The morbidity and mortality of HIV infection and AIDS have dramatically decreased since the introduction of highly active antiretroviral therapy (HAART), now commonly referred to as combination antiretroviral therapy (cART). Thus, HIV infection has become a manageable chronic infectious disease. This significant clinical advancement has resulted from progress in the discovery and development of anti-HIV drugs that exploit novel mechanisms of action, improve efficacy and durability with less inherent toxicity, and are amenable to more convenient dosing regimens for patients. However, there still exist unmet medical needs, including those for patients who have experienced treatment failure, for prevention of transmission via a preexposure prophylaxis (PrEP) approach, and for patients with difficulties in maintaining adherence. The reasons for virologic failure can be complex but are often due to intrinsic characteristics of a drug, which may include the emergence of drug-resistant mutants and can in turn be exacerbated by low drug levels due to pharmacokinetic or adherence issues.
The first integrase strand transfer inhibitor (INSTI), raltegravir (RAL) (MK-0518) (1
), was approved by the U.S. FDA in 2007. The second INSTI, elvitegravir (EVG) (GS-9130, formerly JTK-303), was approved in 2012 as a component of “Stribild,” which is a fixed-dose combination of EVG, cobicistat, tenofovir disoproxil fumarate, and emtricitabine (3
). INSTIs are now recognized as a safe and highly effective class of anti-HIV drugs (6
). However, clinical resistance to RAL and EVG has been observed, and a high degree of cross-resistance between these two agents has been demonstrated (7
). Furthermore, dosing of RAL is done twice daily, while once-daily administration of EVG requires a pharmacokinetic (PK) booster, such as ritonavir or cobicistat. Because these two agents are inhibiters of drug metabolism, their use raises concerns about the levels of concomitantly used drugs. Ritonavir also raises long-term safety concerns (13
). Therefore, new INSTIs should have attributes that address these unmet needs.
The Shionogi and GlaxoSmithKline research collaboration initiated in 2002 has made considerable progress in engineering INSTIs with a distinct resistance profile and low-dose once-daily unboosted regimens. Dolutegravir (DTG) (S/GSK1349572, brand name TIVICAY) was approved by the U.S. FDA in 2013, and GSK1265744 (formerly S/GSK1265744), with the generic name cabotegravir (USAN approved), is in phase 2 clinical trials. They contain a two-metal binding pharmacophore consisting of a carbamoyl pyridone moiety (see Fig. 1
) and were optimized to deliver the attributes that would differentiate them as new INSTIs (14
). Clinical data for GSK1265744 administered to healthy subjects and HIV patients present a PK profile supporting once-daily oral administration from a low-milligram dose, with low PK variability and excellent short-term safety/tolerability, as well as highly effective anti-HIV activity from 10 days of monotherapy (16
The focus of the development of GSK1265744 was a long-acting (LA) injectable formulation due to its favorable attributes: low solubility (0.015 mg/ml in pH 6.8 phosphate buffer at 20°C) and low plasma clearance (0.32 ml/min/kg of body weight in monkeys) for HIV treatment and prevention. The data from a phase 1 single-dose GSK1265744 LA study in healthy volunteers supported once-monthly to once-quarterly dosing (17
). Recently, a repeat-dose coadministration study of GSK1265744 LA and TMC278 LA in healthy subject results was described (19
). LA development is attractive for both treatment and use as a preventative measure in a preexposure prophylaxis (PrEP) setting. Although Truvada (emtricitabine and tenofovir disoproxil fumarate) was approved by FDA for PrEP, a PrEP clinical study data of Truvada suggested that daily compliance was a complicating factor for healthy high-risk individuals, ultimately leading to reduced efficacy of the approach (20
We report herein the in vitro antiretroviral properties of GSK1265744, including its mechanism of action and in vitro resistance characteristics essential for clinical development.
INSTIs are generally recognized as a safe and effective class of anti-HIV drugs. DTG, RAL, and EVG have all been recommended as components for first-line therapy regimens in the most recent U.S. DHHS guidelines (13
). Clinical data indicate that viruses resistant to the earlier INSTIs (RAL and EVG) have arisen (2
). DTG and GSK1265744 are clearly differentiated as new INSTIs. They are highly effective against both wild-type and most INSTI-resistant mutants that have been identified to date. DTG has been clinically demonstrated with significantly less treatment-emergent INSTI or background therapy resistance than RAL in the SAILING study through 48 weeks (33
). Thus, DTG could have a higher barrier to resistance clinically than RAL. GSK1265744 has in vitro
characteristics similar to those of DTG which support its potential for having a high barrier to resistance. Herein, we define “high barrier to resistance” in vitro
as a mutant virus which has more than a 3-fold change as SDM not emerging through more than 100 days in our in vitro
passage study. While the chemical structure of GSK1265744 is related to that of DTG, GSK1265744 has unique attributes (high protein binding, low solubility, and low plasma clearance in animals). Consequently, the human oral PK data for GSK1265744 indicate a longer half-life (t1/2
) of approximately 32 h) (16
). Preliminary evaluation of GSK1265744 LA in animals suggested a possibility of parenteral once-monthly dosing in humans or possibly less frequently. Based on its resistance and pharmacokinetic profiles, GSK1265744 is in full clinical development as an LA formulation along with an oral tablet for use in the induction phase.
The potency shift for GSK1265744 with 100% human serum is 408-fold, which is consistent with a highly protein-bound molecule. Of note is the fact that achieving plasma concentrations above the PA-EC90
was still anticipated due to PK measurements (16
) and high intrinsic antiviral efficacy. The clinical efficacy of GSK1265744 was demonstrated in a 10-day monotherapy trial at doses of 5 and 30 mg once daily (16
); PK measurements showed that trough concentrations above the PA-EC90
were achievable with these very low oral doses.
The in vitro
experiments described here confirmed that the mechanism of action of GSK1265744 is through inhibition of HIV IN at the strand transfer step of viral DNA insertion into host DNA. The inhibitor had no effect on total viral DNA synthesis in the infected cells but blocked the integration of viral DNA into host DNA with the same potency as its antiviral effect (Fig. 2
). In addition, GSK1265744 increased the appearance of viral 2-LTR circles, a known by-product of integrase inhibition with the same potency as other INSTIs (14
). As expected, GSK1265744 had efficacy against mutant viruses resistant to NRTIs, NNRTIs, and PIs, similar to its potency against the wild-type virus and consistent with its acting on a different antiretroviral target. GSK1265744 maintained activity against single and several multiple IN mutants but had decreased efficacy against a small subset of multimutated viruses resistant to other INSTIs (Table 2
). In vitro
combination studies confirmed that GSK1265744 has no antagonistic effect on all measured anti-HIV drugs across all classes, including likely combination agents in the NRTI and NNRTI classes.
It is striking that GSK1265744 had significant efficacy against most double or multiple integrase mutants in our panel of 24 mutants; only 6 mutant viruses (the E138A/Q148R, E138K/Q148K, E138K/Q148R, G140C/Q148R, G140S/Q148R, and Q148R/N155H viruses) had a FC of >10 (Table 2
). This profile is similar to that of DTG, for which only 2 such mutants had a FC of >10. In contrast, RAL and EVG showed FC values of >10 with 20 and 22 such mutants, respectively. Therefore, we conclude that GSK1265744 has a new resistance profile similar to that of DTG.
Importantly, GSK1265744 showed potency against 25 INSTI-resistant single mutants in our panel of INSTI-resistant SDMs that was approximately equivalent to that of the wild type. The Q148 pathway confers a high level of resistance to RAL and EVG. The Q148K (FC = 5.6) and Q148R (FC = 5.1) mutants gave rise to a moderate loss of efficacy for GSK1265744. Given this moderate loss, we were curious as to whether Q148K/R would arise in a passage study. Neither mutant was observed. From these results, we surmise that the potential of resistance to GSK1265744 emerging may be very rare during clinical use for INSTI-naive patients.
