and clinical isolates of HIV type 1 (HIV-1), including viruses with mutations in the protease gene. In addition, serum protein binding was determined to estimate total drug requirements for 90% suppression of virus replication (plasma IC(90)). Two compounds resulting from our studies, designated DPC 681 and DPC 684, are potent and selective inhibitors of HIV protease with IC(90)s for wild-type HIV-1 of 4 to 40 nM. DPC 681 and DPC 684 showed no loss in potency toward recombinant mutant HIVs with the D30N mutation and a fivefold or smaller loss in potency toward mutant variants with three to five amino acid substitutions. A panel of chimeric viruses constructed from clinical samples from patients who failed PI-containing regimens and containing 5 to 11 mutations, including positions 10, 32, 46, 47, 50, 54, 63, 71, 82, 84, and 90 had mean IC(50) values of <20 nM for DPC 681 and DPC 681, respectively. In contrast, marketed PIs had mean IC(50) values ranging from 200 nM (amprenavir) to >900 nM (nelfinavir).

Synthesis and evaluation of novel quinolinones as HIV-1 reverse transcriptase inhibitors.

A series of 4,4-disubstituted quinolinones was prepared and evaluated as HIV-1 reverse transcriptase inhibitors. The C-3 substituted compound 9h displayed improved antiviral activity against clinically significant single (K103N) and double (K103N/L100I) mutant viruses.

Synthesis and evaluation of efavirenz (Sustiva) analogues as HIV-1 reverse transcriptase inhibitors: replacement of the cyclopropylacetylene side chain.

Two series of efavirenz analogues have been developed: one in which the cyclopropane ring has been replaced by small heterocycles and another in which the entire acetylenic side chain has been replaced by alkyloxy groups. Several members of both series show equivalent potency to efavirenz against both wild-type virus and the key K103N mutant.

Genotypic correlates of phenotypic resistance to efavirenz in virus isolates from patients failing nonnucleoside reverse transcriptase inhibitor therapy.

Efavirenz (also known as DMP 266 or SUSTIVA) is a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and of HIV-1 replication in vitro and in vivo. Most patients on efavirenz-containing regimens have sustained antiviral responses; however, rebounds in plasma viral load have been observed in some patients in association with the emergence of mutant strains of HIV-1. Virus isolates from the peripheral blood mononuclear cells (PBMCs) of patients with such treatment failures, as well as recombinant viruses incorporating viral sequences derived from patient plasma, show reduced in vitro susceptibility to efavirenz in association with mutations in the RT gene encoding K103N, Y188L, or G190S/E substitutions. Patterns of RT gene mutations and in vitro susceptibility were similar in plasma virus and in viruses isolated from PBMCs. Variant strains of HIV-1 constructed by site-directed mutagenesis confirmed the role of K103N, G190S, and Y188L substitutions in reduced susceptibility to efavirenz. Further, certain secondary mutations (V106I, V108I, Y181C, Y188H, P225H, and F227L) conferred little resistance to efavirenz as single mutations but enhanced the level of resistance of viruses carrying these mutations in combination with K103N or Y188L. Viruses with K103N or Y188L mutations, regardless of the initial selecting nonnucleoside RT inhibitor (NNRTI), exhibited cross-resistance to all of the presently available NNRTIs (efavirenz, nevirapine, and delavirdine). Some virus isolates from nevirapine or delavirdine treatment failures that lacked K103N or Y188L mutations remained susceptible to efavirenz in vitro, although the clinical significance of this finding is presently unclear.

4,1-Benzoxazepinone analogues of efavirenz (Sustiva) as HIV-1 reverse transcriptase inhibitors.

A series of 4,1-benzoxazepinone analogues of efavirenz (Sustiva) as potent NNRTIs has been discovered. The cis-3-alkylbenzoxazepinones are more potent then the trans isomers and can be synthesized preferentially by a novel stereoselective cyclization. The best compounds are potent orally bioavailable inhibitors of both wild-type HIV-1 and its clinically relevant K103N mutant virus, but are highly protein-bound in human plasma.

Human immunodeficiency virus type 1 mutations selected in patients failing efavirenz combination therapy.

Efavirenz is a potent and selective nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Nucleotide sequence analyses of the protease and RT genes (coding region for amino acids 1 to 229) of multiple cloned HIV-1 genomes from virus found in the plasma of patients in phase II clinical studies of efavirenz combination therapy were undertaken in order to identify the spectrum of mutations in plasma-borne HIV-1 associated with virological treatment failure. A K103N substitution was the HIV-1 RT gene mutation most frequently observed among plasma samples from patients for whom combination therapy including efavirenz failed, occurring in at least 90% of cases of efavirenz-indinavir or efavirenz-zidovudine (ZDV)-lamivudine (3TC) treatment failure. V108I and P225H mutations were observed frequently, predominantly in viral genomes that also contained other nonnucleoside RT inhibitor (NNRTI) resistance mutations. L100I, K101E, K101Q, Y188H, Y188L, G190S, G190A, and G190E mutations were also observed. V106A, Y181C, and Y188C mutations, which have been associated with high levels of resistance to other NNRTIs, were rare in the patient samples in this study, both before and after exposure to efavirenz. The spectrum of mutations observed in cases of virological treatment failure was similar for patients initially dosed with efavirenz at 200, 400, or 600 mg once a day and for patients treated with efavirenz in combination with indinavir, stavudine, or ZDV-3TC. The proportion of patients carrying NNRTI resistance mutations, usually K103N, increased dramatically at the time of initial viral load rebound in cases of treatment failure after exposure to efavirenz. Viruses with multiple, linked NNRTI mutations, especially K103N-V108I and K103N-P225H double mutants, accumulated more slowly following the emergence of K103N mutant viruses.

