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New Details on Resistance to Darunavir and Tipranavir: TMC & Tipranavir not cross-resistant; new tipranavir genotypic resistance profile; TMC114 & amprenavir not cross-resistant; selection of mutations conferring resistance to TMC114
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14th Conference on Retroviruses
February 25-28, 2007, Los Angeles
Approval of darunavir (TMC114) and tipranavir transformed salvage planning for people with virus resistant to earlier protease inhibitors (PIs). But much remains to be learned about how people respond to either of these new drugs after other PIs fail. Several Retrovirus Conference studies filled gaps in this knowledge base, showing that darunavir can work for many people in whom tipranavir, fosamprenavir, or lopinavir flounders.
Susceptibility to darunavir after tipranavir failure
Failure of tipranavir with the signature V82L/T mutations--or with other mutations--did not make virus resistant to darunavir, according to a 39-sample analysis of people enrolled in the RESIST 1 or 2 tipranavir trials [1]. This finding by Boehringer investigators and Daniel Kuritzkes (Brigham and Women's Hospital, Boston) complements results of an earlier study showing that most people with tipranavir-susceptible virus retained susceptibility to tipranavir after darunavir failure [2]. Together these findings suggest that sequential therapy with tipranavir then darunavir, or vice versa, may be possible in certain people, though resistance testing before trying one of these PIs will remain essential.
Kuritzkes and Boehringer colleagues started with 62 matched viral PI-resistant isolates collected before tipranavir therapy and after virologic failure of a tipranavir regimen. They excluded 23 isolates because no protease mutations emerged during failure, because measured susceptibility to tipranavir did not change upon failure, because samples had mixtures of V82 mutant and nonmutant virus, or because samples had V82L/T before treatment with tipranavir.
Judging PI susceptibility with the Antivirogram assay, the researchers found that no isolates fully susceptible to darunavir before tipranavir therapy (fold-change in susceptibility less than 10) became resistant to darunavir (fold-change above 40) after tipranavir failure, regardless of which mutations arose. Among 19 isolates fully susceptible to darunavir at study entry, 3 (16%) were judged partially susceptible to darunavir after tipranavir failure, while 16 remained fully susceptible. Among 13 isolates partially susceptible to darunavir at baseline, 6 were fully susceptible at failure, 5 partially susceptible, and 2 resistant.
Median fold-change in susceptibility to tipranavir rose sharply upon virologic failure, as one would expect (Table 1). But susceptibility to darunavir or to other PIs generally remained the same or improved whether or not V82L/T emerged during failure (Table 1). The one exception was atazanavir, for which high pre-tipranavir resistance rose even farther when V82L/T appeared during tipranavir failure (Table 1).
Simpler score to predict response to tipranavir
With virologist colleagues, Boehringer researchers famously advanced a mutation score involving substitutions at 16 protease positions to predict response to tipranavir/ritonavir in PI-experienced people [3]. At the Retrovirus Conference Anne-Genevieve Marcelin (Pitie-Salpetriere Hospital, Paris) and colleagues at other centers detailed a score with substitutions at only five protease positions that may make predicting response easier [4]. Marcelin and coworkers cautioned, though, that their new mutation formula must be validated in other cohorts.
The new score rests on analysis of 143 people who had tried a median of four PIs (interquartile range three to five) and had a median of three major and nine minor mutations in protease according to the IAS-USA scheme. Everyone had a viral load above 1000 copies after at least 3 months of treatment with tipranavir/ritonavir and no other PIs.
Defining virologic response as at least a 1-log (10-fold) drop in viral load by treatment month 3 or a load below 200 or 50 copies at month 3, Marcelin counted 79 people (55%) with a virologic response to tipranavir/ritonavir. Mutations that appeared in more than 10% of pre-tipranavir genotypes and correlated with response at a P value less than 0.10 underwent further statistical analysis to pick the mutation cluster most strongly tied to virologic response. This statistical test correlated the mutations M36I/L/V, Q58E, H69I/K/N/Q/R/Y, and L89I/M/R/T/V with a worse virologic response and F53L/W/Y with a better response.
The earlier Boehringer score included three of those mutations, at positions 36, 58, and 69 [3]. Mutations at position 89 imperiled virologic response in a data set analyzing phenotype/genotype correlations [3]. And the F53L mutation turned up in a Virco analysis of mutations linked to tipranavir response [5].
Marcelin combined these mutations to yield a score of 36 - 53 + 58 + 69 + 89. All 7 people with a score of -1 were 3-month virologic responders, compared with about 80% of 33 people with a score of 0, 60% of 63 people with a score of 1, 40% of 24 people with a score of 2, 20% of 14 people with a score of 3, and neither of 2 people with a score of 4 (P = 1.47 x 10[-7]). Multivariate analysis determined that a mutation cutoff score of 2 correlated with virologic response at an odds ratio of 6.8 (P < 0.001). Previous enfuvirtide independently raised the risk of nonresponse 3.99 times (P = 0.0015), regardless of enfuvirtide use in the tipranavir regimen. Combining efavirenz with tipranavir independently lowered odds of virologic failure 86% (odds ratio 0.14, P = 0.035).
