Recent Advances And Impact On Treatment Biology Essay


Research and development of more than 20 antiretroviral drugs leads to efficient control of Human immunodeficiency virus 1 replication. Recent research has identified new compounds that could be used to target cellular entry and nuclear integration of virus in addition to common targets reverse transcriptase and protease. These additional targets improved the combination antiretroviral therapy (cART) approaches that added new dimension to our fight against HIV by improving the armamentarium especially when deals with emergence drug resistant HIV. Cellular entry of HIV is a multistep procedure involving a range of cellular and molecular interaction between virus envelope protein and receptors expressed on the surface of the target cells, thus providing many opportunities to block infection. Some of these entry inhibitors are currently being used in the clinic, further more compounds are under various stages of development. Entry inhibitors interfere with the function of the highly variable envelope glycoprotein as it continuously adapts to changing immune pressure and available target cells in the extracellular environment. In this review, we focused on fusion inhibitory peptides that have been developed since the first HIV-1 fusion inhibitor, enfuvirtide (T-20). T-20 is able to suppress HIV-1 replication that could even act against viruses resistant to reverse transcriptase or protease inhibitors. Integration of the HIV-1 DNA is required and essential to maintain the viral DNA in the infected cell. The design and discovery of integrase inhibitors were first focused at targeting the catalytic site of integrase that selectively acting on strand transfer. Integrase inhibitors that were developed clinically so far include two first generation inhibitors, raltegravir and elvitegravir and later two second-generation inhibitors, dolutegravir and MK-2058. In addition, allosteric integrase inhibitors that could interfere with the integrase-LEDGF/p75 interaction also have been designed. Thus, entry and integrase inhibitors present a real added value in combined treatment against HIV infection. In this review, we are providing a summary of recent development of future science in the discovery of inhibitors of HIV entry and integration in for the use in clinical settings.

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Introduction: The entry of HIV-1 into susceptible target cells and its integration to host cell genome are multistep process that leads to the fusion of viral and cell membranes and ultimately productive infection and latency development. Antiretroviral drugs that interact with each step in the HIV-1 entry process have been developed, but only two compounds (maraviroc and enfuvirtide) are currently approved for the usage in clinics. The integrase enzyme facilitates the incorporation of HIV-1 proviral DNA into the host cell genome and catalyses a function vital to replication of the virus. Inhibitors of integrase inhibitors are the newest class of antiretroviral drugs in our effort to treat HIV-1infection. Raltegravir, an integrase strand transfer inhibitor, was the first drug of this class approved by the US FDA; it is a potent and well tolerated antiviral agent. However, numerous limitations exist in the usage of these newly developed compounds mostly depends on its daily dosing, adverse events and genetic barrier to the development of resistance. There is an urgent need for continuing research for developing agents minimal with once daily dosing, a more robust barrier to resistance, and a resistance profile of limited overlap with each other. Given the potential for these agents to block viral entry and itegration, there has been increased interest in using them to prevent acquisition and establishment of HIV-1 infection. Here we summarize progress in the development of HIV-1 entry and integration inhibitors, with an emphasis on molecules in different stages of clinical development and information about its patents. We review a series of HIV entry and integrase inhibitors that are in clinical or advanced pre-clinical studies in humans. Each of these contributes a new benefit to the class and will extend the treatment options for patients with HIV-1 infection.

HIV-1 entry: Binding of the viral envelope to its primary receptor, CD4, on the surface of macrophages or T-helper lymphocytes is the first step in virus entry. Binding to CD4 is mediated by gp120, the surface subunit of the envelope. In its native form, the envelope glycoprotein is a hetero-trimer of three gp120 molecules and three molecules of gp41, the transmembrane subunit, which remain attached through noncovalent interactions [1, 2]. Conformational changes in gp120 triggered by CD4 binding exposes structural elements that engage one of two chemokine receptors, either CCR5 or CXCR4. Coreceptor binding allows the hydrophobic N-terminus, or fusion peptide, of the gp41 ectodomain to insert into the target cell membrane. The anti-parallel association of two helically coiled heptad repeats (HR-1 and HR-2) in the gp41 ectodomain to form a six-helix bundle leads to the close approximation of the cell and virus membranes, resulting in fusion [3].

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Attachment inhibitors: Early attempts to develop specific inhibitors of HIV-1 entry focused on the design and testing of recombinant soluble CD4 molecules. These molecules lack the trans-membrane and cytoplasmic domains of CD4, but retain the ability to bind gp120, thereby functioning as molecular decoys. Although these molecules showed good in vitro activity against tissue culture-adapted strains of HIV-1, activity in early phase clinical trials was disappointing [4-7]. More promising data were generated in preliminary studies of PRO 542, a tetravalent CD4- immunoglobulin fusion protein [8, 9], but no additional studies of PRO 542 are ongoing at this time (www.clini-

Small molecule inhibitors that bind to a specific region within the CD4 binding pocket of gp120 and block the gp120-CD4 interaction are more promising [10, 11]. A proof-of-concept study with the compound, BMS-488043 resulted in 1 log10 reduction in plasma HIV-1 RNA in treatment-naive subjects [12]. Further development of this molecule was discontinued due to suboptimal pharmacokinetics. However, BMS-663068 (a prodrug of the attachment inhibitor BMS-626529) demonstrated improved pharmacokinetics and increased potency against a greater range of HIV-1 subtypes [13]. A recent randomized, open- label, phase 2a study of BMS-663068 with or without ritonavir boosting showed that the medication was well tolerated and resulted in up to a 1.7 log10 reduction in plasma HIV-1 RNA levels after eight days of treatment [14].

Postattachment inhibitors (ibalizumab) The monoclonal antibody (mAb) ibalizumab (formerly TNX-355) is a humanized IgG4 mAb that binds to the second (C2) domain of CD4 [15]. In contrast to attachment inhibitors, ibalizumab does not prevent gp120 binding to CD4, but is thought to decrease the flexibility of CD4, thereby hindering access of CD4-bound gp120 to CCR5 and CXCR4. The mAb is a potent inhibitor of HIV-1 in vitro, shows synergy when combined with gp120 antibodies or the fusion inhibitor enfuvirtide, and does not appear to interfere with immunological functions that involve antigen presentation [16-19]. Phase 1 studies of intravenous ibalizumab showed up to a 1.5 log10 reduction in plasma HIV-1 RNA levels 14-21 days after a single dose [20], but resistance emerged after repeated dosing over nine weeks [21]. A phase 2 study of ibalizumab in highly treatment-experienced patients showed that this mAb plus an optimized background regimen resulted in significantly greater reductions in plasma HIV-1 RNA compared to the background regimen alone [22]. Ibalizumab reduced virus load by 4 log10 in a patient with high-level five-class antiretroviral drug resistance, but the virologic response was lost rapidly after a single missed infusion [23]. Viruses with reduced susceptibility to ibalizumab from phase 1b trials have higher levels of infectivity compared to paired, baseline viruses, but remain susceptible to the small-molecule CCR5 antagonist maraviroc and the fusion inhibitor enfuvirtide [24]. In vitro resistance experiments suggested that reduced susceptibility to ibalizumab is correlated with fewer potential asparagine-linked glycosylation sites in the gp120 variable region 5 (V5), especially at the V5 N-terminus [24, 25]. A phase 1, randomized, placebo-controlled sequential dose escalation study of ibalizumab given by weekly subcutaneous injection in healthy volunteers is currently underway ( The long acting sub-cutaneous injection has been proposed to improve drug adherence in patients who have difficulty taking daily oral regimens, and is an attractive candidate for HIV-1 pre-exposure prophylaxis (PrEP).

Chemokine receptors and HIV-1 co-receptor usage: Viruses that use CCR5 exclusively as coreceptor for entry (termed R5 viruses) predominate in early HIV-1 disease and are primarily responsible for transmission of infection. Viruses that use CXCR4 (X4) or both CCR5 and CXCR4 are rare in early disease, but emerge over time. Mixtures of R5 and X4 viruses can also be found, but because commonly used tropism assays cannot distinguish between dual tropic and a mixture of R5 and X4 viruses, such samples are referred to as having 'dual-mixed' (D/M) coreceptor usage. The prevalence of X4 variants increases with decreasing CD4+ cell count, and several studies show a significantly increased risk of disease progression among patients with D/M or X4 virus [26-28].

CCR5 antagonists: Several approaches have been developed to block interactions between HIV-1 and CCR5, including small molecule antagonists, mAbs, and covalently modified natural CCR5 ligands (e.g. AOP-RANTES). However, only small-molecule CCR5 antagonists are currently approved for use or in later-stages of clinical development. Several orally available compounds aplaviroc, maraviroc, vicriviroc, cenicriviroc, and INCB009471 have progressed to phase 2 or 3 clinical trials. These compounds demonstrate potent inhibition of HIV-1 replication in vitro against laboratory-adapted and primary isolates across all clades of group M HIV-1. Aplaviroc treatment resulted in significant reduction in plasma HIV-1 RNA levels during 10 days of treatment [29], but development was terminated after nonfatal, reversible drug-induced hepatitis occurred in five subjects in phase 2b and 3 trials [30]. Vicriviroc demonstrated potent suppression of HIV-1 in combination with an optimized background regimen in placebo-controlled phase 2b studies in antiretroviral experienced individuals, but increased rates of virologic failure in treatment-naive patients compared with an efavirenz control arm led to the discontinuation of phase 2b study [31-33]. Maraviroc is a CCR5 antagonist with potent in vitro and in vivo anti-HIV-1 activity, and is the only chemokine receptor agonist currently in clinical use. The molecule is a pure CCR5 antagonist that blocks MIP- 1-a and RANTES-mediated signaling at nanomolar concentrations [34]. The efficacy of maraviroc was confirmed in a pair of phase 3 randomized, placebo-controlled trials (MOTIVATE 1 and 2) [35, 36]. In both studies, subjects in the maraviroc arms experienced plasma HIV-1 RNA reductions that were more than twice as great as those in the control arms using optimized background regimens alone [35, 36]. Increases in CD4 cell counts were also higher in the maraviroc arms, and the frequency of adverse events was similar in all groups. Virologic response to maraviroc remained durable through 96 weeks of therapy . The possibility that treatment with CCR5 antagonists would promote emergence of X4 [37] viruses, thereby accelerating disease progression, was a significant concern during early clinical trials with these agents. Virologic failure to maraviroc was associated with emergence of CXCR4-using virus in 57% of subjects in whom a repeat tropism test was obtained at the failure time-point [35]. Although CD4 count increases were smaller in this sub-group than among those with R5 virus at failure, a greater increase in CD4 counts was nevertheless observed among maraviroc recipients with D/M or X4 virus at failure when compared to the placebo group overall. Given the clinical efficacy of maraviroc, its relatively low toxicity profile, and its ability to antagonize viral entry, there has been much interest in using the drug for anti- retroviral treatment intensification and as a component in nucleoside or nucleotide reverse transcriptase inhibitor (NRTI)-sparing regimens; several trials are currently ongoing ( In a small study of 34 patients on suppressive antiviral therapy, the addition of maraviroc did not lead to a target increase of CD4+ T cells/mL, but was associated with decreased markers of inflammation [38]. Maraviroc intensification also led to a decrease in the proviral latent HIV-1 reservoir size in memory T cells in four patients, but there was no change in residual, low-level viremia [39]. Larger studies are needed in order to clarify the effects of maraviroc intensification on immunologic response and viral reservoirs. R5 virus is primarily responsible for establishing acute and early HIV-1 infection, and as a result, there has been interest in using maraviroc as PrEP, either in oral form or as a topical virustatic agent. Maraviroc has showed some promise in protecting macaques from R5 simian-human immunodeficiency virus challenge [40], but further investigation of maraviroc to prevent HIV-1 infection is needed. Cenicriviroc (TBR-652, formerly TAK-652) is an investigational small-molecule CCR5 antagonist currently in clinical development. Cenicriviroc has a longer half-life than maraviroc and is dosed once daily. It also inhibits CCR2, a receptor for monocyte chemoattractant protein-1 that has been associated with various inflammatory diseases. In phase 1 2a studies, cenicriviroc treatment resulted in up to a 1.8 log10 reduction in plasma HIV-1 RNA levels and was generally safe and well tolerated without significant adverse events [41, 42]. A phase 2b study of cenicriviroc in combination with tenofovir/emtricitabine or efavirenz plus tenofovir/emtricitabine in HIV 1-infected, treatment-naı¨ve patients with only R5 virus is underway ( The long-term effects of CCR2 antagonism and subsequent modulation of inflammation are not known and are the subject of ongoing investigation.

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CXCR4 antagonists: In contrast to CCR5, there are no known naturally occurring mutations that lead to absence of CXCR4. As a result, the development of CXCR4 inhibitors has been more challenging. One problem unique to CXCR4 inhibitors is that whereas R5 viruses are found on their own in 50% or more of patients, X4 viruses usually are present as mixtures together with R5 viruses [38, 43]. Inhibition of just the X4 component of the virus population may not lead to measurable declines in overall plasma viremia, thereby complicating assessment of drug activity. Co-administration of CCR5 and CXCR4 antagonists may be effective, but development of CXCR4 antagonists has stalled. Preliminary studies with AMD3100 showed inhibition of the X4 component of the virus population [44]. Interestingly, early studies revealed that CXCR4 blockade releases myeloid and plurioptent (CD34+) stem cells from the bone marrow into the blood [45]. This observation led to the subsequent development of AMD3100 (now called plerixafor) as an adjunct to G-CSF stimulation of the bone marrow, resulting in substantially increased stem cell yields [45]. The safety of long-term CXCR4 blockade is unknown.

Fusion inhibitors: Enfuvirtide (T-20) is a 36-mer synthetic oligopeptide whose sequence corresponds to that of the HR-2 region of the HIV-1 envelope gp41 subunit. Binding of enfuvirtide to the trimeric HR-1 complex prevents the association of HR-1 with HR-2, thereby inhibiting fusion and blocking virus entry [46] . Enfuvirtide became the first entry inhibitor approved for clinical use as a result of phase 3 clinical trials that demonstrated efficacy of the drug when combined with an optimized background regimen [47, 48]. The drug has minimal systemic toxicity, but the frequent occurrence of painful injection site reactions has limited long-term use. Co-administration of enfuvirtide significantly improved response rates to newer agents such as tirapanavir, darunavir, and maraviroc in clinical trials conducted in highly treatment-experienced patients [35, 49, 50]. When given as part of a regimen that does not succeed at fully suppressing HIV-1 replication, however, resistance to enfuvirtide emerges rapidly [51]. Sifuvirtide, a third generation fusion inhibitor that can be injected once daily, has entered early phase human clinical studies in Asia. Sifuvirtide has a longer plasma half-life and greater in vitro antiviral activity compared to enfuvirtide as a result of improved helical structure stability and high affinity for its target N-terminal HR peptide. It also demonstrated activity against HIV-1 strains resistant to enfuvirtide [52-54]. HR-1-based and small molecule fusion inhibitors are being developed, but no clinical data are available for these novel com-pounds.

Integrase inhibitors:

The choices of antiretroviral regimens available to treat HIV-1 infection have improved in number, ease of administration, safety and tolerability. Nonetheless, the development of resistance and persistent toxicities make it desirable to identify additional medications targeting unique and constitutive steps in the HIV-1 viral life cycle.

Integrase, an enzyme critical for retroviral replication, with no human homolog, has been identified as an excellent target for drug development. Integrase is a 288 amino acid protein encoded by the pol gene. The integrase enzyme performs three functions that lead to viral integration into the host cell genome. The first is site-specific endonucleolytic cleavage of the 30-ends of the viral DNA. Secondly, it participates in the assembly of the pre-integration complex (PIC) on the ends of the viral DNA, which migrates into the host nucleus. Lastly, integrase catalyses the insertion of the viral DNA into host chromosomal DNA (the strand transfer step) [55]. Raltegravir was the first US FDA-approved integrase inhibitor in 2007, followed by the recent approval of elvitegravir as part of the fixed-dose combination pill containing tenofovir disoproxil fumarate (tenofovir), emtricitabine, cobicistat and elvitegravir (StribildTM, Gilead Sciences). All integrase inhibitors in clinical development interfere with the strand transfer step of integration . Most bind the catalytic site in the CCD; however, the inhibitors of the LEDGF/p75 binding site of integrase (LEDGINs) and BI 224436 bind alternative sites but still inhibit strand transfer. Mass screening has identified numerous compounds that inhibit integrase by various mechanisms [55]. In this review, we focus on compounds in or near clinical development (for a summary, see Table 1).