Inhibition of clinically relevant mutant variants of HIV-1 by quinazolinone non-nucleoside reverse transcriptase inhibitors.

A series of 4-alkenyl and 4-alkynyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones were found to be potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type-1 (HIV-1). The 4-alkenyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones DPC 082 and DPC 083 and the 4-alkynyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones DPC 961 and DPC 963 were found to exhibit low nanomolar potency toward wild-type RF virus (IC(90) = 2.0, 2.1, 2.0, and 1.3 nM, respectively) and various single and many multiple amino acid substituted HIV-1 mutant viruses. The increased potency is combined with favorable plasma serum protein binding as demonstrated by improvements in the percent free drug in human plasma when compared to efavirenz: 3.0%, 2.0%, 1.5%, 2. 8%, and 0.2-0.5% for DPC 082, DPC 083, DPC 961, DPC 963, and efavirenz, respectively.

Expanded-spectrum nonnucleoside reverse transcriptase inhibitors inhibit clinically relevant mutant variants of human immunodeficiency virus type 1.

A research program targeted toward the identification of expanded-spectrum nonnucleoside reverse transcriptase inhibitors which possess increased potency toward K103N-containing mutant human immunodeficiency virus (HIV) and which maintain pharmacokinetics consistent with once-a-day dosing has resulted in the identification of the 4-cyclopropylalkynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 961 and DPC 963 and the 4-cyclopropylalkenyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 082 and DPC 083 for clinical development. DPC 961, DPC 963, DPC 082, and DPC 083 all exhibit low-nanomolar potency toward wild-type virus, K103N and L100I single-mutation variants, and many multiply amino acid-substituted HIV type 1 mutants. This high degree of potency is combined with a high degree of oral bioavailability, as demonstrated in rhesus monkeys and chimpanzees, and with plasma serum protein binding that can result in significant free levels of drug.

Unsymmetrical cyclic ureas as HIV-1 protease inhibitors: novel biaryl indazoles as P2/P2' substituents.

The preparation of unsymmetrical cyclic ureas bearing novel biaryl indazoles as P2/P2' substituents was undertaken, utilizing a Suzuki coupling reaction as the key step. Compound 6i was equipotent to the lead compound of the series SE063.

Design and selection of DMP 850 and DMP 851: the next generation of cyclic urea HIV protease inhibitors.

BACKGROUND: Recent clinical trials have demonstrated that HIV protease inhibitors are useful in the treatment of AIDS. It is necessary, however, to use HIV protease inhibitors in combination with other antiviral agents to inhibit the development of resistance. The daunting ability of the virus to rapidly generate resistant mutants suggests that there is an ongoing need for new HIV protease inhibitors with superior pharmacokinetic and efficacy profiles. In our attempts to design and select improved cyclic urea HIV protease inhibitors, we have simultaneously optimized potency, resistance profile, protein binding and oral bioavailability. RESULTS: We have discovered that nonsymmetrical cyclic ureas containing a 3-aminoindazole P2 group are potent inhibitors of HIV protease with excellent oral bioavailability. Furthermore, the 3-aminoindazole group forms four hydrogen bonds with the enzyme and imparts a good resistance profile. The nonsymmetrical 3-aminoindazoles DMP 850 and DMP 851 were selected as our next generation of cyclic urea HIV protease inhibitors because they achieve 8 h trough blood levels in dog, with a 10 mg/kg dose, at or above the protein-binding-adjusted IC90 value for the worst single mutant--that containing the Ile84-->Val mutation. CONCLUSIONS: In selecting our next generation of cyclic urea HIV protease inhibitors, we established a rigorous set of criteria designed to maximize chances for a sustained antiviral effect in HIV-infected individuals. As DMP 850 and DMP 851 provide plasma levels of free drug that are sufficient to inhibit wild-type HIV and several mutant forms of HIV, they could show improved ability to decrease viral load for clinically significant time periods. The ultimate success of DMP 850 and DMP 851 in clinical trials might depend on achieving or exceeding the oral bioavailability seen in dog.

Nonsymmetric P2/P2' cyclic urea HIV protease inhibitors. Structure-activity relationship, bioavailability, and resistance profile of monoindazole-substituted P2 analogues.

Using the structural information gathered from the X-ray structures of various cyclic urea/HIVPR complexes, we designed and synthesized many nonsymmetrical P2/P2'-substituted cyclic urea analogues. Our efforts concentrated on using an indazole as one of the P2 substituents since this group imparted enzyme (Ki) potency as well as translation into excellent antiviral (IC90) potency. The second P2 substituent was used to adjust the physical and chemical properties in order to maximize oral bioavailability. Using this approach several very potent (IC90 11 nM) and orally bioavailable (F% 93-100%) compounds were discovered (21, 22). However, the resistance profiles of these compounds were inadequate, especially against the double (I84V/V82F) and ritonavir-selected mutant viruses. Further modification of the second P2 substituent in order to increase H-bonding interactions with the backbone atoms of residues Asp 29, Asp 30, and Gly 48 led to analogues with much better resistance profiles. However, these larger analogues were incompatible with the apparent molecular weight requirements for good oral bioavailability of the cyclic urea class of HIVPR inhibitors (MW < 610).

Resistance to HIV protease inhibitors: a comparison of enzyme inhibition and antiviral potency.

Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR). Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors. Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor. The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity. The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups. The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90. Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold). However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme. Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance. Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene.