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The Activity of the Integrase Inhibitor Dolutegravir Against HIV-1 Variants Isolated From Raltegravir-Treated Adults

Underwood, Mark R. PhD*; Johns, Brian A. PhD*; Sato, Akihiko PhD; Martin, Jeffrey N. MD; Deeks, Steven G. MD§; Fujiwara, Tamio PhD

JAIDS Journal of Acquired Immune Deficiency Syndromes: November 1st, 2012 - Volume 61 - Issue 3 - p 297–301
doi: 10.1097/QAI.0b013e31826bfd02
Clinical Science

Background: Dolutegravir (DTG, S/GSK1349572) is an integrase inhibitor with low nanomolar potency. Susceptibility to dolutegravir and raltegravir was determined for raltegravir-resistant clinical isolates.

Methods: Genotypic and phenotypic susceptibility to integrase inhibitors was examined using 39 clinical isolate samples obtained from 18 adults who had exhibited incomplete viral suppression on a raltegravir-based regimen.

Results: Of 39 samples evaluated, 30 had genotypic and phenotypic resistance to raltegravir. All samples lacking raltegravir resistance retained complete susceptibility to dolutegravir. Of the 30 samples with genotypic evidence of raltegravir resistance, the median level of phenotypic resistance to raltegravir was high (median fold change in inhibitory concentration at 50%, >81; range, 3.7 to >87), while the level of resistance to dolutegravir was close to that of wild-type variants (median fold change, 1.5; range, 0.9–19.0). Longitudinal samples from 5 subjects collected during long-term failure of raltegravir revealed time-dependent general decreases in phenotypic susceptibility to raltegravir, with minimal changes in phenotypic susceptibility to dolutegravir. The median fold change to dolutegravir for isolates containing changes at G140S + Q148H, G140S + Q148R, T97A + Y143R, and N155H (thus including raltegravir signature resistance codons) were 3.75, 13.3, 1.05, and 1.37, respectively.

Conclusions: Dolutegravir retained in vitro activity against clinical isolates obtained from subjects who failed raltegravir-based therapy at near wild-type levels for variants containing the Y143 and N155 resistance mutations. Isolates with Q148 plus additional integrase mutations possessed a broader range of and more reduced susceptibility to dolutegravir.

*Infectious Diseases Therapy Area Unit, Medicines Discovery & Development, GlaxoSmithKline, Research Triangle Park, NC

Medicinal Research Laboratories, Shionogi & Co., Ltd, Osaka, Japan

Department of Epidemiology and Biostatistics. University of California, San Francisco, San Francisco, CA

§Positive Health Program, Department of Medicine, San Francisco General Hospital, San Francisco, San Francisco, CA

Global Development, Shionogi & Co., Ltd, Osaka, Japan.

Correspondence to: Mark R. Underwood, PhD, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709 (e-mail:

Supported by the National Institutes of Health Grants P30 AI027763, UL1 RR024131, and R24 AI067039 and by Shionogi-ViiV Healthcare LLC.

M.R. Underwood and B.A. Johns are employees of GlaxoSmithKline. A. Sato and T. Fujiwara are employees of Shionogi & Co., Ltd.

Portions of these data were presented at the 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention; July 19–22, 2009; Cape Town, South Africa (abstract WEPEA098).

M.R. Underwood and B.A. Johns are employees of GlaxoSmithKline. A. Sato and T. Fujiwara are employees of Shionogi & Co., Ltd. The remaining authors have no conflicts of interest to disclose.

Received April 10, 2012

Accepted July 26, 2012

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With the registration of raltegravir in 2007, the HIV-1 integrase enzyme became the most recent drug target for which an antiretroviral drug has been approved. As with all antiretroviral drug classes, the long-term in vivo utility of integrase inhibitors is limited by the development of drug resistance. The genotypic and phenotypic characteristics of raltegravir resistance have been well described. In clinical studies of raltegravir, subjects with virologic failure and reduced integrase inhibitor susceptibility typically harbor virus with one of 3 signature mutational pathways: Y143, Q148 (typically Q148H in combination with G140S), or N155.1–4

Although many characteristics of a drug class can improve as new agents emerge, a key feature for the expanded utility of new drugs against an existing target is a substantially different resistance profile. Dolutegravir (DTG, S/GSK1349572) is a novel integrase inhibitor with a distinct in vitro resistance profile that includes substantial activity against HIV with Y143 or N155H plus secondary raltegravir-associated mutations or against the Q148 mutations alone; a broader activity range is observed for viruses with Q148 pathway genotypes with fold change (FC) resistance generally increasing as the number of secondary mutations increases.5,6 Among integrase inhibitor–naive subjects, dolutegravir taken once daily has exhibited potent antiretroviral activity in phase 1/2 studies.7,8 Dolutegravir has demonstrated that a predictable well-characterized exposure-response relationship and low pharmacokinetic variability does not require a pharmacokinetic boosting agent9 and is currently in phase 3 clinical development. On the basis of extensive experience testing the phenotypic susceptibility of drugs from other antiretroviral drug classes, it is expected that the dolutegravir phenotypic susceptibility of samples from subjects exhibiting virologic failure on raltegravir will predict how well this novel drug will work in raltegravir-experienced subjects. In this report, we describe our initial investigation of the activity of dolutegravir against clinical isolates from subjects experiencing virologic failure while on raltegravir therapy.

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HIV Isolates

The HIV-1 samples evaluated were from 18 adults and included 8 clinical isolates containing integrase inhibitor resistance mutations from a Monogram Biosciences, Inc (South San Francisco, CA) library set and 31 clinical isolate samples from the University of California, San Francisco Study of the Consequences of Protease Inhibitors Era (SCOPE) cohort.10 SCOPE is an observational prospective study of HIV-1–infected adults designed to provide a specimen bank of samples with carefully characterized clinical data. Of 39 clinical isolate samples examined, 30 had integrase coding region mutations, and 21 of these were longitudinal samples from 9 subjects. All 8 clinical isolate samples obtained from Monogram Biosciences had evidence of raltegravir resistance, while 22 of the SCOPE samples had evidence of raltegravir resistance. In addition, we studied 11 site-directed mutant control HIV-1 samples (integrase sequences based on NL43).

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Viral Genotyping and Phenotyping Assays

Integrase-resistant HIV-1 sample phenotypes were evaluated using the PhenoSense IN assay at Monogram Biosciences, Inc.2,11,12 Dolutegravir and raltegravir were tested side by side, and inhibitory concentration at 50% (IC50) and FC-IC50 versus wild type were generated. Briefly, 1.6 kb of the HIV-1 pol sequence containing the C-terminal domain of reverse transcriptase, RNase H, and integrase was amplified from subject plasma by reverse transcriptase polymerase chain reaction and transferred into a resistant test vector containing a luciferase reporter gene. Cotransfections of HEK293 cells with integrase-specific resistant test vectors and an amphotropic murine leukemia virus envelope expression vector were performed to produce pseudovirus stocks that contain patient-derived integrase sequences. Virus stocks were used to infect fresh HEK293 cells in the absence or the presence of serial dilutions of the integrase inhibitor test compounds. Susceptibility was calculated by plotting the percent inhibition of virus replication (luciferase activity) versus the log10 drug concentration to derive the IC50. The integrase genotypes of isolates were determined using the GeneSeq IN assay (Monogram Biosciences, San Francisco, CA).

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The median IC50 for dolutegravir against 9 wild-type isolates was 1.07 nM (range, 0.8–1.6 nM) compared with 9.15 nM for raltegravir (range, 5.2–13.6 nM). Phenotypic resistance to raltegravir and dolutegravir is reported here as the FC-IC50 of the test sample compared with the wild-type sample. The FC for representative raltegravir-resistant clinical isolate virus compared with wild-type HIV-1 is presented in Table 1. Dolutegravir has essentially wild-type levels of activity against the N155H (median FC-IC50, 1.37) and the T97A + Y143R mutants (median FC-IC50, 1.05). Dolutegravir susceptibility is diminished by HIV-1 isolates carrying G140S + Q148H (median FC-IC50, 3.75) and is further diminished by isolates containing G140S + Q148R (median FC-IC50, 13.3). Overall, for the 30 clinical isolate samples with integrase coding region mutations examined, the dolutegravir FC-IC50 ranged from 0.9 to 19.0; the raltegravir FC-IC50 ranged from 3.7 to >87 (a maximum within assay measureable FC-IC50 for raltegravir). As previously reported,2 resistance to raltegravir is moderate with N155H (median FC-IC50, 19.0) and high with the other representative genotypes (median FC-IC50, >87 for the combinations of G140S + Q148H, G140S + Q148R, and T97A + Y143R).



Longitudinal samples from 5 subjects receiving raltegravir plus optimized background therapy were analyzed for changes in susceptibility to dolutegravir and raltegravir (Fig. 1). Susceptibility to dolutegravir remained at virtually wild-type levels throughout, whereas raltegravir resistance emerged, with some fluctuation, over time.



