Review CCR5 Antagonists: Host-Targeted Antivirals for the Treatment of HIV Infection

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Antiviral Chemistry & Chemotherapy 16:339–354 Review CCR5 antagonists: host-targeted antivirals for the treatment of HIV infection

Mike Westby* and Elna van der Ryst

Pfizer Global R&D, Kent, UK

*Corresponding author: Tel: +44 1304 649876; Fax: +44 1304 651819; E-mail: mike.westby@pfizer.com

The human chemokine receptors, CCR5 and suggest that these compounds have a long plasma CXCR4, are potential host targets for exogenous, half-life and/or prolonged CCR5 occupancy, which small-molecule antagonists for the inhibition of may explain the delay in viral rebound observed HIV-1 infection. HIV-1 strains can be categorised by following compound withdrawal in short-term co-receptor tropism – their ability to utilise CCR5 monotherapy studies. A switch from CCR5 to (CCR5-tropic), CXCR4 (CXCR4-tropic) or both (dual- CXCR4 tropism occurs spontaneously in approxi- tropic) as a co-receptor for entry into susceptible mately 50% of HIV-infected patients and has been cells. CCR5 may be the more suitable co-receptor associated with, but is not required for, disease target for small-molecule antagonists because a progression. The possibility of a co-receptor natural deletion in the CCR5 gene preventing its tropism switch occurring under selection pressure expression on the cell surface is not associated by CCR5 antagonists is discussed. The completion with any obvious phenotype, but can confer of ongoing Phase IIb/III studies of maraviroc, resistance to infection by CCR5-tropic strains – the aplaviroc and vicriviroc will provide further insight most frequently sexually-transmitted strains. into co-receptor tropism, HIV pathogenesis and The current leading CCR5 antagonists in clinical the suitability of CCR5 antagonists as a potent development include maraviroc (UK-427,857, new class of antivirals for the treatment of HIV Pfizer), aplaviroc (873140, GlaxoSmithKline) and infection. vicriviroc (SCH-D, Schering-Plough), which have demonstrated efficacy and tolerability in Keywords: co-receptor, chemokine, entry, HIV, HIV-infected patients. Pharmacodynamic data also antiretroviral

Introduction

Human immunodeficiency virus (HIV), the retrovirus that discontinued HAART did so for reasons related to drug causes acquired immune deficiency syndrome (AIDS), is a toxicity, which mainly occurred within 3 months of starting major cause of death worldwide. In 2004 alone, AIDS therapy (d’Arminio et al., 2000). A further limitation of resulted in the death of an estimated 3.1 million people HAART is the development of viral resistance, which has (WHO, 2004). Highly active antiretroviral therapy limited the effectiveness of many antiretroviral drugs (HAART) regimens introduced in the late 1990s (Martinez-Picado et al., 2000). In one large study of HIV- profoundly reduced morbidity and mortality due to HIV positive adults who received treatment yet were viraemic infection in developed countries. These regimens generally with >500 HIV RNA copies/ml it was estimated that include at least three drugs selected from four classes: approximately 76% of patients had resistance to one or nucleoside/nucleotide reverse transcriptase inhibitors more HIV drugs within 3 years (Richman et al., 2004). (NRTIs), non-nucleoside reverse transcriptase inhibitors These limitations highlight the continuing unmet medical (NNRTIs), protease inhibitors (PIs) and fusion inhibitors. need for anti-HIV agents with novel mechanisms of action. Although effective in reducing plasma viral load, delaying Until recently, all licensed anti-HIV drugs have inhib- disease progression to AIDS and prolonging survival, ited viral replication by acting on intracellular targets. HAART has two major limitations. Firstly, drug toxicity Only one currently licensed drug, enfuvirtide (Fuzeon®, (reviewed by Carr, 2003) can often lead to poor treatment Trimeris/Roche; formerly known as T-20) acts at the compliance and treatment failure and facilitates emergence point of virus entry into cells. The licensed approval of of resistance. In one study, 58% of patients who enfuvirtide in 2003 demonstrated that HIV entry is a viable

