The development of resistance of HIV-1 to zalcitabine.
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Amino Acids↗
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Sequencing antiretroviral drugs.
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Alkynes↗
Pharmacokinetics and resistance mutations affect virologic response to ritonavir/saquinavir-containing regimens.
The authors assessed the impact of protease and reverse transcription (RT) mutations and individual pharmacokinetic parameters on virologic response to a four-drug regimen including ritonavir/saquinavir. Treatment was given at the start of the study (M0) to 22 HIV-1 protease inhibitor-naive or pretreated patients. Protease and RT genes were sequenced at M0, at the time of virologic failure, or at the end of the follow-up. Plasma ritonavir and saquinavir peak C(max), C(min), and area under the curve (AUC) were determined based on samples taken 0, 1, 2, 3, 4, 6, 8, and 12 hours after administration. HIV-1 RNA decreased to less than 50 copies/mL in 11 patients (group 1). At M0, five of them had no RT mutation and 10 had three or fewer secondary protease mutations with no new mutation during follow-up. Ritonavir and saquinavir pharmacokinetics showed wide interindividual variability. Treatment failed in 11 patients (group 2): 9 had three to eight protease mutations and a mean of 5.8 RT mutations at M0, with emergence of new mutations during follow-up. Pharmacokinetics was similar to those of group 1. The other two patients with virologic failure showed no baseline primary mutation but were the only patients with insufficient saquinavir and ritonavir AUC. The authors showed the complementarity between drug-resistance genotype and individual pharmacokinetics and the potential utility of AUC and Cmax to manage treatment.
CD4 Lymphocyte Count↗
S-adenosylhomocysteine hydrolase inhibitors interfere with the replication of human immunodeficiency virus type 1 through inhibition of the LTR transactivation.
Various analogues of adenosine have been described as inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, and some of these AdoHcy hydrolase inhibitors (e.g., 3-deazaadenosine, 3-deazaaristeromycin, and 3-deazaneplanocin A) have also been reported to inhibit the replication of human immunodeficiency virus type 1 (HIV-1). When evaluated against HIV-1 replication in MT-4 cells, macrophages, or phytohemagglutinin-stimulated peripheral blood lymphocytes infected acutely or chronically with HIV-1IIIB or HIVBaL strains, a wide range of adenosine analogues did not inhibit HIV-1IIIB replication for 50% at subtoxic concentrations. However, they inhibited HIV-1 replication in HeLa CD4+ LTR-LacZ cells at concentrations well below cytotoxicity threshold. A close correlation was found among the inhibitory effect of the compounds on AdoHcy hydrolase activity, their inhibition of HIV-1 replication in Hela CD4+ LTR-LacZ cells, and their inhibition of the HIV-1 Tat-dependent and -independent transactivation of the long terminal repeat, whereas no inhibitory effect was seen on HIV-1 reverse transcription or a Tat-independent cytomegalovirus promoter. Our results suggest that AdoHcy hydrolase and the associated S-adenosylmethionine-dependent methylation mechanism play a role in the process of long terminal repeat transactivation and, hence, HIV replication.
Adenosylhomocysteinase↗
Influence of stereochemistry on antiviral activities and resistance profiles of dideoxycytidine nucleosides.
beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.
Animals↗
Extent of cross-resistance between agents used to treat human immunodeficiency virus type 1 infection in clinically derived isolates.
The phenomenon of cross-resistance to antiretroviral agents used to treat human immunodeficiency virus type 1 infection is well known but so far has been only qualitatively described. Here, we quantitate the degree of cross-resistance among all commonly prescribed antiretroviral agents in almost 5,000 clinically derived recombinant isolates collected in the United States since January 2000.
Anti-HIV Agents↗
Prevalence of genotypic resistance to nucleoside analogues in antiretroviral-naive and antiretroviral-experienced HIV-infected patients in Spain.
OBJECTIVE: To determine the prevalence of genotypic resistance to nucleoside analogues (NA) in a large group of HIV-infected individuals in Spain, some of whom had no previous treatment with antiretroviral drugs (antiretroviral-naive) and some of whom had such experience (antiretroviral-experienced). SETTING: Cross-sectional study in out-patient clinics in three reference hospitals for HIV/AIDS located in Barcelona, Madrid and Seville, Spain. PATIENTS AND METHODS: Primary mutant genotypes were examined in plasma HIV RNA collected from 150 antiretroviral-naive subjects, half in 1993 and the other half in 1997. Furthermore, drug resistance mutations were analysed in plasma collected from another 150 antiretroviral-experienced patients who had received 2 NA for longer than 1 year, either in sequence as monotherapy or as combination therapy. A line probe assay was used for recognizing mutations conferring resistance to zidovudine (ZDV), didanosine (ddI), zalcitabine (ddC), and lamivudine (3TC). A point-mutation nested-PCR assay was used for examining a codon 151 mutation associated with multiple drug resistance. RESULTS: One or more mutations associated with primary resistance to NA were seen in 10 antiretroviral-naive (13.3%) patients in 1993 and in nine (12%) in 1997. In all but two cases, they were associated with ZDV resistance. In contrast, all but six (96%) of the antiretroviral-experienced subjects harboured drug-resistant mutant viruses. The codon 184 mutation (associated with resistance to 3TC) was detected in 92% of patients treated with 3TC, but also in 18% of those treated with only ddI or ddC. The codon 215 mutation was found in 67.3% of patients who had been exposed to ZDV; the codon 69 mutation was found in 15% of patients treated with ddC; and the codon 74 mutation was found in only 7.2% of patients treated with ddI. Finally, the codon 151 multidrug resistant mutation was found in four (2.7%) of 150 patients with a long-term exposure to NA. CONCLUSIONS: Overall, the prevalence of drug-resistant HIV-1 genotypes was 12.7% in antiretroviral-naive patients, most of whom had ZDV-resistant mutants. There is no evidence of an increase during the last 5 years. However, multidrug-resistant HIV genotypes are currently circulating in Spain.
Adult↗
Antagonism between the HIV-1 reverse-transcriptase mutation K65R and thymidine-analogue mutations at the genomic level.
Prior virologic and biochemical studies have shown phenotypic antagonism between K65R and multiple thymidine-analogue mutations (TAMs) in site-directed mutants tested in vitro. We hypothesized, on the basis of this observed antagonism, that K65R and T215Y/F with multiple TAMs would not be selected on the same human immunodeficiency virus type 1 genome in vivo. We searched a large database of patient genotypes (n=59,262) for the frequency of K65R in combination with >or=3 TAMs as determined by standard population sequencing. K65R and multiple TAMs were rarely detected (<0.1%) in the same plasma sample. Samples with both K65R and >or=3 TAMs (n=21) were further analyzed by use of single-genome sequencing. K65R was never found on the same genome with T215F/Y and >or=2 other TAMs, except in the presence of the Q151M multiple nucleoside reverse-transcriptase inhibitor (NRTI)--resistance complex. These results indicate that antagonism between the K65R and T215Y/F pathways of NRTI resistance occurs at the genomic level. Therapy with NRTI combinations that select both pathways simultaneously may delay the emergence of NRTI resistance and prolong treatment response.
Amino Acid Substitution↗
Cellular and molecular mechanisms of antiretroviral effects of HPA23.
HPA23 is an antimonio-tungstate that exhibits numerous antiviral activities both in vivo and in vitro. It has been described as a competitive inhibitor of human immunodeficiency virus (HIV) reverse transcriptase (RT). Patients treated with daily injections of HPA23 show an inhibition of HIV RT activity in cell culture in 60% of the cases. Using biophysical (electronic spin resonance [ESR]), ultrastructural (microspectroscopic analysis), chemical (spectroscopy), and biological (cell culture) assays, HPA23 cellular and molecular mechanisms may be summarized as follows: 1) competitive inhibition of HIV-RT, 2) no or slight effect on cells infected with HIV in culture, 3) interactions with the cell membranes when long incubations are performed, and 4) antiviral activity possibly mediated by immune modulator effect of the drug.
Antimony↗
Abacavir: a review of its clinical potential in patients with HIV infection.
UNLABELLED: Abacavir is a carbocyclic 2'-deoxyguanosine nucleoside analogue. It is metabolised intracellularly to a 2'-deoxyguanosine nucleoside analogue which competitively inhibits HIV reverse transcriptase and terminates proviral DNA chain extension. In double-blind trials in antiretroviral therapy-experienced or -naive patients, reductions in HIV RNA levels were greater and more prolonged in patients receiving abacavir in combination with other antiretroviral drugs than in those receiving placebo in combination with the same agents. Furthermore, abacavir in combination with lamivudine and zidovudine reduced viral load to below detectable levels in a proportion of patients, and to a similar extent to the protease inhibitor indinavir in combination with lamivudine and zidovudine. Greatest viral load reductions were seen in antiretroviral therapy-naive patients. Preliminary results suggest that the viral suppression achieved with a protease inhibitor plus 2 nucleoside reverse transcriptase inhibitors (NRTIs) can be maintained as effectively with abacavir in combination with 2 NRTIs as it can be by continuing the protease inhibitor-containing treatment regimen. Initial virological data from studies of combination regimens including abacavir and protease inhibitors appear promising but larger controlled trials are required to confirm these observations. Nausea is the most frequently reported adverse event in patients receiving abacavir-containing combination therapy. Adverse events tend to be reported most frequently soon after starting treatment; the majority of events are mild or moderate in intensity and transient. Other adverse events reported in >5% of patients include vomiting, malaise and fatigue, headache, diarrhoea, sleep disorders, cough, anorexia and rash. A major cause of abacavir treatment discontinuation is the development of a hypersensitivity reaction which has been reported in 3 to 5% of patients. The reaction usually occurs within 6 weeks of commencing treatment, shows evidence of multiorgan system involvement and typically includes fever and/or rash. Symptoms resolve rapidly after discontinuation of treatment. Continuing treatment or rechallenge can result in more severe symptoms, life-threatening hypotension and even death. CONCLUSION: Abacavir used in combination with other antiretroviral drugs effectively reduces viral load in both adults and children with HIV infection. Although these responses are greatest in individuals with little or no previous antiretroviral treatment, useful responses are still sometimes achieved in heavily pretreated individuals. Abacavir in combination with lamivudine and zidovudine provides a simple and convenient dosage regimen which is generally well tolerated, able to produce sustained suppression of viral replication and has the advantage of sparing other classes of antiretroviral drugs for subsequent use. This triple combination represents an alternative antiretroviral regimen for patients intolerant to protease inhibitors or those wishing to retain the option of protease inhibitors for later use. Further clinical studies are needed to define the activity of abacavir in combination with protease inhibitors and non-nucleoside reverse transcriptase inhibitors.
Animals↗
Nucleotide analogue binding, catalysis and primer unblocking in the mechanisms of HIV-1 reverse transcriptase-mediated resistance to nucleoside analogues.
Nucleoside analogues play a key role in the fight against HIV-1. Unfortunately, under therapeutic pressure, HIV-1 inevitably develops resistance to these inhibitors. This resistance correlates with specific pol gene mutations giving rise to specific substitutions in reverse transcriptase that are responsible for the loss of efficacy of the corresponding analogue. This work is an overview of the molecular mechanisms of HIV-1 drug resistance as judged by the analysis of chemical reactions at play at the reverse transcriptase active site. One class of mechanism involves nucleotide analogue discrimination either at the binding step or at the catalytic step, the latter being by far the most common mechanism. The other class of mechanism involves repair of the analogue-terminated DNA chain. The mechanisms were elucidated using purified reverse transcriptase and biochemical assays aimed at correlating resistant HIV-1 phenotypes to enzymatic data. The elucidation of these molecular mechanisms of drug-resistant reverse transcriptase is important for effective and rational combination therapies as well as for the conception of second-generation drugs that do not confer nucleotide resistance to reverse transcriptase or are active against pre-existing resistant viruses.
