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  12th International HIV Drug Resistance Workshop
June 10-14, 2003, Los Cabos, Mexico
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Tenofovir Resistance at the HIV Drug Resistance Workshop
Reported by Jules Levin
  Tenofovir (Viread) has been available in the pharmacy for about 1.5 years. Study 903 was the first large study reporting on tenofovir resistance in treatment-naive patients. 48-week resistance and efficacy data was reported at the 2002 November Glasgow HIV Conference. At the Resistance Workshop (June 10-14, 2003, Cabos, Mexico) Mike Miller from Gilead Sciences reported 96 weeks on data from Study 903, and researchers at the conference focused discussion on tenofovir resistance. In addition there were 2 relevant studies related to the tenofovir K65R resistance mutation at the conference. The studies are discussed below following the data reported on study 903 by Miller. Study authors reported low but increased prevalence of the K65R mutation in the last few years, and shed some light on the development and implications of this mutation. When HIV antiretroviral drugs become available resistance research follows. We have been following resistance research for years regarding other HIV drugs. The reports discussed below are some of the first reports on the tenofovir K65R resistance mutation. I expect we will see ongoing research so we can gain further needed understanding of tenofovir resistance. The researchers here reported on resistance seen when tenofovir was used in combination with a few other NRTIs. But clearly we need continued follow-up on tenofovir resistance issues as we see with all other HIV antiretroviral drugs.
First here is a brief summary of Study 903 from Mike Miller. Study 903 is a 3 year, randomized, double-blind study in treatment-naive patients. 600 patients received either tenofovir (TDF) or d4T with 3TC and efavirenz (EFV). The objective of this analysis was to examine resistance development in patients in the 903 study. Gilead reported week 48 data at the Glasgow HIV Conference and here reports week 96 data. For the week 96 analysis HIV was analyzed genotypically by Virco and phenotypically for replication capacity and drug susceptibility by Virologic.
Baseline viral load was 81,000 copies/ml; mean CD4 count was 275; 42% had >100,000 copies/ml and 38% had <200 CD4 count. So, this was a relatively advanced patient group. At Glasgow, Gilead reported 80% of patients in both the d4T and the TDF groups had <50 copies/ml. Here at the Workshop Miller reported that 80% in both patient groups had <400 copies/ml at week 96 (ITT). Clearly, this regimen is potent and overall in the study the failure rate was low. Tenofovir has the benefit of being dosed once a day with 1 pill and appears to be tolerable relative to other HIV drugs.
The percent of resistant patients did not differ much between week 48 and 96 in Study 903. At week 96, there were 12% of virologic failures in the TDF group (36/299) and there were 12.6% virologic failures in the d4T group (38/301). 8 of the 36 viral (22%) of EFV/TDF/3TC viral failures had K65R mutation. 2 of 38 failures in d4T/EFV/3TC group developed the K65R. No patient in either group had the K65R mutation alone. The K65R mutation only emerged along with EFV or EFV/3TC mutations.
5 patients developed EFV-resistance+3TC (M184) resistance+K65R resistance. And 3 patients developed K65R+EFV-resistance. Gilead reported phenotypic resistance susceptibility (Virologic) for patients who developed K65R. For patients with the M184+K65R mutation 2/4 patients were called "intermediate susceptibility" to ddI and 2/4 to abacavir; 1/4 was called "resistant" to abacavir. All 4 patients were called "resistant" to 3TC. And all 4 were called fully susceptible to AZT, d4T, and TDF. For patients with K65R without M184, susceptibility was below the cutoff for resistance to d4T, there were 3 patients bordering at the cutoff for ddI resistance, and 1 patient bordering at the cutoff to abacavir. Resistance researchers pretty much agree that phenotypic cutoffs for ddI and d4T are difficult to interpret.