therapeutic target for future drug development. Several binding of gp120 to CD4 causes a reconfiguration of the new classes of anti-HIV agents have since emerged, acting V1/V2 and V3 loops of gp120 to expose the bridging sheet on either viral or human target proteins. This review and form a co-receptor binding site (Kwong et al., 1998; outlines the processes involved in HIV entry and discusses Rizzuto et al., 1998; Wyatt et al., 1998). Once this has the development of several promising compounds termed occurred, co-receptor binding triggers conformational HIV co-receptor antagonists. In particular, the class of changes in gp41, which drive the remaining steps in fusion co-receptor antagonists that act on the human chemokine and entry of the viral core (reviewed by Chan & Kim, receptor, CCR5, is discussed in detail. 1998). According to a recent study of HIV-1 entry kinetics, the entry efficiency of cell-attached virus is mainly The process of HIV entry controlled by three kinetic processes: a lag phase caused in part by the reversible, concentration-dependent association The process by which HIV-1 attaches to and enters host of virus with CD4 and a co-receptor; a lowering of the acti- cells has been studied extensively (reviewed by Pierson et vation energy barrier for a co-receptor-dependent confor- al., 2004). The interactions between the virus and the cell mational change in gp41; and a relatively rapid and surface required for entry are mediated by the viral enve- kinetically dominant process of viral inactivation, possibly lope protein, Env. During viral replication, Env is expressed involving endocytosis, which competes with viral entry as a 160kDa precursor protein, termed glycoprotein (Platt et al., 2005). (gp)160, which is later cleaved by a host cell protease into surface and transmembrane subunits, termed gp120 and Targeting HIV-1 entry gp41, respectively. The gp120 subunit is required for specific binding to host-cell receptors and possesses five The development of agents targeting discrete stages of the variable regions (V1–V5) and five constant regions HIV-1 entry process has been facilitated by two lines of (C1–C5) that are conserved among different HIV-1 research, namely: the discovery of the cellular receptors strains. In mature, free virions, gp120 is arranged as inner required for HIV infection, and an understanding of how and outer domains connected by a bridging sheet with viral components interact with these receptors. A potential conserved and functionally important regions hidden from advantage of targeting HIV-1 entry is that the site of host immune recognition (Kwong et al., 1998; Wyatt et al., inhibitory action is likely to be extracellular. An extracel- 1998). The native gp41 subunit has an N-terminal, glycine- lular target is potentially more accessible and, unlike rich ‘fusion peptide’ which is concealed in a non-fusogenic NRTIs, there is no requirement for intracellular processing state until specific interactions between gp120 and host- of the agent. Therefore, there is no mechanistic reason for cell receptors have occurred. Each gp120 unit is non-cova- non-receptor-linked intracellular toxic effects such as mito- lently associated with a gp41 unit and these heterodimers chondrial toxicity. A further advantage of entry inhibitors is are arranged as trimers on the outer surface of the mature that there is no cross-resistance with existing agents that virion. The first mandatory step in the process of HIV-1 act on intracellular targets, as demonstrated by the potency entry is the specific binding of gp120 to CD4, the primary of co-receptor antagonists AMD3100 (Este et al., 1996), receptor for HIV-1 (Figure 1). The CD4 receptor is maraviroc (UK-427,857) (Westby et al., 2003) and expressed mainly on T-lymphocytes and macrophages and aplaviroc (Maeda et al., 2004), and the fusion inhibitors is a member of the immunoglobulin (Ig)-like protein enfuvirtide and T-1249 (Sista et al., 2001) against viruses superfamily. However, the binding of gp120 to CD4 alone resistant to other classes of anti-HIV drugs. is not sufficient for HIV-1 entry (Maddon et al., 1986). However, there are potential disadvantages of entry The observation that human chemokines are capable of inhibition. Firstly, Env is the most sequence-variable of the inhibiting HIV-1 infection of T-lymphocytes (Cocchi et HIV-1 proteins (Griffin, 2003). Hence, differences in drug al., 1995) and the identification of polymorphisms in the sensitivity between HIV-1 strains are possible, as has been CCR5 gene leading to resistance to HIV infection (Liu et described for the gp120 inhibitor, BMS-806, which has a al., 1996), led to the discovery that a human chemokine wide range of activities against panels of B-clade and non- receptor is an essential co-receptor for HIV-1 infection B-clade viruses (Lin et al., 2003). Secondly, targeting host (Feng et al., 1996). Chemokines are a large family of receptors may inhibit their natural function. If this inter- secreted chemoattractant proteins that regulate leukocyte rupts essential host processes then compounds may have activation and migration to sites of inflammation via inter- unwanted secondary pharmacological effects. action with a family of chemokine receptors. The The inhibition of binding of gp120 to CD4 has been chemokine receptors most commonly utilised by HIV-1 in explored with mixed results. Early attempts to inhibit vivo are CCR5 and/or CXCR4 (Choe et al., 1996; Deng et this interaction with soluble, recombinant CD4 were al., 1996; Dragic et al., 1996; Feng et al., 1996). The abandoned because of lack of activity against primary

Figure 1. A model for HIV entry

A B gp41 gp120

CD4

Co-receptor (CCR5 or CXCR4)

Host cell membrane

C D

HIV gp120 binds to CD4 (A). This induces conformational changes in gp120 and exposure of the co-receptor binding site (B), which is a complex domain comprising the V3 loop and specific amino acid residues in C4, collectively termed the ‘bridging sheet’. Exposure of the co-receptor binding site permits binding of gp120 to the co-receptor (C). Co-receptor antagonists inhibit this step by binding the co-receptor and changing its shape such that gp120 cannot recognise it. Co-receptor binding induces conformational changes in gp41 and insertion of a ‘fusion peptide’ into the host cell membrane (D), ultimately resulting in fusion of viral and cell membranes. Multiple gp120-co-receptor interactions are required to form a fusion pore through which the viral core can pass and infect the cell.

HIV-1 isolates. More recent developments include parenterally. Two orally bioavailable compounds, BMS-806 PRO-542 (Progenics Pharmaceuticals, USA), which is a and BMS-043 (Bristol-Myers Squibb, USA), target gp120 tetravalent CD4-IgG2 fusion protein incorporating four rather than CD4 (Lin PF, Ho HT, Gong YF, Dicker I, copies of the Env-binding domains of CD4 ( Jacobson et Zhou N, Fan L, McAuliffe B, Kimmel B, Nowicka-Sans al., 2000), and TNX-355 (Tanox, USA), a humanized B, Wang T, Kadow J, Yamanaka G, Lin Z, Meanwell N & IgG4 monoclonal antibody targeted towards CD4 rather Colonno R [2004] Characterization of a small molecule than gp120 to prevent post-binding entry (Kuritzkes et al., HIV-1 attachment inhibitor BMS-488043: virology, resis- 2004). Both of these agents are in Phase II development tance and mechanism of action. 11th Conference on and resemble enfuvirtide in that they must be administered Retroviruses and Opportunistic Infections. San Francisco,

Antiviral Chemistry & Chemotherapy 16.6 341 M Westby & E van der Ryst

CA, USA, February 2004. Abstract 534; Madani et al., Although several distinct chemokine receptors have been 2004). Both compounds demonstrate variable activity shown to function as co-receptors for HIV-1 infection in when tested in vitro against panels of virus isolates (Lin et vitro, the vast majority of HIV-1 clinical isolates studied to al., 2003; Lin et al., 2004: see above), although BMS-043 date use CCR5, CXCR4 or both to infect human periph- has shown efficacy in short-term monotherapy in HIV-1 eral blood mononuclear cells (Berger et al., 1998; Berger et infected individuals (Hanna G, Lalezari J, Hellinger J, al., 1999). Historically, HIV-1 strains were classified as Wohl D, Masterson T, Fiske W, Kadow J, Lin P, Giordano either T-tropic or M-tropic based on their ability to infect M, Colonno R & Grasela D [2004] Antiviral activity, T-cells or macrophages, respectively, in vitro. The T-tropic safety, and tolerability of a novel, oral small-molecule HIV- phenotype was later associated with the ability to induce 1 attachment inhibitor, BMS-488043, in HIV-1 infected syncytium formation in MT-2 cells, an in vitro cytopathic subjects. 11th Conference on Retroviruses and Opportunistic effect which was not apparent with M-tropic strains. This Infections. San Francisco, CA, USA, February 2004. led to the phenotypic nomenclature of syncytium-inducing Abstract 141). (SI) and non-syncytium-inducing (NSI) strains. Following As mentioned previously, enfuvirtide is currently the the discovery of the CXCR4 and CCR5 co-receptors, the only licensed HIV-1 entry inhibitor (Arasteh et al., 2004; SI phenotype was shown to most often be associated with Lalezari et al., 2003; Lazzarin et al., 2003). Enfuvirtide is a virus that could utilise CXCR4, while most primary NSI 36-amino acid synthetic peptide that targets gp41 to isolates use CCR5 for entry. However, there is not an prevent the conformational changes required for fusion absolute correlation between cellular tropism, SI/NSI (Clotet et al., 2004). It is particularly effective when added phenotype and co-receptor tropism (Hendrix et al., 2004; to optimized background therapy in viraemic patients with Todd et al., 1995). Since CCR5 antagonists target CCR5- a history of multiple treatment failures (Arasteh et al., using strains, references to virus tropism in this review will 2004; Lalezari et al., 2003; Lazzarin et al., 2003). Apart focus on the definition based on co-receptor usage. from injection site reactions, it has a favourable safety In the current system of nomenclature HIV-1 strains are profile and no clinically significant drug–drug interactions. categorised as R5 (CCR5-tropic), X4 (CXCR4-tropic) or However, enfuvirtide use is currently limited to extensively R5X4 (strains using both CCR5 and CXCR4; also referred pre-treated patients who have few treatment options and to as ‘dual-tropic’) (Berger et al., 1998). CCR5 antagonists this is largely due to its inconvenient route of administra- only inhibit strains which are obligate users of CCR5, tion (Clotet et al., 2004). while X4 and R5X4 strains can be described collectively as A similar agent, T-1249 (Roche, Switzerland), which “CXCR4-using” to indicate their ability to infect cells in was widely regarded as the leading next generation fusion the presence or absence of a CCR5-specific antagonist. A inhibitor and a successor to enfuvirtide, is no longer in patient serum sample may also contain a heterogeneous development despite having activity against enfuvirtide- population of viruses with different tropism, termed ‘mixed resistant virus (Eron et al., 2004; Lalezari et al., 2005). tropism’. There is a complex association between HIV-1 According to its developers, the formulation of T-1249 co-receptor tropism, transmission and pathogenesis which would not be suitable for use in large-scale clinical trials is not yet fully understood (reviewed by Moore et al., 2004 (Martin-Carbonero, 2004). Small-molecule inhibitors and Philpott et al., 2003). Generally, strains that are trans- targeting fusion have been described, although these mitted and establish new infections in a host are R5 represent interesting leads rather than clinical candidates (Schuitemaker et al., 1991; Shankarappa et al., 1999; Zhu ( Jiang et al., 2004; Salzwedel K, Crisafi K, Jackson T, et al., 1993). In some individuals, CXCR4-tropism evolves Castillo A, Kilgore N, Reddick M, Allaway G & Wild C over time and the emergence of X4 virus has been associ- [2004] Identification of small molecule HIV-1 fusion ated with rapid CD4 T-lymphocyte decline and accelerated inhibitors. 11th Conference on Retroviruses and Opportunistic disease progression. Although increasing prevalence of X4 Infections. San Francisco, CA, USA, February 2004. virus and decreasing prevalence of R5 virus have been asso- Abstract 311). The possibility of inhibiting HIV-1 entry by ciated with increasing viral load and decreasing CD4 cell targeting the co-receptors, specifically CCR5, has been the counts (Brumme et al., 2005; Moyle et al., 2005), a switch subject of increased interest in recent years and will be to CXCR4-tropism is not exclusively required for the discussed in more detail below. development of AIDS. R5 variants are often the only variants detected in the circulating virus of HIV-1 infected HIV co-receptor tropism patients. This is true at all disease stages (acute, asymptomatic and symptomatic/advanced). Dual/mixed-tropic Tropism can be defined as the affinity or specificity for a virus is more likely to be detected in patients with advanced target. In the case of HIV-1, tropism is largely determined disease, but the identification in patients of a circulating by differential co-receptor usage of different HIV strains. virus pool that is entirely CXCR4-tropic is rare (Moyle et

