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Two New Protease Inhibitors; Novel Mechanism Leading to PI Resistance
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Reported by Jules Levin
At today's session at the XIII International HIV Drug Resistance Workshop, early data was presented on two new protease inhibitors effective in vitro against proteaseresistanr virus. One PI is from Pzifer/Agouron and the other is from GlaxoSmithKline. An interesting study reported in an oral presentation today found that protease inhibitor resistance may occur from other ways besides the typical way, by drug mutation. Monique Nijhuis from Charles Boucher's group (University Medical Centre, The Netherlands) found resistance developed to a protease inhibitor without mutations in the viral protease enzyme. It was suggested and felt by a number of researchersat this conference that perhaps viral failure to Kaletra and Fosamprenavir/r may be caused by this mechanism. As you may know, studies looking at viral failures to these two ritonavir boosted protease inhibitors do not show mutations, as of yet in research findings.
"Novel HIV Drug Resistance Mechanism Leading to Protease Inhibitor (PI) Resistance in Response to a High Genetic Barrier PI In Vitro"
In vitro experiments were performed to identify novel drug resistance pathways. HIV-1 HXB2 was cultured in SupT1 cells in the presence of increasing levels of a high genetuc barrier PI Ro-033-4649. Genotypic and phenotypic drug susceptibility analysis was performed.
After more than a year in the presence of increasing drug concentrations the researchers were ableto selecta viral population displaying 6-8-fold resistance to Ro-033-4649 and to other PI. Interestingly, the viral population harbored no mutation in the viral protease enzyme. Sequence analysis of the viral gag gene demonstrated several nucleotide mixtures [1126, (matrix), 1460, (capsid) and 2180 (p6gag0]: gag mutations were found. Two nucleotide changes were observed at position 2095 and 2099. Repeat experiments also demonstrated nucleotide changes in the same regions, nucleotide: 2093, 2098, or 2099. These changes are located in the ribosomal frameshift site, responsible for the production of the pol gene products. In addition, the same region codesfor the transframe protein (TFP) and the p7/p1, p7/TFP and TFP/p6pol protease cleavage sites.
The authors concluded that this demonstrates that a novel alternative drug resistance mechanism was identified. Reroducible nucleotide changes in the region coding for the ribosomal frameshift site, the TFP and protease cleavage sites were observed that may be responsible for the reduced drug susceptibility. Charles Boucher's lab is developing resistance assay to evaluate and identify this mechanism of resistance.
"Antiviral Activity and Resistance Profile of AG-001859, a Novel HIV-1 Protease Inhibitor with Potent Activity Against Protease Inhibitor-Resistant Strains of HIV"
Jennifer Hammond (Pfizer, La Jolla, CA) reported that they identified a series of novel allophenylnorstatin-containing HIV-1 protease inhibitors, which demonstrate potent antiviral activity against srrains of HIV resistant to the currently approved protease inhibitors. Hammond reports AG-001859 is a potent, tight binding inhibitor of both wild-type HIV protease and an I84V/L90M mutant HIV protease, exhibiting a Ki of <0.1 nM against both enzymes. In cell-based assays, AG-001859 demonstrated potent antiviral activity against several strains of wild-type HIV, including laboratory adapted and primary isolates, with EC50 values ranging from 14 to 60 nM.