According to the Boehringer score, four or more mutations at the 16 protease positions mean virus is resistant or possibly resistant to tipranavir/ritonavir. Fewer than four mutations at those mutations predict the virus will be sensitive to tipranavir/ritonavir. Comparing the new score with the Boehringer score in 473 people with virologic failure in 2006, Marcelin found that the new score calls 74% of the viral samples sensitive to tipranavir and 26% resistant. The Boehringer score calls 68% of the same isolates sensitive to tipranavir and 32% resistant or possibly resistant. Thus the new score is simpler and eliminates the gray zone of "possible resistance." Lack of any major PI mutations in the new score, Marcelin proposed, may explain why tipranavir retains activity against viruses containing major mutations.
Besides Marcelin's group, Boehringer, and Virco, the FDA also recently proposed a pre-tipranavir resistance score to predict response to tipranavir/ritonavir [6]. Analyzing genotypes from the RESIST trials, the FDA found that five or more mutations at positions 13, 32, 36, 47, 58, 60, 82, and 84 foretold a worse 24-week response to tipranavir/ritonavir. People with five or more of these mutations and not combining enfuvirtide with tipranavir had a median 24-week viral load drop of 0.86 log, compared with more than a 1.5-log drop with five or more of these mutations in people who took both enfuvirtide and tipranavir.
Mutations at only one protease position, 36, overlap in the FDA and Marcelin recipes. These divergent results underline the difficulty of predicting response with mutation scores, which vary considerably with the patient populations and statistical methods used to derive them.
Fears quelled on amprenavir-darunavir cross-resistance
Because darunavir is chemically related to amprenavir, mutations that reduce susceptibility to one of these drugs also reduce susceptibility to the other. This mutational coincidence raised concerns that people in whom amprenavir or fosamprenavir failed would not respond to darunavir. But two conference studies allayed this worry.
Classifying 1631 viral samples as sensitive, partially sensitive, or resistant to amprenavir or darunavir based on lower and upper clinical cutoffs,[7] Monogram's Neil Parkin and Tibotec colleagues confirmed extensive overlap between mutations conferring resistance to these two PIs [8]. Darunavir and amprenavir shared 38 of 65 protease mutations, including all designated darunavir-linked mutations--V11I, V32I, L33F, I47V, I50V, I54L or M, G73S, L76V, I84V, and L89V. Nevertheless, all 305 samples partially sensitive to amprenavir retained full susceptibility to darunavir, and only 11% of 301 samples resistant to amprenavir proved resistant to darunavir. If D30N, I50L, or N88S appeared as the only major protease mutation, the affected viral sample had increased susceptibility to darunavir.
Parkin and coworkers proposed that the "predicted incidence of clinically meaningful cross-resistance [between amprenavir and darunavir] is low" because of "differences in clinical cut-offs, which are higher for darunavir." The apparently greater potency of darunavir than amprenavir in PI-experienced people, Parkin added, probably results from the 16-fold lower 50% inhibitory concentration of darunavir in the PhenoSense assay and the approximately 2-fold higher level of free darunavir in plasma. A unique darunavir cross-resistance profile appears not to explain the differing potency of the two PIs.
What Parkin and colleagues found in clinical samples of PI-treated people, Tibotec investigators found in 48-week results of people taking 600/100 mg of darunavir/ritonavir twice daily in the POWER 1, 2, and 3 trials: Pre-darunavir resistance to amprenavir or fosamprenavir had little impact on response to darunavir [9]. The same held true for people with resistance to lopinavir when they started darunavir.
The Tibotec study involved 449 POWER participants with VircoTYPE assay results and 438 with Antivirogram assay readouts. Gaston Picchio and Tibotec colleagues analyzed 48-week virologic responses to 600/100 mg of darunavir/ritonavir twice daily in six groups:
1. People using amprenavir or fosamprenavir when screened for a POWER study
2. People who used amprenavir or fosamprenavir at any time before darunavir
3. People using amprenavir or fosamprenavir when screened for a POWER study and having a VircoTYPE amprenavir fold-change in susceptibility above the clinical cutoff of 9.6
4. People with a VircoTYPE amprenavir fold-change in susceptibility above the clinical cutoff of 9.6
5. People with an Antivirogram amprenavir fold-change in susceptibility above 2.5
6. Everyone taking 600/100 mg of darunavir/ritonavir twice daily in a POWER study
Regardless of which method Tibotec used to rate pre-darunavir resistance to amprenavir or fosamprenavir, 48-week change in viral load and percentage of people with a 48-week load below 50 copies with darunavir did not vary from 48-week responses of all people taking the licensed darunavir/ritonavir dose. For example, a 48-week noncompleter-equals-failure analysis figured that 45% of all people taking 600/100 mg of darunavir/ritonavir had a viral load under 50 copies. Rates for darunavir takers in the five amprenavir-resistant groups defined above were (1) 38%, (2) 43%, (3) 36%, (4) 35%, and (5) 43%. In a similar way response to darunavir/ritonavir did not vary in five groups with exposure or resistance to lopinavir and the overall darunavir/ritonavir group at 48 weeks.