al., 2005; Whitcomb JM, Huang W, Fransen S, Wrin T, Mezzatesta J, Assumma A, Czarnecky R, Maddon P, Paxinos E, Toma J, Greenberg M, Sista P, Melby T, Kremer A & Isreal R [2005] First-in-humans trial of Matthews T, DeMasi R, Heilek-Snyder G, Cammack N, PRO 140, a humanized CCR5 monoclonal antibody for Hellmann N & Petropoulos C [2003] Analysis of baseline HIV-1 therapy. 3rd International AIDS Society Conference, enfuvirtide (T20) susceptibility and co-receptor tropism in Rio de Janeiro, Brazil, July 24–27, 2005. Abstract two-phase III study populations. 10th Conference on WePe6.2C04). Retroviruses and Opportunistic Infections. Boston, USA, The concept of targeting GPCRs for therapeutic inter- 10–14 February 2003. Abstract 557). Whether emergence vention is not a new one. The GPCR superfamily is the of X4 strains is a marker for disease progression rather than most prominent class of drug target; approximately 60% of the cause may only be answered following carefully all commercially available prescription drugs work by selec- controlled multi-centre clinical trials of CCR5 antagonists tive modulation of a GPCR (Gudermann et al., 1995) and (Moore et al., 2004), such as those discussed below. new agents are constantly emerging to target GPCRs involved in a variety of disease processes (reviewed by CCR5 as a therapeutic target Gurrath, 2001). Of the two GPCRs acting as the principal HIV-1 co-receptors, CCR5 may be the most suitable CCR5 is a 352-amino-acid protein with a basic structure candidate target for novel therapeutic agents. The potential that is consistent with other members of the seven- efficacy of such agents is highlighted by the observation transmembrane G protein-coupled receptor (GPCR) that individuals with a mutation in their CCR5 gene superfamily. It consists of three extracellular loops (ECL1, showed resistance to HIV-1 infection (Dean et al., 1996; ECL2 and ECL3) and the N terminus, which are involved Huang et al., 1996; Liu et al., 1996; Samson et al., 1996). in chemokine binding, and three intracellular loops and the Approximately 1% of the Caucasian population is homo- C terminus, which participate in G protein-mediated zygous for a natural 32-base-pair deletion in the CCR5 signal transduction. Chemokines are the most important gene (CCR5-∆32), which results in complete absence of regulators of leukocyte trafficking. The typical cellular CCR5 expression on the cell surface. Furthermore, HIV- response to stimulation of chemokine receptors is chemo- positive patients who are heterozygous for the CCR5-∆32 taxis up or down a concentration gradient of the allele have delayed disease progression (Dean et al., 1996; chemokine. This is suspected also for CCR5 in light of Huang et al., 1996), although these patients may show a chemotaxis observations using recombinant cell lines relatively rapid decline in CD4+ T-cells following AIDS (Strizki et al., 2001). The activation of CCR5 also causes a diagnosis (Garred et al., 1997). number of cellular responses that are typical of many Confidence in safety for the development of a CCR5 GPCRs, including inhibition of cAMP production, stimu- antagonist for the treatment of HIV/AIDS was suggested lation of Ca2+ ion release and activation of MAP kinase and by the observation that there is no overt phenotype associ- Jun-N-terminal kinase (Thelen, 2001). The natural ligands ated with CCR5-∆32 homozygosity (a natural knockout in of CCR5 (reviewed by Mueller & Strange, 2004) include humans) (Samson et al., 1996). R5 strains are the most the chemokines known as macrophage inflammatory commonly transmitted (Schuitemaker et al., 1991; protein (MIP)-1α, MIP-1β and ‘regulated on activation, Shankarappa et al., 1999; Zhu et al., 1993), they usually normal T-cell expressed and secreted’ (RANTES). The predominate throughout asymptomatic infection and observation that these ligands can inhibit HIV-1 entry 50% or more of patients who develop AIDS carry only (Cocchi et al., 1995) led to the identification of CCR5 as a R5 strains (Fitzgibbon et al., 1998; Roda Husman et al., co-receptor for HIV-1 (Alkhatib et al., 1996). Mutational 1999; Tuttle et al., 2002). Several compounds in analyses and the inhibition of chemokine and gp120 development that target CCR5 have been shown to be binding to CCR5 by monoclonal antibodies have identified effective inhibitors of HIV-1 infection in mouse and ECL2 and a tyrosine-rich region within the N terminus of macaque models (Nakata et al., 2005; Strizki et al., 2001; CCR5 as the major components involved in interactions Veazey et al., 2003) and in humans (Fätkenheuer et al., with gp120 during HIV-1 entry (Dragic et al., 1998; 2004; Lalezari J, Thompson M, Kumar P, Piliero P, Davey Farzan et al., 1998; Farzan et al., 1999; Farzan et al., 2000; R, Murtaugh T, Patterson K, Shachoy-Clark A, Adkison Farzan et al., 2002; Lee et al., 1999; Olson et al., 1999). K, Demarest J, Sparks S, Fang L, Lou Y, Berrey M & These findings prompted the development of an early Piscitelli S [2004] 873140, a novel CCR5 antagonist: drug candidate, PRO-140 (Progenics Pharmaceuticals, antiviral activity and safety during short-term monotherapy USA), a murine monoclonal antibody that recognises in HIV-infected adults. 44th Interscience Conference on multiple extracellular domains of CCR5 (Olson et al., Antimicrobial Agents and Chemotherapy. Washington DC, 1999; Trkola et al., 2001). PRO-140 is currently under USA, 30 October–2 November 2004. Abstract H-1137b; investigation in Phase I clinical trials (Olson P, Doshan H, Reynes J, Rouzier R, Kanouni T, Baillat V, Baroudy B,

Antiviral Chemistry & Chemotherapy 16.6 343 M Westby & E van der Ryst

Keung A, Hogan C, Markowitz M & Laughlin M [2002] bioavailable in rats and monkeys. TAK-220 inhibited the SCH C: safety and antiviral effects of a CCR5 receptor in vitro replication of R5 HIV-1 clinical isolates, antagonist in HIV-1-infected subjects. 9th Conference on including mutants resistant to reverse transcriptase and

Retroviruses and Opportunistic Infections. Seattle, USA, protease inhibitors, with a mean EC50 of 1.1 nM (Iizawa February 2002. Abstract 1; Schurmann D, Rouzier R, Y, Kanzaki N, Takashima K, Miyake H, Tagawa Y, Nougarede R, Reynes J, Fätkenheuer G, Raffi F, Michelet Sugihara Y & Baba M [2003] Anti-HIV-1 activity of C, Tarral A, Hoffmann C, Kiunke J, Sprenger H, van Lier TAK-220, a small molecule CCR5 antagonist. 10th J, Sansone A, Jackson M & Laughlin M [2004] SCH-D: Conference on Retroviruses and Opportunistic Infections. antiviral activity of a CCR5 receptor antagonist. 11th Boston, USA, 10–14 February 2003. Abstract 11). The Conference on Retroviruses and Opportunistic Infections.San most recent developments at Takeda involve another Fancisco, USA, 8-11 February 2004. Abstract 140LB). The candidate CCR5 antagonist, TAK-652, which inhibits more promising compounds are discussed in detail below. the binding of RANTES, MIP-1α, and MIP-1β to

CCR5 with an EC50 of 3.1, 2.3, and 2.3 nM, respectively, CCR5 antagonist discovery and inhibits the replication of R5 HIV-1 clinical isolates

with a mean 50% inhibitory concentration (IC50) of Several major pharmaceutical companies have sought to 0.06 nM (Baba M, Kanzaki N, Miyake H, Wang X, realise the potential of CCR5 antagonism as a new thera- Takashima K, Teshima K, Shiraishi M & Iizawa Y [2005] peutic strategy for HIV infection. The following is an TAK-652, a novel small molecule CCR5 antagonist with overview of the development status of the key CCR5 potent anti-HIV-1 activity. 12th Conference on antagonists described to date. Key clinical data relating to Retroviruses and Opportunistic Infections. Boston, USA, the three leading compounds are presented in Table 1. 22–25 February 2005. Abstract 541). TAK-652 has also One of the first small-molecule CCR5 antagonists to be demonstrated favourable in vitro antiviral interactions described was TAK-779 (Takeda Chemical Industries, with a range of marketed antiretroviral drugs (Tremblay Japan; Figure 2) (Baba et al., 1999). Although active CL, Giguel F, Chou TC, Dong H, Lizawa Y, Shiraishi M against R5 HIV-1 in vitro (50% effective concentration & Hirsch M [2005] TAK-652, a novel small molecule

[EC50] <4 nm), development of TAK-779 was discon- inhibitor of CCR5 has favorable anti-HIV interactions tinued due to a lack of oral bioavailability. Takeda then with other antiretrovirals in vitro. 12th Conference on began development of TAK-220, which inhibits binding Retroviruses and Opportunistic Infections. Boston, USA, of MIP-1α to CCR5, but not MIP-1β, and is orally 22–25 February 2005. Abstract 542).

Table 1. Comparison of three CCR5 antagonists in clinical development

Maraviroc Aplaviroc Vicriviroc (UK-427,857) (873140) (SCH-D/SCH-417690)

Efficacy (10–14 day monotherapy; –1.84 log10 copies/ml –1.66 log10 copies/ml –1.62 log10 copies/ml mean HIV RNA change from baseline) (300 mg BID) at nadir (600 mg BID) at nadir (50 mg BID) at day 15

–1.60 log10 copies/ml (300 mg BID) at day 11

–1.35 log10 copies/ml (300 mg QD) at day 11

Adverse reactions at clinical dose Headache, flatulence Abdominal cramps, Headache, nausea nausea nausea, diarrhoea

Dose(s) evaluated in Phase IIb/III 300 mg QD and BID 400 mg BID 5, 10, 15 mg QD clinical trials (150 mg with PI)* (with ritonavir) (with ritonavir)

T1/2 16–23 h 2 h >24 h

Doses of aplaviroc and vicriviroc being evaluated in Phase IIb/III clinical trials were obtained from www.clinicaltrials.gov. *150 mg maraviroc dose used in regimens containing HIV-1 protease inhibitors (PIs), with the exception of tipranavir. (Adkison K, Shachoy-Clark A, Fang L, Lou Y, Otto V, Berrey M & Piscitelli S (2005a) The pharmacokinetic interaction between the CCR5 antagonist 873140 and lopinavir/ritonavir in healthy subjects. 12th Conference on Retroviruses and Opportunistic Infections, Boston, USA. February 2005. Abstract 664; Adkison et al., 2005b; Fätkenheuer et al., 2004; Lalezari et al., 2004: see text; McHale et al., 2005: see text; Sansone A (2005) Pharmacokinetics of SCH 417690 administered alone or in combination with ritonavir and efavirenz in healthy volunteers. 6th International Workshop on Clinical Pharmacology of HIV Therapy, Quebec City, Quebec, Canada. April 28–30 2005. Abstract 79; Schurmann et al., 2004: see text.)

Figure 2. Chemical structures of TAK-779, SCH-C, vicriviroc, maraviroc and aplaviroc

TAK-779 SCH-C/SCH-351125

H C O 3 N CH H N 3 N CI- CH3 N H3C + H C O + H3C O N O 3 N – CH3 O

Vicriviroc/SCH-D/SCH-417690

H3CO CH3

N CH H C N 3 3 N F3C N N

O CH3

Maraviroc/UK-427,857 Aplaviroc/873140/AK602

O CH F 3 O H3C F H N HO N N NN O N OH O N

H3C CH3 N O

Este, 2002; Maeda et al., 2004; Tagat et al., 2004; Takashima et al., 2001; Wood & Armour, 2005. Aplaviroc, GlaxoSmithKline, UK; Maraviroc, Pfizer, UK; TAK-779, Takeda Chemical Industries, Japan; Vicriviroc and SCH-C, Schering-Plough Corporation, USA.

Merck has described detailed structure activity prevention of R5 infection in animal models of HIV relationships of a series of agents with CCR5 antagonist infection (Veazey et al., 2003; Wolinsky et al., 2004). activity (Dorn et al., 2001; Shen et al., 2004; Willoughby Schering-Plough has been developing CCR5 antago- et al., 2001). However, to date, Merck has not yet reported nists for a number of years. Its first major candidate, SCH- on an agent that has progressed into clinical development, C (SCH-351125; Figure 2), was the first CCR5 antagonist although their agents have proved useful tools for to be studied for clinical efficacy. SCH-C is a small, understanding how small-molecule antagonists bind to the non-peptidic oxime-piperidine based on a compound iden- receptor (Castonguay et al., 2003) and for investigating tified by high throughput screening of inhibitors of

Antiviral Chemistry & Chemotherapy 16.6 345 M Westby & E van der Ryst

Figure 3. A model for CCR5 antagonist binding RANTES-CCR5 binding and was confirmed to be a specific CCR5 antagonist by receptor binding and signal transduction assays. The separate binding domains on N-terminus CCR5 for SCH-C and RANTES (Blanpain et al., 2003; Extracellular loops Tsamis et al., 2003; Wu et al., 1997) strongly suggested that gp120 SCH-C, and potentially other small-molecule CCR5 antagonists, act by an allosteric mechanism that alters the CCR5 transmembrane conformation of CCR5. In the allosteric model of CCR5 region antagonism, binding of the antagonist in the transmem- Cell surface brane domain of CCR5 (Figure 3) induces a conformational change in ECL2 that prevents its interaction with the V3 crown, thus inhibiting viral entry (Tsamis et al., Antagonist 2003). In vitro infectivity assays using SCH-C and primary