Under our isolation conditions, the lamivudine-resistant M184V virus emerged as expected on day 14 (14
). Our RAL data suggested that there was not sufficient pressure below 6.4 nM RAL to effectively select resistant viruses. However, above that concentration, RAL-resistant mutants were isolated, and the minimum culture period required for resistance to RAL to develop was 28 days under our experimental conditions.
In the case of GSK1265744, four mutant viruses (the Q146L, S153Y, T124A/S153Y, and T124A/I162Mvariants) were isolated (Table 3
). Of note is the fact that T124A is polymorphic and has no effect on GSK1265744 activity and that it was present in the starting inoculum of the passage virus (35
). However, as mentioned above, no Q148K/R or any other RAL/EVG signature mutant virus was isolated up to 112 days. Among the four isolated mutant viruses, we confirmed that three site-directed molecular clones maintained sensitivity to GSK1265744 (FCs were less than 2.9) (Table 2
), although the T124A/I162M virus could not be evaluated because of its low replication capacity. These data were consistent with the fact that we could not maintain viruses in a GSK1265744 concentration at 32 nM (Fig. 3a
). Interestingly, the Q146L virus was a new resistant variant for EVG (FC = 13), although Q146P has been reported (36
The GSK1265744 data herein are consistent with our definition of a high barrier to resistance in vitro
. However, the high barrier to resistance ultimately must be proven in controlled clinical trials. In a clinical situation, there are many factors to consider, such as the drug plasma trough concentration (Ctau
), drug binding affinity for the IN enzyme, and adherence to drug therapy. One of the key factors is the inhibitory quotient (IQ), which is defined as follows: IQ = Ctau
. In the case of DTG, the mean Ctau
for a 50-mg once-daily dose measured in the SPRING-1 and SPRING-2 treatment-naive studies was 1.1 μg/ml (reference, Tivicay package insert); therefore, the IQ was 17. For GSK1265744, the 30-mg oral daily dose gave a geometric mean Ctau
of 3.28 μg/ml, for an IQ of 19.8 (16
). The IQ at 4 weeks after GSK1265744 LA 800-mg single-dose intramuscular injection was approximately 12 (16
). Thus, because the IQ for GSK1265744 is similar to that of DTG, which has a proven clinically high barrier to resistance (33
), in conjunction with its encouraging in vitro
resistance profile, we expect little if any clinical failure due to resistance for the GSK1265744 treatment regimens planned.
Recently, Quashie et al. reported that R263K and G118R were isolated in vitro
as potential DTG resistant viruses (40
). Also, although fold changes were less than 2 in a phenotype assay with isolated viruses, the R263K and R263R/K variants were identified from two DTG-treated virologic failure patients (33
). Therefore, we evaluated the activity of GSK1265744 against these mutant viruses. Our preliminary data are that the fold change against the R263K variant was 1.3 ± 0.49 (n
= 3) and the fold change against the G118R variant was 8.9 ± 0.86 (n
= 3). We concluded that the R263K virus is not a resistant mutant for GSK1265744 and the G118R variant is a potential resistant mutant. However, the possibility of emergence of G118R during clinical use might be very low because the replication capacity of G118R virus is very poor (41
In conclusion, GSK1265744 has a markedly more distinct profile than RAL and EVG in terms of cross-resistance and in vitro resistance passage studies. These characteristics in combination with its excellent antiviral efficacy and safety profile, as well as suitable PK from both oral and LA injection regimens, should provide a novel treatment option for patients. Taken together, the attributes of GSK1265744 may combine to allow for durable efficacy in difficult-to-treat patient populations, with improvement of compliance and quality of life, and provide an attractive new option for both treatment and PrEP indications.