K65R Studies
There were two additional studies on K65R resistance, one by Parikh and a second by Winston. Parikh's in vitro research suggests (1) K65R reduces susceptibility to certain NRTIs; (2) AZT plus TDF may delay development of K65R; (3) the prevalence of the K65R mutation increased from 0.8% in 1999 to 3.8% in 2003; (4) she proposes a mechanism of action. Further clinical research of patients needs to be conducted to confirm these in vitro findings. If you do in fact use AZT+TDF you should consider monitoring development of mutations and viral load closely to see if TAMS (thymidine analalogue, d4T and AZT, mutations) are emerging. You don't want too many TAMS to develop. This could lead to reduced response to other NRTIs.
Winston's study found the prevalence of the occurrence of the K65R mutation at Chelsea and Winstminter Hospital in London increased from 1.7% before 2000 to 4% in the period between 2000-2002. He also found K65R less likely t occur when multiple TAMS are present. K65R was more likely to occur with TDF/ddI and TDF/ddI/ABC, and less likely to occur when used in conjunction with thymidine analogues.
The prevalence of the K65R mutation appears to be low now but it's important to understand the implications of the presence of this mutation, which is discussed by the studies below. In studies of patients with treatment-experience conducted by Gilead 6 patients had the K65R before the studies and none of the 6 patients responded to tenofovir.
K65R: a multi-nucleoside resistance mutation of low but increasing frequency
Urvi Parikh (University of Pittsburgh, Pittsburgh) reported results from this study in oral presentation in Clinical Implications of Resistance Session.
The K65R mutation in the fingers domain of HIV-1 reverse transcriptase (RT) is commonly selected in vitro by D-nucleosides but is paradoxically rare in vivo, although recent reports suggest that its frequency may be increasing. In the GS-903 trial, 24% of virological failures in the tenofovir/lamivudine(3TC)/efavirenz arm developed 65R (Miller et al., 6th Intl Congress on Drug Therapy in HIV Infection). The effect of 65R on susceptibility and response to approved and investigational nucleoside RT inhibitors (NRTI) is uncertain. To gain further insights about 65R, we examined recent trends in the frequency of 65R, determined the resistance profile of 65R alone and in combination with zidovudine (AZT) mutations, and analysed the effect of 65R on the primer unblocking activity of RT. The K65R is commonly selected in vitro by ddC, PMEA (adefovir), PMPA (tenofovir), abacavir, 3TC, FTC, and d4T.
We searched the Virco and Stanford data-bases for the frequency of 65R and its association with other NRTI mutations. We examined the effect of 65R alone and with different AZT resistance mutations on HIV-1 susceptibility to a large panel (n=36) of D- , L-and acyclic NRTIs using a single cycle replication assay (P4/R5 cells) and/or a multiple cycle assay (MT-2, p24). The primer unblocking activity of RTs was determined by measuring the formation of [32P] Ap 4 ddA from ATP-catalysed removal of ddAMP from ddAMP-terminated primer.
Among the >62,222 samples submitted to Virco for testing from 1998 to 2003 that contained any NRTI mutation, the frequency of 65R increased from 0.6% in 1998, 0.8% in 1999, 0.9% in 2000, 0.9% in 2001 to 2.1% in 2002. Among samples received in 2003, 3.8% had 65R. A molecular clone of HIV-1 encoding 65R showed 2.5-fold to >>10-fold reduced susceptibility to all D-, L- and acyclic NRTIs tested (including 3TC, FTC, ddI, d4T, tenofovir, abacavir) except those containing a 3-azido moiety (AZT and AZA), which showed wild-type susceptibility. In examining the frequency of other NRTI mutations that occurred with 65R, a strong negative relation was noted with AZT resistance mutations in the Virco database. For example, 10.3% of mutations containing the AZT mutation 41L also had the K65R mutation, but 49% of the 41L mutations did not contain the K65R mutation. This same neagtive correlation occurred with other AZT mutations 67N (8.7% vs 37%), 210W (4% vs 28%), 215Y (7.4% vs 47%), and 215F (0.7% vs 12.3%). They did not see this negative correlation with 2 AZT mutations, 70R and 219 Q and E.