R5 HIV-1 isolates demonstrated a mean IC50 of less than 9 nM, while potent in vivo activity was demonstrated in a murine model (Strizki et al., 2001). Although a study in 12 HIV-infected patients demonstrated good oral bioavailability and a threefold or greater reduction in viral load at 25 mg BID, observations of QT prolongation in healthy volunteers receiving 600 mg QD led to the termination of further studies of SCH-C (Este, 2002; Reynes et al., 2002: see above). The QT interval represents the duration of ventricular depolarization and repolarization – a delay in cardiac repolarization can lead to the development of cardiac arrhythmias. Schering-Plough has continued to develop agents related to SCH-C, most notably vicriviroc Binding of the antagonist within the transmembrane domain is (SCH-D, SCH-417690; Figure 2), which is reported to be thought to alter the conformation of the N-terminus and extracellular loops of CCR5 such that HIV-1 gp120 can no longer bind. more potent than SCH-C and has a more favourable safety profile with respect to QTc prolongation. In a clinical study in HIV-infected patients, more than 80% of patients who received vicriviroc 50 mg BID achieved at least a Whitlock L, Ridgway C, McHale M & Abel S [2003]

1 log10 copies/ml mean reduction in viral load after 14 days Multiple dose study to investigate the safety of of treatment (Schurmann et al., 2004: see above). Phase IIb UK-427,857 [100 mg or 300 mg] BID for 28 days in studies of vicriviroc are ongoing. healthy males and females. 43rd Interscience Conference on A CCR5 antagonist in development by Pfizer, Antimicrobial Agents and Chemotherapy. Chicago, IL, USA, maraviroc (UK-427,857; Figure 2), has also been shown to September 2003. Abstract H-874). In Phase IIa studies, be active in vitro against a wide range of clinical R5 isolates treatment-naive HIV patients received maraviroc

(geometric mean IC90=2.0 nM) (Macartney MJ, Dorr P, monotherapy at doses ranging from 25 mg QD to 300 mg Smith MW et al. [2003] In vitro antiviral profile of BID for 10 days. At 300 mg BID a maximum mean viral

UK-427,857, a novel CCR5 antagonist. 43rd Interscience load reduction of 1.84 log10 copies/ml was demonstrated Conference on Antimicrobial Agents and Chemotherapy. (McHale M, Abel S, Russell D, Gallagher J & Van der Chicago, USA, 14–17 September, 2003; Abstract H-875). Ryst E [2005] Overview of Phase 1 and 2a safety and effi- Pharmacokinetic studies have demonstrated good oral cacy data of maraviroc [UK-427,857]. 3rd International bioavailability and a terminal half-life of 16–23 h following AIDS Society Conference. Rio de Janeiro, Brazil, 24-27 July multiple dosing, which does not alter significantly with 2005. Abstract TuOA0204). Dosing with food had no dose. Single doses of up to 900 mg and multiple doses of up significant effect on efficacy (Fätkenheuer et al., 2004) and to 300 mg BID for 28 days were well tolerated (Abel S, there was no significant difference in efficacy between Van der Ryst E, Muihead GJ, Rosario A, Edgington A & 150 mg BID and 300 mg QD dosing regimens. The drug Weissgerber G [2003] Pharmacokinetics of single and is also well tolerated, with headache, nausea and flatulence multiple oral doses of UK-427,857 – a novel CCR5 antag- the only adverse events occurring at a rate higher than onist in healthy volunteers. 10th Conference on Retroviruses placebo at doses up to and including 300 mg BID and Opportunistic Infections, Boston, USA. February 2003. (Fätkenheuer et al., 2004; McHale et al., 2005: see above). Abstract 547; Russell D, Bakhtyari A, Jazrawi RP, Phase IIb/III trials with maraviroc in both treatment-naive