9.1. First-Generation Integrase Inhibitors

9.1.1 Raltegravir (MK-0518): Developed by Merck & Co., Inc., is a diketo acid (DKA) that inhibits the strand transfer step of integration. It was approved by the FDA for treatment of HIV in 2007 and, until recently, was the only integrase inhibitor approved for clinical use. Raltegravir has potent antiviral activity with a 95 % inhibitory concentration (IC95) of 33 nM [56] and a more rapid time to viral suppression than efavirenz [57]. Its efficacy as an antiretroviral agent has been supported by several studies. Studies in Treatment-Naive Subjects: In a multi-centre double blind study, subjects were given raltegravir monotherapy at various doses (100, 200, 400 or 600 mg twice daily) versus placebo for 10 days. On day 10, the mean change in HIV RNA from baseline was -1.9, -2.0, -1.7 and -2.2 log10 copies/mL, respectively. More than 50 % of subjects in each active group achieved an HIV RNA level below 400 copies/mL. The STARTMRK trial [58-60] was a phase III, double-blind, randomized, non-inferiority trial in antiretroviral-naı¨ve subjects. Subjects received tenofovir and emtricitabine and were randomized to take either raltegravir or efavirenz. Those in the raltegravir group had faster time to virologic suppression, equivalent rates of virologic suppression at week 48, and significantly fewer adverse events. These results were maintained at weeks 96 and 156. At weeks 192 and 240 (5 years), the raltegravir group had statistically superior virologic suppression compared with the efavirenz group; however, a larger number of subjects in the efavirenz arm were counted as non-completers due to clinical adverse events (9 vs. 5 %) [61]. Additionally, raltegravir was equally efficacious as efavirenz in suppressing viraemia regardless of age, sex, race, hepatitis status, baseline viral HIV RNA level, baseline CD4 T-cell count or HIV-1 subtype [62, 63]. A limitation of raltegravir is its twice-daily dosing, and this led to the QDMRK trial [64, 65]. A total of 770 antiretroviral-naı¨ve HIV-positive subjects were randomized to receive tenofovir and emtricitabine, plus either once-daily or twice-daily administration of raltegravir (800 mg total daily dose). In the once-daily group, 83 % achieved viral suppression, compared with 89 % of the twice-daily group; the difference in these rates was statistically significant and was consistent with inferiority of the once-daily dosing arm. However, a low trough drug level and a high baseline viral load (100,000 copies/mL) were associated with an increased risk of failure. Studies in Treatment-Experienced Subjects The P005 study [66, 67] was a multi-centre, randomized, triple-blind, placebo-controlled trial of HIV-positive, antiretroviral-experienced subjects with resistance to multiple classes of antiretrovirals. Subjects were randomized to receive an optimized background regimen (OBR) ± raltegravir (200, 400, 600 mg twice daily) for 24 weeks. It was noted that adding raltegravir to an OBR improved the virologic response at all doses studied. Subjects were all offered open-label raltegravir after week 24; at 96 weeks, the improved virologic response was sustained. BENCHMRK 1 and 2 [66, 68-70] were phase III, randomized, double-blind studies conducted in Europe, Asia, Australia and North and South America. The studies enrolled treatment-experienced HIV-1-infected subjects with resistance to at least one drug in each of three classes; these subjects were randomized to receive an OBR based on resistance testing and treatment history with or without raltegravir. Suppression of HIV RNA levels to<50 copies/mL was achieved in 62.1 %of the subjects in the raltegravir arm versus 32.9 % of those in the placebo arm at week 48. The addition of raltegravir remained virologically superior at weeks 96 and 156. As part of the SHCS (Swiss HIV Cohort Study), the 'real world' therapeutic use of raltegravir in treatment experienced patients was described [71]. Results showed that those with an undetectable baseline viral load had a 95.8 % rate of viral suppression on raltegravir, while those with detectable HIV RNA levels showed 71.8 % viral suppression. Failures were associated with very low genotypic sensitivity of the background regimen, very low plasma raltegravir concentrations, poor adherence, and high baseline viral load. Additionally, higher rates of virologic suppression were seen in raltegravir-based salvage regimens with two nucleoside reverse transcriptase inhibitors (NRTIs), even if genotype testing revealed partial to highgrade resistance to this class [72]. The REALMRK study was a phase III, multi-national, single-arm, observational trial that enrolled a diverse cohort of HIV-positive subjects whoo are both treatment-naı¨ve and treatment-experienced and administered raltegravir 400 mg twice daily in addition to a background antiretroviral therapy (ART) regimen [73]. Raltegravir had potent efficacy and was generally well tolerated regardless of sex or race.

Enfuvirtide, previously a staple of salvage therapy for HIV-1, has the distinct disadvantages of being a twicedaily injectable with a complicated preparation of the dose and adverse reactions that include painful subcutaneous nodules at the site of injection. A small, open-label, uncontrolled trial [74] was conducted, in which 35 HIV-infected treatment-experienced subjects elected to switch from enfuvirtide to raltegravir while leaving the remainder of their regimens intact. After 7 months, 34 subjects maintained virologic suppression with resolution

of the enfuvirtide-induced injection site reactions. EASIER ANRS 138 [75, 76] was a randomized, open-label study in which half of the subjects switched from enfuvirtide to raltegravir. At week 24, 88-89 % of subjects in both arms had HIV viral load \50 copies/mL; the control arm was then permitted to switch to raltegravir. At week 48, 90 % of subjects in both arms were virologically suppressed. All the studies support support the use of raltegravir as a potent antiretroviral agent in both treatment-naı¨ve patients and as part of salvage regimens in those with multi-class resistance. Notable failures of raltegravir include (a) the mixed results after an attempt to switch to raltegravir from a PI-based regimen and (b) the trial of once-daily raltegravir. This drug is very well tolerated, but limitations of this medication are its twice-daily dosing schedule and its relatively low genetic barrier to viral resistance. These constraints have prompted the search for other integrase inhibitors.