Anti-HIV Agents↗
Antiretroviral treatment. HIV infection in adults: better-defined first-line treatment.
(1) There is still no cure for HIV infection. The short-term treatment aims are to drive viral load below the current detection limit and to increase the CD4+ T cell count, in order to reduce morbidity and prolong survival. (2) Early initiation of treatment has both advantages and disadvantages. If the patient is symptomatic or if the CD4+ T cell count is below 200 per mm3, antiretroviral treatment should be started immediately. If the patient is asymptomatic, the CD4+ T cell count is above 350 per mm3, and viral load is below 50 000 copies/ml, antiretroviral treatment can often be deferred. Other situations should be considered case by case. (3) The benefits of treating symptomatic primary infection have not been adequately documented, and current recommendations diverge. (4) The advent of new antiretroviral drugs has increased the choice, but cross-resistance often limits treatment options. The new antiretroviral family to have emerged since 1999 is the fusion inhibitor; the only representative of this class, enfuvirtide, is reserved for patients with multiple treatment failure. (5) There is broad agreement on the principles of first-line antiretroviral treatment. It should combine at least two nucleoside (or nucleotide) inhibitors of HIV reverse transcriptase and one non nucleoside inhibitor, or at least one HIV protease inhibitor. Comparative studies have now identified the most effective combinations in terms of virological efficacy and tolerability. The combination should be chosen according to its established efficacy, adverse effects, risks of interactions, and convenience. There is no reference combination suitable for all patients. Among the combinations containing a non nucleoside inhibitor, those based on efavirenz are the most effective after 48 weeks of follow-up. There is less agreement on the optimal treatment of pregnant women. (6) Among the HIV protease inhibitor-based combinations, those containing nelfinavir or the lopinavir + ritonavir combination must be taken during meals. The lopinavir + ritonavir combination showed better virological efficacy than nelfinavir in a comparative trial. Experience and safety evaluation are in favour of nelfinavir, but recent American guidelines issued in November 2003 recommend the lopinavir + ritonavir combination. (7) There is no major difference in virological efficacy between non nucleoside inhibitors and protease inhibitors as first line therapy. (8) In combination with protease inhibitors or non nucleoside inhibitors, the best-assessed nucleoside inhibitors (effective for several years) are lamivudine + zidovudine and lamivudine + stavudine. For first-line treatment, combinations consisting of only three nucleoside (or nucleotide) inhibitors are less effective than combinations containing HIV protease inhibitors or non nucleoside inhibitors. (9) Efficacy is monitored on the basis of changes in viral load and the CD4+ T cell count, one month after the beginning of treatment, then about every three months. Attention must be paid to adverse effects, which may necessitate the replacement of the causative antiviral drug, treatment of the adverse effect, treatment modification or, in extreme cases, treatment withdrawal. (10) Treatment failures must be carefully investigated: the cause(s) may include poor adherence, drug interactions, and inadequate plasma drug concentrations. Ongoing antiretroviral regimens do not always have to be modified if treatment failure occurs. (11) Resistance tests can help to determine the most effective alternative in case of virological failure due to drug resistance. The choice of back-up treatments is complex, and is limited by cross-resistance. Multiple lines of treatment fail in about 3% to 4% of patients.
Adult↗
Screening anti-HIV Chinese materia medica with HIV and equine infectious anemic virus reverse transcriptase.