and treatment-experienced patients are ongoing (McHale vicriviroc (Schurmann et al., 2004: see above) and aplaviroc et al., 2005: see above). (Lalezari et al., 2004: see above; Sparks S, Adkison K, GlaxoSmithKline (GSK) is currently developing small- Shachoy-Clark A, Piscitelli S & Demarest J [2005] molecule CCR5 antagonists based on spirodiketopiper- Prolonged duration of CCR5 occupancy by 873140 in azine (SDP) derivatives. Aplaviroc (licensed from Ono HIV-negative and HIV-positive subjects. 12th Conference Pharmaceuticals, Japan; Figure 2) is effective at inhibiting on Retroviruses and Opportunistic Infections. Boston, USA, laboratory-adapted and primary R5 HIV-1 isolates in vitro 22–25 February 2005. Abstract 77) to up to 5 days for with an IC50 ranging from 0.1 to 0.6 nM (Maeda et al., maraviroc (Fätkenheuer et al., 2004), although these 2004) and is currently in Phase IIb/III development. apparent differences may be because of differing frequen- According to mode-of-action studies, aplaviroc does not cies of viral load measurement among these studies. There inhibit RANTES or MIP-1β binding, yet it does inhibit are several possible reasons for the rebound delay. A long RANTES-mediated signalling and chemotaxis (Maeda et plasma half-life would result in drug levels remaining high al., 2004; Watson et al., 2005). Together with the observa- enough to inhibit viral entry for some time after drug tion that the antagonism of CCR5 by aplaviroc is saturable, discontinuation. A similar phenomenon has also been seen these findings are consistent with the allosteric model of with some of the approved antiretroviral drugs, such as the CCR5 antagonism proposed by Tsamis et al. (2003). NNRTIs efavirenz (Taylor S, Allen S, Fidler S, White D, Indeed, co-administration studies of aplaviroc with SCH- Gibbons S, Fox J, Clarke J, Weber J, Cane P,Wade A, Smit C, vicriviroc, TAK-779 and maraviroc support the theory E & Back D [2004] Stop Study: after discontinuation of that all five of these CCR5 antagonists bind to an allosteric efavirenz, plasma concentrations may persist for 2 weeks or site on CCR5 to alter ECL2 conformation but, as longer. 11th Conference on Retroviruses and Opportunistic evidenced by their differential effects on RANTES Infections. San Francisco, CA, USA, February 2004. binding, they do not exert the same allosteric effect Abstract 131) and nevirapine (Muro et al., 2005). This (Watson et al., 2005). This observation highlights the explanation may be sufficient to account for the 1–2 day importance of infectivity assays when assessing new small- delay seen with vicriviroc, which has a plasma half-life of molecule CCR5 antagonists because, unlike orthosteric approximately 24 h (Schurmann et al., 2004: see above). inhibitors of receptor binding, the efficacy of a non- However, for maraviroc, a plasma half-life of 16–23 h is not competitive, allosteric antagonist is more related to the sufficient to account for a 5-day prolongation of virological specific conformational changes it induces in the target response. An alternative explanation is that prolonged receptor rather than a simple function of its receptor- receptor occupancy may result in CCR5 molecules binding efficiency. In a Phase IIa study, patients receiving remaining blocked with respect to HIV entry after drug an aplaviroc regimen of 600 mg BID for 10 days demon- levels decline. For example, in all patients treated with strated a mean maximum viral load reduction from baseline maraviroc during the monotherapy studies there was a high of 1.66 log10 copies/ml (Lalezari et al., 2004: see above). degree of CCR5 occupancy during the dosing period The drug appears to be well tolerated when taken orally, (mean ≥85%, pre-dose on days 5 and 10) and, at all doses apart from mild/moderate gastrointestinal side effects except 25 mg QD, CCR5 occupancy remained >60% including abdominal cramps, nausea and diarrhoea 5 days after drug discontinuation (Fätkenheuer et al., 2004: (Adkison et al., 2005b). In vivo CCR5 occupancy suggests see above) (Figure 4). Similarly, 95% CCR5 occupancy by a long receptor binding half-life (Demarest J, Adkison K, aplaviroc during dosing in vivo has been reported, with an Shachoy-Clark A, Schell K, Reddy S, Fang L, O’Mara K, estimated half-life of between 69 and 152 h (Sparks et al., Shibayama S & Piscitelli S [2004] Single and multiple dose 2005: see above). In vitro data generated using membranes escalation study to investigate the safety, pharmacokinetics, purified from Chinese hamster ovary cells in a MIP-1α and receptor binding of GW873140, a novel CCR5 displacement assay suggested a CCR5 binding half-life of receptor antagonist, in healthy subjects. 11th Conference on approximately 190 h for maraviroc and 200 h for aplaviroc Retroviruses and Opportunistic Infections. San Francisco, (Watson et al., 2005). The methods used to measure CCR5 USA, 8–11 February 2004. Abstract 139). occupancy are still under development and can vary widely; the relevance of measurements taken in vitro to the in vivo Pharmacodynamics situation is not clear. For example, whilst it is most practical to study receptor occupancy ex vivo in peripheral blood In monotherapy trials of CCR5 antagonists the rebound in samples, the majority of virus replication within an infected viral load after treatment discontinuation does not appear individual occurs in the secondary lymphoid tissue, such as to be immediate and viral load may continue to fall after the gut associated lymphatic tissue (GALT) (Brenchley et cessation of the drug. This delayed rebound appears to be a al., 2004; Veazey et al., 1998). Also, factors such as the class effect and its duration varies from 1 to 2 days for degree of general immune activation in the HIV-infected

Antiviral Chemistry & Chemotherapy 16.6 347 M Westby & E van der Ryst

Figure 4. Maraviroc CCR5 saturation in treatment-naive, HIV-infected subjects receiving various regimens of maraviroc monotherapy or placebo for 10 days

25 mg QD

) 50 mg QD

% 120 100 mg BID ( 300 mg BID n Placebo 1007 o

i 100

t 150 mg BID fed a

r 150 mg BID

u 80 t 100 mg QD

a 300 mg QD s 60 Placebo 1015 5 R

C 40 C

0 0 5 10 15 20 25 30 35 40 45 Time (day)

Fätkenheuer et al., 2004.

individual will influence the rate of de novo CCR5 expres- assays use patient-derived envelope sequences amplified sion during the dosing interval (Ostrowski et al., 1998). from plasma to infect CD4+ cell lines expressing either Receptor occupancy will therefore be affected both by the CXCR4 or CCR5. Co-receptor usage is determined by the compound’s ability to remain bound to the receptors presence of viral replication in these cell lines, as indicated present at the time of dosing as well as its concentration by the expression of a reporter gene. The HIV-1 genotypic being sufficiently high during the entire dosing interval to methodologies described to date are based on the principle bind to any CCR5 molecules subsequently expressed. The that the V3 loop is a major determinant of co-receptor combination of prolonged CCR5 occupancy and a long usage. For example, an association was identified between half-life may therefore be required for a convenient and SI/X4 phenotypes and positively charged amino acids at efficacious dosing schedule. position 11 or 25 in the V3 loop, known as the ‘11/25 rule’. A study in 1191 patients to investigate this relationship Tropism and virus evolution in the context demonstrated a strong association between the X4 pheno- of CCR5 antagonist therapy type and the 11/25 genotype (P<0.0001) but, conversely, the 11/25 genotype was not a particularly sensitive (33%) There is a need for reliable assays to assess HIV co-receptor predictor of X4 phenotype (Brumme et al., 2005). Other tropism in patient samples now that three CCR5 antago- algorithms exist (for example position-specific scoring nists have progressed from in vitro development to Phase matrices, PSSM; Jensen et al., 2003), the aim of these being IIb/III efficacy studies as components of multi-drug regi- to incorporate more information from other amino acid mens. Phenotypic and genotypic-based tropism assays have residues to improve their predictive power. A DNA-based both been described (reviewed by Coakley et al., 2005). assay using a different approach to sequencing has also Promising phenotypic assays have been developed and been described by Nelson et al. (1997), who used a V3- there are two available commercially (Phenoscript, specific heteroduplex-tracking assay (V3-HTA) to study VIRalliance, France; and PhenoSense HIV, Monogram V3 loop sequences associated with the SI phenotype. In Biosciences Inc., formally ViroLogic Inc., USA). Both this assay a heteroduplex is formed between a patient-