Selection of mutations conferring resistance to darunavir
Antiretroviral research veteran Hiroaki Mitsuya (US National Institutes of Health) and colleagues in Japan and the US used classic in vitro passage techniques to select protease mutations rendering virus resistant to darunavir [10]. Notably, though, the experiment worked only with a combination of viral samples already resistant to other PIs; it did not work when Mitsuya used wild-type (nonmutant) virus. The results confirm darunavir's high intrinsic barrier to resistance but suggest resistance could be a problem in PI-naive people who start darunavir/ritonavir then get superinfected with PI-resistant virus.
Exposing a wild-type HIV-1 strain to increasing concentrations of darunavir up to 0.1 micromol, Mitsuya and colleagues ran through 90 passages before mutations arose in protease. The mutations that did emerge, including R41I, L63P, and V82I, replicated poorly on their own--and replicated not at all when treated with about 0.1 micromol of darunavir.
Next Mitsuya exposed a mixture of eight multi-PI-resistant viruses to increasing doses of darunavir. This time the virus mixture became 170-fold resistant to darunavir after 39 passages at a darunavir dose of 1.0 micromol. At that point Mitsuya cataloged 14 PI mutations, including several cross-resistance classics (in bold): L10I, I15V, K20R, L24I, V32I, L33F, M36I, M46L, I54M, L63P, K70Q, V82A, I84V, and L89M. This multimutant virus replicated well despite darunavir treatment and proved cross-resistant to amprenavir, indinavir, nelfinavir, ritonavir, and lopinavir.
References
1. Elston RC, Kuritzkes DR, Bethell R. An investigation into the influence of the tipranavir-associated V82L/T mutations on the susceptibility to darunavir and brecanavir. 14th Conference on Retroviruses and Opportunistic Infections. February 25-28, 2007. Los Angeles. Abstract 602.
2. De Meyer S, Cao-Van K, Lathouwers E, et al. Phenotypic and genotypic profiling of TMC114, lopinavir and tipranavir against PI-resistant HIV-1 clinical isolates. 4th European HIV Drug Resistance Workshop. March 29-31, 2006. Monte Carlo. Abstract 42.
3. Baxter JD, Schapiro JM, Boucher CAB, et al. Genotypic changes in human immunodeficiency virus type 1 protease associated with reduced susceptibility and virologic response to the protease inhibitor tipranavir. J Virol. 2006;80:10794-10801.
4. Marcelin AG, Masquelier B, Descamps D, et al. Mutations associated to response to boosted tipranavir in HIV-1 infected protease inhibitor experienced patients. 14th Conference on Retroviruses and Opportunistic Infections. February 25-28, 2007. Los Angeles. Abstract 612.
5. Bacheler L, Vermeiren H, Winters B, et al. Clinically relevant phenotypic resistance and cross resistance to tipranavir among recent routine clinical isolates. 4th European HIV Drug Resistance Workshop. March 29-31, 2006. Monte Carlo. Abstract 40.
6. Naeger LK, Struble KA. Food and Drug Administration analysis of tipranavir clinical resistance in HIV-1-infected treatment-experienced patients. AIDS. 2007;21:179-185.
7. Coakley E, Chappey C, Benhamida J, et al. Defining the upper and lower phenotypic clinical cut-offs for darunavir/ritonavir by the PhenoSense assay. 14th Conference on Retroviruses and Opportunistic Infections. February 25-28, 2007. Los Angeles. Abstract 610.
8. Parkin N, Stawiski E, Chappey C, Coakley E. Darunavir/amprenavir cross-resistance in clinical samples submitted for phenotype/genotype combination resistance testing. 14th Conference on Retroviruses and Opportunistic Infections. February 25-28, 2007. Los Angeles. Abstract 607.
9. Picchio G, Vangeneugden T, Van Baelen B, et al. Prior utilization or resistance to amprenavir at screening has minimal effect on the 48-week response to darunavir/r in the POWER 1, 2, and 3 studies. 14th Conference on Retroviruses and Opportunistic Infections. February 25-28, 2007. Los Angeles. Abstract 609.
10. Koh T, Towata T, Ghosh A, Mitsuya H, Mitsuya H. Selection in vitro of HIV-1 variants highly resistant to darunavir using a mixture of HIV-1 isolates resistant to multiple PI. 14th Conference on Retroviruses and Opportunistic Infections. February 25-28, 2007. Los Angeles. Abstract 606.
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