Parikh said that this data suggests that 65R is antagonistic to AZT resistance mutations. She found the addition of 65R to two different AZT-resistant clones (41L/210W/215Y and 67N/70R/215F/219Q) reduced AZT resistance >10-fold (>30-fold to ~threefold). Similarly, the addition of 65R to AZT-resistant RT reduced primer unblocking activity 10-fold to wild-type levels; the addition of K65R to 2 AZT resistant clones (41L/210W/215Y, 67N/70R/215F/219Q) reduced excision of AZTMP about 10-fold)--measured ability of RT to restore chain elongation after removal of AZT from an AZT-terminated primer template.
Parikh summarized:
--that 65R reduced susceptibility to all NRTIs tested except those containing a 3-azido moiety (AZT and AZA)
--the K65R is negatively associated with TAMS, thymidine analogue mutations (AZT, d4T). The addition of K65R to two different AZT-resistant clones described above reduced AZT resistance >10-fold and reduced primer unblocking activity to wild-type virus
--65R appears to be counter-selected by AZT because 65R antagonizes ATP-catalysed primer unblocking, which is the primary mechanism of AZT resistance
--no significant difference between HIV-wild-type and K65R -HIV were observed in replication capacity in a single cell assay or in replication kinetics measured by p24 production over time although comparison in primary cells is needed
--65R can be an important mechanism of resistance to NRTIs for which primer unblocking is not required
--NRTIs that can select 65R may need to be given with AZT to prevent 65R from emerging (suggesting a combination of tenofovir plus AZT may delay K65R from developing)
Which nucleoside and nucleotide backbone combinations select for the K65R mutation in HIV-1 reverse transcriptase?
Alan Winston (Chelsea and Westminster Hospital, London, UK) reported an oral talk also in the Clinical Implications of Resistance session.
BACKGROUND: The K65R mutation has been reported to be associated with the use of abacavir (ABC), didanosine (DDI) and tenofovir (TDF). With the advent of TDF this study assesses the change in prevalence of the K65R mutation and its association with TDF, DDI and ABC therapy.
METHODS: In Chelsea and Westminster Hospital individuals failing therapy have a resistance test. Genotypes were analysed on individuals failing nucleoside and nucleotide containing regimens from October 2000 to October 2002 (time 2). This was compared to genotypes pre-October 2000 (time 1) in TDF-naive individuals.
RESULTS: The K65R was identified in viruses from 28 of 705 individuals having resistance tests for virological failure during time 2 (prevalence 4.0%). The prevalence of this mutation during time 1 was significantly lower at 1.7% (P=0.004). Winston reported that for patients with the K65R or without the K65R there were no differences in CD4 count, viral load, or in time on ART. When adjusting for the use of other drugs, neither ABC, DDI or TDF alone was associated with the development of the K65R mutation.
However certain combinations were associated with the mutation. The use of TDF and DDI was associated with the development of the K65R variant (14% of isolates with the K65R were on dual use of TDF, DDI versus 3.8% of isolates, which did not develop the mutation, P=0.007). Current thymidine analogue use protected from the development of the mutation, those on thymidine analogues were 76% less likely to develop the mutation.
115 individuals were taking ddI, abacavir (ABC), and TDF. 93 patients did not have a resistance test. A total of 22 individuals receiving this combination had a resistance test. 13 patients did not have the K65R mutation and 9 did have the K65R mutation. The combination of ABC, DDI and TDF was associated with the mutation. 32% of individuals who developed the K65R were on this combination (32% vs 1.8%, p<0.001), those not on this combination were 96% less likely to develop the K65R. None of the patients on TDF/ddI/ABC had the K65R mutation. The K65R developed in nine isolates (41%).
Winston summarized:
--The prevalence of the K65R mutation remains low but has significantly increased over a 2-year period
--Although not associated with a specific drug, the K65R mutation is more common in those receiving concomitant TDF and DDI, and is less common in those on thymidine analogues
--In individuals failing ABC, DDI, TDF there is a high prevalence of the K65R, which may be due to all components of this regimen selecting for the development of this mutation. For patients on combinations of this drug K65R is less likely to occur when these drugs are used in conjunction with thymidine analogues