derived V3 loop sequence and a B-clade consensus V3 loop (Kitrinos et al., 2005). The clinical relevance of these find- sequence and the presence of evolutionary variants is ings in the context of an optimized multi-drug regimen is measured by divergence from the consensus. Genotypic not clear. It is important to note that the emergence of X4 assays are generally cheaper and easier to run than their variants has been observed in patients receiving HAART phenotypic counterparts, although the relative specificity for 5 years with undetectable viral load (Delobel et al., and sensitivity of the approaches in predicting clinical 2005). Hence, X4 emergence is likely to occur over time outcome will ultimately determine their utility in conjunc- with all HAART regimens and, therefore, it will be impor- tion with CCR5 antagonists. tant to understand the net effect of CCR5 antagonists In an individual identified as harbouring only R5 virus, added to or as part of a HAART regimen on the X4 virus could emerge under drug selection pressure emergence of CXCR4-using variants. through one of two possible mechanisms: the outgrowth of a previously undetectable minority reservoir of X4 virus, or Future directions the selection of mutations in an R5 virus leading to a switch to CXCR4 tropism. Interestingly, there have been several It will be interesting to see the results of Phase IIb and independent observations in vitro of the selection of virus IIb/III studies to determine the efficacy of CCR5 antago- that is resistant to a CCR5 antagonist but has retained nists in HIV-infected patients, including patients with CCR5 tropism (Kuhmann et al., 2004; Maeda et al., 2004; dual/mixed-tropic HIV infections, who are receiving back- Maeda et al., 2000; Trkola et al., 2002). In contrast, there is ground HAART.These data should be available in the near only one published report of R5 virus switching tropism in future, from the large ongoing Phase IIb/III studies of vitro in the presence of a CCR5 antagonist (Mosier et al., maraviroc, aplaviroc and vicriviroc in combination with 1999). Indeed, the findings of that study may not be rele- optimized background therapy in HIV-1-infected, treat- vant to treatment with small-molecule CCR5 antagonists, ment-experienced patients. Resistance to CCR5 antagonists since the agents used by Mosier and colleagues were has been described (Maeda et al., 2000; Marozsan et al., analogues of the agonist, RANTES. The co-receptor shift 2005; Trkola et al., 2002), although resistance does not could, therefore, have been selected as a result of internal- appear to be generated easily (Westby M, Mori J, Smith- ization of CCR5. Studies showing selection for CXCR4- Burchnell C, Lewis M, Mosley M, Perruccio F, Mansfield tropic variants by serial passage of R5 variants in cell R, Dorr P & Perros M [2005] Maraviroc [MVC, UK- cultures expressing low CCR5 levels suggest that the path- 427,857]-resistant HIV-1 variants, selected by serial ways to tropism shift involve sequential accumulation of passage, are sensitive to CCR5 antagonists [GW873140, gp120 mutations in both the V1/V2 and V3 regions Schering-C, Schering-D] and T-20 [enfuvirtide]. 14th (Pastore et al., 2004). Therefore, emergence of X4 variants International Resistance Workshop, Québec, Canada. 7–11 during CCR5 antagonist treatment in vivo may be more June 2004. Abstract 65). In those reports, resistance did not likely from pre-existing reservoirs than via mutation of involve a switch in co-receptor usage, while Pastore and co- circulating R5 virus. workers (2004) have suggested that co-receptor switching is The emergence of X4 virus during treatment with an inefficient process in many cases. With multiple agents CCR5 antagonists has been described for a minority of in advanced clinical development, the issue of cross-resis- treated patients undergoing short-term monotherapy. In tance between CCR5 antagonists will be of interest to the Phase IIa trials of maraviroc CXCR4-tropic virus was determine whether drug sequencing within this promising detected in two of 63 patients with CCR5-tropic virus at new class is possible, providing, of course, that clinical baseline following 10 days of monotherapy. Phylogenetic failure is not always mediated by emergence of or switch to analysis indicated that the CXCR4-using variants probably CXCR4-using species. Furthermore, simultaneously emerged from a pre-existing CXCR4-using reservoir, targeting more than one stage of the process of rather than via co-receptor switch of a CCR5-tropic clone HIV-1 entry using drug combinations could be a valuable under selection pressure from maraviroc (Lewis M, Van der treatment option in the future, as implied by early in vitro Ryst E, Youle M, Jenkins T, James I, Medhurst C & findings demonstrating synergistic activity of SCH-C with Westby M [2004] Phylogenetic analysis and co-receptor the fusion inhibitor, enfuvirtide (Tremblay et al., 2002), and tropism of HIV-1 envelope sequences from two patients by a more recent demonstration of synergy between a with emergence of CXCR4 using virus following treatment CCR5 antagonist (AMD887, AnorMED, Canada) and a with the CCR5 antagonist UK-427,857. 44th Interscience CXCR4 antagonist (AMD070) (Schols D, Vermeire K, Conference on Antimicrobial Agents and Chemotherapy. Hatse S, Princen K, De Clercq E, Calandra G, Fricker S, Washington DC, USA, 30 October–2 November 2004. Nelson K, Labrecque J, Bogucki D, Zhou Y, Skerlj R & Poster H-584b). Similar findings have been presented for Bridger G [2004] In vitro anti-HIV activity profile of vicriviroc (Schurmann et al., 2004: see above) and aplaviroc AMD887, a novel CCR5 antagonist, in combination with

Antiviral Chemistry & Chemotherapy 16.6 349 M Westby & E van der Ryst

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Received 10 August 2005, accepted 6 October 2005