The inhibitory activities of human immunodeficiency virus reverse transcriptase (HIV-RT) inhibitors PFA and Suramin against HIV-RT and equine infectious anemic virus reverse transcriptase (EIAV-RT) were studied in this paper. The 50% inhibitory concentration (IC50) of HIV-RT and EIAV-RT treated by PFA and Suramin were 0.2 mumol, 9.8 mumol and 17 mumol, 19.9 mumol, respectively. More than thirty Chinese medicines, including recipes, herbs, extracts of traditional materia medica and isolated compounds were tested using HIV-RT and EIAV-RT as target enzymes. It was found that 5 crude extracts such as Rhizoma Polygoni Cuspidati, and Radix Notoginseng, 7 isolated compounds like Flavone of Ramulus Visci, Flavone of Ajuga Decumbens Thumb, and Aristolochic acid, as well as the extract of the complex prescription Xiao Chai Hu Tang have shown various inhibitory actions on these two enzymes. The activities of the two enzymes were comparable, but HIV-RT was more sensitive, suggesting that HIV-RT can be used as index for the screening of anti-aids drugs.
Animals↗
A rapid and sensitive real-time PCR assay for the K65R drug resistance mutation in SIV reverse transcriptase.
Macaques infected with simian immunodeficiency virus (SIV) provide a suitable model for assessing the efficacy of antiretroviral (ARV) drug interventions and drug resistance selection associated with treatment. Resistance to the HIV reverse transcriptase inhibitor tenofovir continues to be examined in different treatment strategies in the macaque model. Evaluations of treatment interventions and drug resistance are hampered by the limited sensitivity of conventional population sequencing and the substantial effort involved in testing various tissue compartments in which viruses may reside. Therefore, a sensitive assay that permits simple and rapid testing for drug-resistant viruses would benefit appraisals of ARV treatments using in vivo models. To have this capability, we developed a real-time PCR-based assay for the detection of the SIV K65R reverse transcriptase mutation, a key marker for reduced susceptibility to tenofovir. Evaluations of SIV sequences yielded an assay detection limit mean of 0.4% mutant virus (range = 0.1-2%) in a wild-type background. In testing longitudinal plasma specimens from four SIV-infected macaques that received an active daily regimen of 30 mg/kg of tenofovir subcutaneously, the assay was able to detect K65R-positive viruses in all animals within 1-7 weeks after treatment began. The emerging mutants were initially present at frequencies estimated between 0.4% and 3%, below the detection capability of population sequencing. We propose the SIV K65R real-time PCR assay provides improved sensitivity and simplicity in studying tenofovir resistance in macaque models.
Adenine↗
Equilibrium distribution of HIV antiviral drugs into human peripheral blood mononuclear cells (PBMC) is controlled by free drug concentration in the extracellular medium.
Effect of protein binding on the equilibrium distribution of selected HIV antiviral drugs into isolated human peripheral blood mononuclear cells (PBMC, mainly lymphocytes) was investigated. Human PBMC from a single healthy human donor were isolated, purified, and cryopreserved. Uptake of non-peptide HIV-1 protease inhibitors PNU-96988 and PNU-103017 by these cells in vitro was evaluated as a function of increasing concentration of human serum in the cell incubation media. Both PNU-96988 and PNU-103017 were extensively bound to serum proteins. Uptake/efflux kinetics were very rapid such that accumulation by the cells was thermodynamically, not kinetically, controlled. Accumulation by human PBMCs in vitro was directly proportional to the free and not the total drug concentration in the media. For comparative purposes, the serum protein binding effect on the distribution of two HIV reverse transcriptase (RT) inhibitors, delavirdine (RESCRIPTOR) and zidovudine (AZT), was also evaluated. Like the HIV-1 protease inhibitors, delavirdine was found to be extensively associated with serum proteins and its accumulation by human PBMCs in vitro to be proportional to the free and not total drug concentration. In contrast, AZT was not bound to serum proteins to any significant extent. The uptake of this drug by human PBMCs in vitro was independent of serum concentration. However, the intrinsic cellular accumulation of PNU-96988, PNU-103017 and delavirdine were all greater than AZT. Thus, the extent to which drugs uptake by cells is affected by serum appears proportional to the binding affinity of the serum proteins for the drug.
Adult↗
Detection of HIV-1 RNA in heparinized plasma of HIV-1 seropositive individuals.