9.1.2 Elvitegravir (GS-9137/JTK-303)

Elvitegravir (Fig. 2b) is a monoketo acid strand transfer inhibitor [22, 77] developed by Gilead Sciences. It is a potent antiretroviral drug with an in vitro EC90 (90 % effective dose) of 1.7 nM and 30 % oral bioavailability in dogs. It is excreted predominantly in the faeces. Elvitegravir versus a Boosted Protease Inhibitor Zolopa et al. [78] described a phase II, non-inferiority trial of ritonavir-boosted elvitegravir (20, 50 or 125 mg) versus a boosted PI (PI/r) added to an OBR (consisting of NRTIs ± enfuvirtide) in treatment-experienced HIV-positive subjects. At week 8, the Data Safety Monitoring Board (DSMB) stopped the elvitegravir 20 mg arm due to virologic failures, and allowed subjects in the other groups to electively add a PI/r. The time weighted change in HIV RNA level from baseline through week 24 was -1.19 log10 copies/mL for the PI/r group, -1.44 log10 copies/mL for the elvitegravir 50 mg arm (non-inferior) and -1.66 log10 copies/mL for the elvitegravir 125 mg arm (superior, p = 0.021). Durability of virologic response was highly dependent upon the activity of the OBR. Recently, Gilead Sciences co-formulated a once-daily combination pill containing elvitegravir, cobicistat, tenofovir and emtricitabine and this combination was been approved by the FDA for treatment of HIV-1 infection in antiretroviral naı¨ve individuals and is being marketed as StribildTM. GS-236-0103 was a phase III, randomized, multinational, non-inferiority trial comparing elvitegravir/cobicistat/tenofovir/emtricitabine with boosted atazanavir/tenofovir/emtricitabine (atazanavir/r/tenofovir/emtricitabine) in treatment-naı¨ve, HIV-positive subjects [79]. A total of 708 subjects were randomized; 89.5 % of the elvitegravir/cobicistat/tenofovir/emtricitabine arm and 86.8 % of the atazanavir/r/tenofovir/emtricitabine arm achieved the primary outcome of HIV RNA<50 copies/mL maintained through week 48 [80].

2.2.1 Elvitegravir versus Raltegravir GS-US-183-0145 directly compared elvitegravir versus raltegravir in addition to an OBR (containing a PI/r plus at least one other drug) in subjects who were treatment experienced and/or resistant to at least two classes of antiretrovirals [80]. Fifty-nine percent of subjects in the elvitegravir arm and 58 % in the raltegravir arm achieved and maintained an HIV viral load\50 copies/mL by week 48. At week 96, 47.6 % of the elvitegravir arm and 45.0 % of the raltegravir arm were virologically suppressed [81]. Upon direct comparison with raltegravir, the elvitegravir arm showed more diarrhoea, but fewer LFT changes and serious adverse events; it may be preferable in patients with concurrent liver disease [82]. Overall, since earlier elvitegravir trials were conducted with ritonavir, data are more limited regarding the safety profile of cobicistat. Elvitegravir, both alone and as part of the elvitegravir/cobicistat/tenofovir/emtricitabine fixed-dose combination pill, will be a convenient and potent antiretroviral medication in treatment-naı¨ve and treatment-experienced HIV-positive patients. With its once-daily dosing, it overcomes one of the limitations of raltegravir, and elvitegravir/cobicistat/tenofovir/emtricitabine contributes another complete regimen in one pill once per day in treatment-naı¨ve HIV-infected individuals. However, the cross-resistance with raltegravir may limit its usefulness in heavily treatment-experienced patients. Additionally, the need for a boosting agent raises concerns for drug-drug interactions. There has been a continued search for an integrase inhibitor with a greater genetic barrier to resistance and a resistance profile that does not overlap highly with first-generation integrase inhibitors.

9.2. Second-Generation Integrase Inhibitors

9.2.1 Dolutegravir (S/GSK1349572)

This search for a once-daily integrase inhibitor that has minimal cross-resistance with first-generation drugs in this class has led to the discovery and development of dolutegravir by Shionogi-ViiV Healthcare and Glaxo-SmithKline (GSK) [83]. It, too, is a potent antiretroviral agent with an EC90 of 2 nM and activity against HIV-2 in addition to all clades of HIV-1 [84]. In a phase IIa, placebo-controlled, dose-ranging study, subjects were randomized to either dolutegravir monotherapy (2, 10 or 50 mg) or placebo for 10 days [85]. Mean change in plasma viral load from baseline in the dolutegravir arms was -1.51 to -2.46 log10 copies/mL, which varied in a dose-dependent fashion. Of the subjects in the dolutegravir 50 mg arm, 70 % chieved an HIV RNA level\50 copies/mL by day 10. The VIKING-1 cohort [86] consisted of 27 subjects with resistance to raltegravir and at least two other classes of antiretroviral drugs who were on a failing regimen at the time of study entry. They were maintained on their failing background regimen and given dolutegravir 50 mg daily for 11 days, at which point their background regimens were optimized if available. Response varied based on the baseline integrase genotype: 16/16 subjects with single integrase mutations achieved a viral load <400 copies/mL. From day 1 to day 11, there was little change in genotypic or phenotypic resistance changes to dolutegravir [87]. The VIKING-2 cohort had the same qualities as cohort 1 and received the same schedule of intervention, except that they were given dolutegravir 50 mg twice daily rather than once daily [88]. A plasma HIV RNA <400 copies/mL or reduction by -0.7 log10 copies/mL was achieved by 96 % of cohort 2 subjects. required to have at least one fully active antiretroviral drug.

