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Antimicrobial Agents and Chemotherapy, April 2009, p. 1683-1685, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01168-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Apricitabine Does Not Select Additional Drug Resistance Mutations in Tissue Culture in Human Immunodeficiency Virus Type 1 Variants Containing K65R, M184V, or M184V plus Thymidine Analogue Mutations

Maureen Oliveira,¹ Daniela Moisi,¹ Bonnie Spira,¹ Susan Cox,^2* Bluma G. Brenner,¹ and Mark A. Wainberg¹

McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, 3755 Côte-Ste-Catherine Road, Montreal, Quebec QC H3T 1E2, Canada,¹ Avexa Ltd., 576 Swan Street, Richmond, Victoria 3121, Australia²

Received 1 September 2008/ Returned for modification 17 October 2008/ Accepted 30 January 2009

Top ABSTRACT INTRODUCTION REFERENCES

 
Human immunodeficiency virus type 1 containing the reverse transcriptase mutation M184V or K65R or mutations M41L, M184V, and T215Y did not accumulate additional resistance mutations in the reverse transcriptase when increasing amounts of apricitabine drug pressure were applied. The original mutations were maintained by the presence of apricitabine but were lost when cultured without drug pressure.

Top ABSTRACT INTRODUCTION REFERENCES

 
Apricitabine (ATC; previously AVX754, SPD754, and BCH-10618) is a deoxycytidine analogue nucleoside reverse transcriptase (RT) inhibitor (NRTI) that has shown good activity in vitro against both wild-type human immunodeficiency virus type 1 (HIV-1) and HIV-1 containing RT mutations that confer resistance to other NRTIs, e.g., M184V and thymidine analogue mutations (TAMs) (1, 6, 8, 12, 19). Clinical trials have demonstrated the promising activity and safety of ATC in both treatment-naïve and treatment-experienced HIV-1-infected patients; ATC is effective at reducing viral load and increasing CD4 cells in treatment-experienced patients with HIV-1 with the M184V mutation and with or without additional TAMs (3, 4, 5). Resistance to ATC is slow to develop. In vitro studies found that ATC selected M184V, V75I, or K65R in separate experiments from a wild-type background following 18 to 20 serial drug passages of this drug, with the resulting virus showing, respectively, only a 1.1-, 1.6-, and 3.6-fold change in susceptibility to ATC (11). The aim of this study was to investigate whether HIV-1 that already contains the M184V or K65R mutations, or M184V plus TAMs, could develop additional resistance mutations when challenged with ATC drug pressure in vitro.

The ability of ATC to select resistance mutations was examined using a method similar to that described by Petrella et al. (15). Recombinant HXB-2D viruses containing the M184V or K65R mutation or the M184V, M41L, and T215Y mutations were prepared by site-directed mutagenesis. Cord blood mononuclear cells were infected with the test virus and plated into 24-well plates using a starting concentration of ATC that was fivefold lower than the 50% inhibitory concentration (IC₅₀). The cells were cocultured with fresh donor cells each week, and the ATC concentration was increased in two- or 2.5-fold increments at each passage unless the pressure on viral survival was too great, in which case the drug concentration was not increased. Emerging viral variants were sequenced at key time points to determine any genotypic changes, using the TRUGENE HIV-1 genotyping test and OpenGene automated DNA sequencing system (Siemens Diagnostics, Toronto, Canada). The presence of a viral population containing mutations that were present at a level of 20% or more of the total viral population were identified. The IC₅₀s for the HXB-2D wild-type and mutant viruses are shown in Table 1.

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TABLE 1. Baseline IC50s of HXB-2D wild-type virus and mutant virusesa

The M184V mutation is rapidly selected by the deoxycytidine analogue lamivudine and is associated with high-level resistance to lamivudine and emtricitabine and low-level resistance to didanosine, zalcitabine, and abacavir (2, 10, 17, 20, 21). Culture of HXB-2D virus containing M184V in increasing concentrations of ATC up to 60 µM (week 38) maintained this mutation (Table 2), consistent with previous observations (15). Maintenance of M184V by ATC has also been observed clinically in treatment-experienced patients infected with HIV-1 with the M184V mutation; after 21 days of functional monotherapy with ATC, the M184V mutation was maintained in all 24 patients receiving ATC whose virus could be genotyped; at week 12, M184V was maintained in the majority of patients receiving ATC (in combination with optimized background antiretroviral therapy) in whom a genotype could be obtained (4, 7).