The interference of reverse transcription by heparin was removed by heparinase. When the HIV-1 RNA in the presence of heparin was detected by a combination of reverse transcription and the polymerase chain reaction (PCR), heparinase treatment followed by removal of Ca2+ before the reverse transcription step permitted the efficient detection of HIV-1 RNA. Prior treatment with heparinase revealed HIV-1 RNA in 68% (13/19) of heparinized plasma samples from HIV-1 carriers, whereas only 26% (5/19) of the same specimens were positive without the heparinase step. Heparinase removed the inhibition of reverse transcription by heparin and is highly recommended when detecting low levels of viral RNA in heparinized plasma.
HIV Reverse Transcriptase↗
Phenotypic and genotypic resistance to nucleoside reverse transcriptase inhibitors in HIV-1 clinical isolates.
OBJECTIVE: To assess phenotypic and genotypic cross-resistance to nucleoside reverse transcriptase inhibitors in patients treated with a combination including zidovudine, who were switched to a combination including stavudine. METHODS: We analysed 24 clinical HIV-1 isolates from 12 patients before and several months after therapeutic switching. Plasma HIV-1 RNA was measured using quantitative polymerase chain reaction (Roche). Genotypic resistance was measured by sequencing the reverse transcriptase gene from plasma HIV-1 RNA. Phenotypic resistance was measured using a recombinant assay (Virco). RESULTS: Patients were treated with a combination including zidovudine for a mean (+/- SEM) period of 21.8 +/- 3.5 months and had a plasma viral load of 4.1 +/- 0.2 log HIV-1 RNA copies/mL (time 1). After a mean period of 19.3 +/- 1.6 months following the therapeutic change, the plasma viral load was 3.6 +/- 0.1 log copies/mL (time 2). At time 1, genotypic resistance to zidovudine was found in all cases (41L: four cases; 41L, 215Y: five cases; 41L, 210W, 215Y: two cases; K70R: one case) with a mean 6.6 +/- 1.6-fold increase in the median inhibitory concentration (IC50) to zidovudine and 1.7 +/- 0.4-fold to stavudine. At time 2, genotypic resistance to zidovudine was found in 11 out of 12 cases (41L: two cases; 41L, 215Y: six cases; 41L, 210W, 215Y: two cases; M41L, D67N, L210W, T215Y: one case) with a mean 18.9 +/- 8.8-fold increase in the IC50 to zidovudine and 1.4 +/- 0.4-fold to stavudine CONCLUSIONS: In this clinical series of patients with suboptimal control of plasma HIV-1 RNA using a combination including zidovudine, the presence of zidovudine-related mutations was associated with a decreased phenotypic sensitivity to this drug. Despite persistent HIV-1 replication, switching to stavudine was associated with a further decrease in phenotypic sensitivity to zidovudine but not to stavudine after 19 months. These data suggest that stavudine remains a possible option in zidovudine-experienced patients.
Adult↗
Analysis of Primer Extension and the First Template Switch during Human Immunodeficiency Virus Reverse Transcription.
Reverse transcription by human immunodeficiency virus (HIV) reverse transcriptase (RT) entails several distinct, early steps in the synthesis of double-stranded DNA from viral RNA templates. These steps include tRNA(lys3) priming of RNA-dependent DNA polymerization and template switching for synthesis of near full-length (-) DNA. However, HIV RT lacks the fluent processivity of other viral reverse transcriptases or cellular polymerases. Previous studies in our laboratory showed that RT reactions primed with tRNA(lys3) had higher efficiencies of template switching than those primed by synthetic oligonucleotides. To further study primer extension, pausing, and template switching, we utilized an in vitro reconstituted reverse transcription/template switching reaction consisting of HIV RNA templates, recombinant HIV RT and primer tRNA(lys3). We observed initial pause sites within the first five nucleotides of the primer terminus; these were extended efficiently to full-length (-) strong-stop DNA with prolonged incubation. Other pause sites occurred in the R/U5/PBS template adjacent to homopolymeric sequences and regions thought to be involved in secondary structure. This assay provides a sensitive means of assessing template switching at times between 10 s and 3 h. Copyright 1995 S. Karger AG, Basel
Journal Article↗