The antiviral The antiviral activity of the drug was also evaluated against a panel of 44 PI-resistant HIV-1 variants (PhenoSense) containing a variety of primary and secondary amino acid substitutions that ci=onfer broad cross-resistance to the approved protease inhibitors. Viruses in the panel contained an average of 5 PI-resistance mutations (range=2 to 11). 52%, 36%, and 5% of viruses in the panel contained one, two, and three major PI-resistance mutations (D30N, G48V, I50V, V82A/T, I84V, L90M). 82% of viruses demonstrated >10-fold resistance to at least one approved PI. 52% of viruses demonstrated >10-fold resistance to 2 or more approved protease inhibitors. The median IC50 (amount of drug to suppress replication) for AG-001859 against the 80% of the viruses was 75 nM, but it was higher for the other drugs: 278 Nm for nelfinavir, 119 nM for Kaletra, 92 nM for saquinavir, 130 nM for amprenavir, and 323 nM for indinavir. The median fold change relative to wild-type HIV was 1.6 (range 0.3-19.8). In contrast viruses in the panel demonstrated median fold-change values of 17 for nelfinavir, 15 for lopinavir, 17 for saquinavir, 3.4 for amprenavir, and 9.3 fold for indinavir.
AG-001859 demonstrated potent activity against all primary and secondary PI-resistant substitutions evaluated, and there was no correlation bewteen the antiviral activity of AG-001859 and the number of PI-resistant substitutions present. Hammond reported that no major PI-resistance mutation is associated with reduced susceptibility to AG-001859. The median fold-change for AG-001859 against D30N was 4.9 (n=5), 2.4 vs G48V (n=3), 2.9 vs T50V (n=2), 1.6 vs V82A/T (n=21), 4.0 vs I84V, and 1.6 vs L90M. There was a range in fold-change against each mutation ranging anywhere from 0.3 to 19.8, depending on the mutation.
AG-001859 susceptibility against isolates (strains) with >10-fold fold change in resistance to approved protease inhibitors: indinavir 3.1 fold-change, lopinavir 1.8 fold-change, nelfinavir 2.6: amprenavir 4.8 fold-change; saquinavir 3.2 fold-change. In other words, the isolates were >10-fold resistant to these protease inhibitors but there was only a 1.8 to 4.8 fold change in IC50 (resistance) for AG-001859 against these isolates. The presence of 50% human serum has a minimal impact on the antiviral activity of AG-001859.
Hammond summarized: AG-001859 is one compound from this series of allophenynorstatin-containing inhibitors of HIV protease that demonstrates: favorable aqueous solubility and metabolic stability; potent in vitro antiviral activity against strainsof HIV which demonstrate genotypic and phenotypic resistance to the currently approved protease inhibitors; potent in vitro activity against wild-type lab adapted and clinical strains of HIV- andHIV-2; potent antiviral activity in the presence of 50% human serum. Hammond was asked if ritonavir will be needed to boost this PI and she responded that they were not sure yet & I think she said they will be exploring both unboosted & boosted. No PK data was presented yet.
New HIV Protease Inhibitor GW640385
Its' too late in the evening now for me to write a report on the GSK protease inhibitor (I want to go to sleep now), GW640385, but I sent out yesterday a GSK press release on the two poster presentations about this drug. I'll report more details later but in brief one study examined 30 clinical isolates from patients experiencing viral failureto amprenavir-containing regimen. The clinical isolates were derived primarily from subjects receiving amprenavir as first linePI or second linePT regimen and had at least 1 amprenavir associated resistance mutation (V32I, I47V, I50V, I54L/M, I84V) present. In the 33% (10/30) viruses with the I50V mutation the mean fold change to amprenavir was 13.4 and 2.7 fold change to GW640385. For the 13% of viruses with the I84V mutation the mean fold change to amprenavir was 13.4 and to "385" it was 3.4. None of the isolates containing I54L/M or V32I/I47V mutations had a >2 fold decrease in susceptibility to GW6400385. In a second poster they found GW640385 appeared to have a unique resistance profile. High level pressure passage selected the A28S protease mutation, which was associated with dramatic reduction in relpicative capacity of the virus and high level resistance to "385". Lowlevel passage selected A71V and Q58E, and viruses with these two mutations had only low level resistance to "385" despite having other multiple mutations (not theusual ones). 5 clinical isolates with multiple PI mutations including major PI mutations 7 to 300 fold resistance to APV, LPV, IDV, and NFV, but 2.5 to 4.8 fold resistance to 385.
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