SPRING-1 was a phase IIb, randomized, dose-finding study comparing dolutegravir (10, 25 or 50 mg daily) with efavirenz (600 mg daily) [89] co-administered with either tenofovir/emtricitabine or lamivudine/abacavir. Subjects received at least one dose of study drug. At week 16, 90 % of the dolutegravir arms and 60 % of the efavirenz arm had achieved a viral load <50 copies/mL. By week 48, 90 % of the dolutegravir arms and 82 % of the efavirenz arm had plasma HIV-1 RNA levels below detection [90]. The results of SPRING-1 were used to support a dolutegravir 50 mg daily dosing schedule for SPRING-2, a phase III, multi-centre, study of dolutegravir versus raltegravir [91]. Antiretroviral-naı¨ve subjects were randomized to receive either dolutegravir 50 mg daily or raltegravir 400 mg twice daily on top of a backbone of tenofovir/emtricitabine or lamivudine/abacavir. At week 48, 88 % in the dolutegravir group and 85 % in the raltegravir arm achieved a viral suppression below 50 copies/ml copies/mL. Overall, dolutegravir has less resistance overlap with raltegravir than does elvitegravir. Raltegravir failure infrequently selects for mutation combinations that would cause resistance to dolutegravir [92, 93]. This illustrates the importance of not maintaining a patient on a failing raltegravir- or elvitegravir-containing regimen, as the accumulation of mutations can hinder future attempts to use the secondgeneration integrase inhibitors. Dolutegravir appears to have a higher genetic barrier to resistance than raltegravir [94]. This may be due to its longer half-life, or to its slower dissociation from HIV-integrase complexes [95]. Should dolutegravir be approved by the FDA for the treatment of HIV, it would provide the advantage of once daily dosing and a comparatively higher genetic barrier to resistance. Multiple studies have demonstrated the relatively narrow overlap in resistance mutations that dolutegravir has with first-generation integrase inhibitors. Finally, the drug has been well tolerated in studies performed to date.

9.2.2. S/GSK1265744 and 744 Long-Acting Parenteral S/GSK1265744: Another strand transfer inhibitor originally developed by Shionogi-ViiV Healthcare/GSK. The drug is very potent, with an in vitro half maximal inhibitory concentration (IC50) of 0.34 nM. However, it is highly protein bound to serum albumin and has a protein-adjusted IC90 of 166 ng/mL. It has a plasma half life of approximately 40 h in its oral formulation, allowing for once-daily dosing. A long-acting parenteral (LAP) nanosuspension preparation was created, which has a t1/2 of 21-50 days. Oral S/GSK1265744 has been studied in a three-part series of phase I/IIa trials [96]and there were no serious adverse events in either of the preceding two parts. HIV RNA levels decreased by a median of 2.6 log10 copies/mL and suppression of viral load was maintained through day 14 in 88 % of subjects. Thus far, there has not been clinical evidence of viral resistance to S/GSK1265744, although few HIV-positive subjects have been treated. Viral isolates from subjects with high-level raltegravir resistance remained sensitive to S/GSK1265744 in vitro [97]. Overall, S/GSK1265744 has been well tolerated, and the long-acting formulation displays excellent pharmacokinetics.

9.3.Other inhibitors of integrase in development

9.3.1. Dual Reverse Transcriptase and Integrase Inhibitors

The integrase protein of HIV-1 and the RNAse-H domain of RT share structural and functional similarities. Integrase mutations also affect the function of RT, and an RT binding site on integrase has been proposed [98-101]. DKAs have been found that bind both enzymes, and these may be developed as a hybrid class of compounds [102].