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TABLE 2. Effect of increasing ATC drug pressure on HXB-2D virus with the M184V mutation

Passage of the M184V-containing virus under ATC drug pressure did not result in the appearance of any new NRTI resistance mutations (Table 2). Polymorphism changes in the M184V-containing virus during culture included F87L, V106I, D177N, and E194G, but no evidence that any of these substitutions had any significant effect on drug IC₅₀s was obtained. Culturing the M184V-containing virus without ATC drug pressure led to the loss of the M184V mutation (Table 2).

The K65R mutation can be selected by abacavir, didanosine, stavudine, and tenofovir and is associated with decreased susceptibility to these drugs and with decreased replication capacity (9, 13, 22, 24). Culturing the K65R-containing virus in increasing concentrations of ATC up to 60 µM (week 38) maintained the K65R mutation and did not result in any new NRTI resistance mutations (Table 3). At week 17 (10 µM ATC) and week 38 (60 µM ATC), some viruses contained the S68G mutation that is thought to partially compensate for K65R by improving viral replication capacity; thus, its appearance under ATC drug pressure is likely to be related to the K65R-associated impairment of replicative capacity (16, 18). When cultured without ATC drug pressure, the K65R mutation was lost (Table 3).

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TABLE 3. Effect of increasing ATC drug pressure on HXB-2D virus containing the K65R mutation

TAMs are selected by and cause decreased susceptibility to the thymidine analogues zidovudine and stavudine. Accumulation of TAMs progressively decreases HIV susceptibility to all NRTIs (23). When viruses containing M184V plus the TAMs M41L and T215Y were grown in increasing ATC concentrations (up to 50 µM ATC; week 28), no new NRTI resistance mutations emerged (Table 4). The M184V, M41L, and T215Y mutations were all maintained in the presence of ATC drug pressure. When viruses were cultured without ATC drug pressure and assessed at weeks 32 and 53, partial reversion of the M184V mutation and a C/Y mixture at position 215 were observed, while the M41L mutation was maintained (Table 4). An E/K mixture was observed at position 224 at week 32 and an I/V mixture was observed at position 108 at week 53 (Table 4). Antagonism between the TAM and K65R pathways may have some bearing on the outcome of ATC selection studies (14).

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TABLE 4. Effect of increasing ATC drug pressure on HXB-2D virus with the M184V, M41L, and T215Y mutations

In a parallel experiment to select ATC-resistant variants with wild-type HXB-2D, we were obliged to increase both the concentration of the drug and the duration of the study in order to successfully generate mutant viruses (Table 5). The M184V mutation emerged initially, followed by K65R, which eventually became the dominant variant, probably due to the increased fitness of K65R-containing viruses compared with M184V-containing viruses. These findings show that ATC pressure on a naïve virus results in the emergence of both M184V and K65R variants which compete for survival. However, when either of these mutations is present individually prior to initiation of drug pressure, there is little chance that other minority species will develop and challenge the dominant variant.

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TABLE 5. In vitro selection of HXB-2D wild-type recombinant clone for resistance with ATC drug pressure

These findings, suggesting a low propensity of ATC to select NRTI resistance mutations in vitro, are similar to data obtained in clinical trials. In a phase 2b study of ATC in treatment-experienced patients infected with HIV-1 with an existing M184V mutation, with or without additional TAMs, there were few changes in genotype in patients treated with ATC for 21 days as functional monotherapy or for up to 48 weeks in combination with background-optimized antiretroviral therapy (4, 7; data not shown).

Our study found that sustained ATC drug pressure for up to 38 weeks did not result in any additional known NRTI resistance mutations in HIV-1 viruses with the M184V, K65R, or M41L M184V T215Y mutations. In addition, these resistance mutations were maintained under sustained ATC drug pressure but were mostly lost in the absence of drug pressure. ATC therefore appears to maintain mutations in the HIV-1 RT associated with reduced viral fitness, such as M184V, while not selecting additional resistance mutations. The continued presence of mixtures of mutant and wild-type variants in the TAM recombinant is most likely a reflection of the difficulty for the virus to change at many sites compared to the occurrence of single-point mutations. These results extend previous studies of the efficacy of ATC against HIV-1 with M184V and TAMs and provide support for ATC as a potential treatment option for HIV-1-infected patients with NRTI resistance.

 
This work was supported by the Canadian Institutes of Health Research, and the study was sponsored by Avexa Ltd.

 
* Corresponding author. Mailing address: 576 Swan St., Richmond, Victoria 3121, Australia. Phone: 61 3 9208 4300. Fax: 61 3 9208 4004. E-mail: scox{at}avexa.com.au

Published ahead of print on 17 February 2009.

Top ABSTRACT INTRODUCTION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, April 2009, p. 1683-1685, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01168-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Oliveira, M. Articles by Wainberg, M. A. Search for Related Content PubMed PubMed Citation Articles by Oliveira, M. Articles by Wainberg, M. A.