The FC-IC50 of dolutegravir and raltegravir against integrase site-directed mutants and clinical isolates were examined for a more thorough profile of the susceptibility of specific mutant pathways. As shown in Figure 2A, for all single mutants examined, dolutegravir maintained activity to a greater extent than raltegravir (FC-IC50 range for dolutegravir, 0.51–2.45; FC-IC50 range for raltegravir, 1.81–36), with the exception of S153Y; against S153Y, dolutegravir had an FC-IC50 of 2.45 and raltegravir had an FC-IC50 of 1.81. Raltegravir had an FC-IC50 >5 for all single mutants except S153Y. Isolated mutations at the Q148 position (generally considered the most significant pathway for raltegravir resistance) seemed to confer small but measurable decreases in susceptibility to dolutegravir. As shown in Figure 2B, dolutegravir had near wild-type activity against isolates examined with ≥2 mutations and without 148H/K/R (range, 0.87–2.25), whereas raltegravir had an FC-IC50 >5 (range, 3.74 to >81) for all but one (T97T/A + N155N/H; FC-IC50 of 3.74) of this set of mutants. Dolutegravir maintained activity across the isolates examined with combined 140S and 148H/R to a greater extent (FC-IC50 range, 1.38–19.0) than raltegravir (FC-IC50 range, 3.74 to >87; Fig. 2C). Dolutegravir maintained activity to a greater extent across all isolates examined with the grouping of 138K + 148K/R or with ≥3 mutations (FC-IC50 range, 1.58–6.9) than raltegravir (FC-IC50 range, 12 to >81; Fig. 2D).



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Although raltegravir has exhibited substantial efficacy in clinical trials, it is expected that over time a significant number of individuals in clinical practice will exhibit incomplete viral suppression on this drug and will generate raltegravir-resistant variants. Hence, there is an obvious need for drugs in the integrase inhibitor class that retain activity against isolates containing clinically relevant raltegravir-associated mutations. Elvitegravir (EVG, GS-9137), another integrase inhibitor that is in clinical development, has a resistance profile similar to that of raltegravir.5,13–16 Preliminary cross-resistance data for the prototype second-generation integrase inhibitor MK-2048, designed to retain activity against HIV with resistance to raltegravir, indicate that it has a distinct resistance profile17–19; however, MK-2048 remains in very early clinical development. In vitro data have demonstrated that dolutegravir retains substantial activity against Y143 and N155H pathway virus with additional secondary mutations and against virus with Q148 mutations alone.5 Dolutegravir activity has a broader range of FC resistance against Q148 pathway virus with additional raltegravir secondary mutations; resistance generally increases with increasing number of mutations.5,6 Using clinically derived samples, we report that dolutegravir often retains full or near-full activity against variants that possess genotypic and phenotypic resistance to raltegravir. This is particularly true for isolates containing common raltegravir-associated mutations at positions 143 and 155 in the integrase open-reading frame. Single mutations at Q148 apparently conferred small but measurable susceptibility to dolutegravir. Isolates containing combined mutations at 140 and 148 have less susceptibility to dolutegravir than those isolates with mutations at positions 143 and 155, although in both cases, there is substantially greater susceptibility to dolutegravir than to raltegravir.

From a theoretical perspective, the problem of cross-resistance within the class of strand-transfer–specific integrase inhibitors has been considered a major concern because of the overlapping binding orientation of key pharmacophore elements in the integrase active site of the integrase inhibitors currently in development.20 However, a close structural comparison of the scaffolds for raltegravir, elvitegravir, and dolutegravir indicates that dolutegravir has a more “streamlined” scaffold.21,22 A comparison of the position of raltegravir, elvitegravir, and dolutegravir within the catalytic pocket of an HIV-1 model demonstrates that dolutegravir occupies less space between the Mg2+ metals at the base of the catalytic pocket and the Y143 at the top of the catalytic loop compared with both raltegravir and elvitegravir.21–23 This model renders dolutegravir more independent of the signature mutations that typically impact both of these integrase inhibitors. Overall, the data reported here indicate that although dolutegravir FC resistance is substantially lower than that of raltegravir for all clinical isolates obtained from subjects failing raltegravir-based therapy, there is a decrease in dolutegravir susceptibility for some HIV-1 virus, particularly with mutations at Q148 plus at least one additional raltegravir secondary mutation.

These data, combined with other recent virologic data,5,6,24 suggest that dolutegravir has a virologic resistance profile distinct from that associated with raltegravir, with some mutation combinations that have a greater decrease in dolutegravir activity. When combined with the potent virologic responses observed during 10-day dolutegravir monotherapy given once daily7 and the good efficacy rates and rapid virologic responses observed in a phase 2b study of antiretroviral-naive subjects,8 these data suggest that there is a need to continue to study dolutegravir in subjects who exhibit raltegravir resistance and to support the development of dolutegravir for subjects across the treatment spectrum.

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All listed authors meet the criteria for authorship set forth by the International Committee for Medical Journal Editors. The authors would like to thank the subjects from the SCOPE study. The authors wish to acknowledge the following individuals for their editorial assistance during the development of this manuscript: Christine Levesque and Chris Lawrence.

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Dolutegravir; DTG; S/GSK1349572; integrase inhibitor; raltegravir resistance; UCSF SCOPE cohort

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