9.3.2. Non-Catalytic Site Integrase Inhibitors LEDGINs : LEDGINs are small molecules, designed to be potent inhibitors of the integrase-LEDGF/p75 interaction, that allosterically block catalytic integrase activities. This activity is promoted by the stabilization of the dimer interface of integrase upon LEDGIN binding [103-105]. These compounds have been shown to retain potency in vitro against an array of clades and against viruses harbouring mutations against integrase strand transfer inhibitors [106]. BI 224436: BI 224436 is an integrase inhibitor currently licensed by Gilead (originally developed by Boehringer- Ingelheim) that operates at a non-catalytic site to interfere with the interaction between integrase and the chromatin targeting the LEDGF/p75 protein; it inhibits 30 processing and HIV replication [107]. This is a potent antiviral with an EC95 of 78 ± 18 nM. Against raltegravir-resistant viral isolates, BI 224436 had a mean fold change in EC50 of 1.4 ± 0.8 relative to a wild-type control virus. BI 224436 has now been entered to first phase I, doubleblind, placebo-controlled, first-in-human trial [108].

10 Conclusion

Although a variety of inhibitors that target different steps in HIV-1 entry have been developed, only maraviroc, a small-molecule CCR5 antagonist, and enfuvirtide, an oligopeptide fusion inhibitor, are approved for clinical use. Several other drugs are currently active in the development pipeline, and entry inhibitors may play an important role in preventing acquisition of HIV-1infecton. Since the approval of raltegravir in 2007, its clinical performance has supported the data from its phase III clinical trials. This is a potent, well tolerated antiretroviral medication. Its twice-daily dosing and low genetic barrier to resistance has spurred the search for other integrase inhibitors. Elvitegravir, recently approved as part of the fixed-dose combination pill elvitegravir/cobicistat/tenofovir/emtricitabine and currently in phase IIIb trials, can be administered only once daily when given concurrently with a potent CYP3A inhibitor (ritonavir or cobicistat). However, there is extensive cross-resistance between the two drugs. Another well tolerated, once-daily integrase inhibitor is dolutegravir. As the second-generation integrase inhibitor with the most advanced development, this drug is also very well tolerated and has only a narrow overlap in resistance profile with raltegravir. The 'backup' drug to dolutegravir, S/GSK1265744,also demonstrates favourable tolerability and a relatively high genetic barrier to resistance in the limited testing to date. The long-acting formulation, 744-LAP, is being studied with monthly or quarterly administration, and this dosing schedule is exciting from both a treatment and an HIV-1 prevention standpoint. Lastly, BI 224436 represents the first integrase inhibitor to bind at a non-catalytic site; viral isolates with high-grade raltegravir resistance retained sensitivity to this drug. The second-generation integrase inhibitors face the challenges of competing with and/or complementing the currently available arsenal of antiretroviral medications. Those in development seem to have good tolerability, and the fact that integrase has no human homolog will likely limit the toxicities of future candidates. They will need to have a maximum dosing frequency of once-daily administration.

Importantly, increased stability of binding integrase and new binding targets on the enzyme will minimize the level of intra-class cross-resistance. While elvitegravir resistance mutations mirror most of those for raltegravir, dolutegravir has already demonstrated a higher genetic barrier to

resistance and a much more narrow overlap with raltegravir resistance mutations. Studies are very early in S/GSK1265744/744-LAP, but no resistance was seen so far in vivo, and in vitro experiments support a high barrier to resistance; raltegravir-resistant viral isolates retained sensitivity to S/GSK1265744. BI 224436 binds a separate site on the integrase protein and retained effectiveness in vitro against isolates with high-level raltegravir resistance.

Newer compounds have been identified whose half-lives and potency allow for oncedaily dosing in the absence of the need for a pharmacological enhancing agent. Raltegravir has been a productive addition to our arsenal of antiretroviral drugs since 2007. Its novel target and tolerability have made it a useful drug in both treatment naıve and heavily treatment-experienced patients with HIV-1. It can also boast the most complete safety profile of the members of this drug class. Raltegravir is best suited for the naı¨ve or experienced patient with the ability to comply with a twice-daily dosing schedule. StribildTM (elvitegravir/cobicistat/tenofovir/emtricitabine) is a convenient, one-pill, once-daily antiretroviral regimen that will certainly be a welcome addition to treatment options for the antiretroviral naı¨ve, HIV-1-infected patient, now that it has been approved by the FDA. Dolutegravir and S/GSK1265744 appear to offer higher barriers to resistance, limited cross-resistance with raltegravir and elvitegravir, and once-daily dosing. If they continue to have favourable safety profiles, these agents show promise both as first-line antiretrovirals and in deep salvage regimens for patients with prior integrase inhibitor exposure. BI 224436 binds to a different site in integrase and is unlikely to have cross-resistance with the other integrase inhibitors. The LEDGINs will require further development prior to clinical testing. Integrase is a target that will continue to be exploited in the treatment, and possibly the prevention, of HIV-1 infection.