Chemokine Antagonists as Therapeutics: Focus on HIV-1

ANRV299-ME58-30 ARI 5 December 2006 20:52

Chemokine Antagonistsas Therapeutics:Focus on HIV-1Athe M.N. Tsibris1,2 and Daniel R. Kuritzkes2,3

1Infectious Disease Unit, Massachusetts General Hospital, Boston,Massachusetts 02114;2Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115;3Section of Retroviral Therapeutics, Brigham and Women’s Hospital, Boston,Massachusetts 02115; email: [email protected]

Annu. Rev. Med. 2007. 58:445–59

First published online as a Review in Advance onSeptember 7, 2006

The Annual Review of Medicine is online athttp://med.annualreviews.org

This article’s doi:10.1146/annurev.med.58.080105.102908

Copyright c© 2007 by Annual Reviews.All rights reserved

0066-4219/07/0218-0445$20.00

Key Words

virus entry, CCR5, CXCR4, viral tropism, G protein–coupledreceptors

AbstractHuman immunodeficiency virus type 1 (HIV-1) entry into targetcells is a multistep process involving the interaction of viral envelopeproteins with cell surface receptors. Binding to CD4 is followed byengagement of specific chemokine receptors (CCR5 or CXCR4),triggering molecular rearrangements in the envelope transmem-brane subunit that result in membrane fusion. Chemokine recep-tor antagonists that block the interaction of the HIV-1 envelopewith CCR5 or CXCR4 potently inhibit HIV-1 in vitro. Pilot studiesof orally bioavailable small-molecule CCR5 inhibitors in HIV-1-infected subjects have provided proof of concept for this novel drugclass; phase III safety and efficacy trials are under way.

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INTRODUCTION

The use of potent combination antiretrovi-ral therapy in the developed world has sub-stantially reduced morbidity and mortalityfrom HIV-1 infection. To date, the threemajor classes of drugs—nucleoside reversetranscriptase inhibitors, non-nucleoside re-verse transcriptase inhibitors, and proteaseinhibitors—have targeted two viral enzymes:reverse transcriptase and protease. Althoughpotent viral suppression can be achieved withthese regimens, the emergence or acquisi-tion of drug-resistant HIV-1 results in treat-ment failure and can significantly limit fu-ture therapeutic options. Moreover, use of an-tiretroviral therapy has been associated witha variety of adverse effects. In an effort toprovide additional therapeutic options to pa-tients with drug-resistant virus or treatment-limiting toxicities on existing therapy, newdrugs with potent antiretroviral activity arebeing developed that target HIV-1 entry intocells. A new drug class, the chemokine recep-tor antagonists, targets cellular proteins onthe surface of CD4+ T cells and macrophagesthat serve as coreceptors for HIV-1, therebyblocking their interaction with the viral en-velope glycoprotein (gp120). Small-moleculeantagonists of both CCR5 and CXCR4 arebeing tested for their therapeutic potential; asof this writing, at least one CCR5 antagonisthas advanced to phase III clinical trials.

We review the structure and function ofCCR5 and note the effect of CCR5 allelicvariants and promoter polymorphisms on sus-ceptibility to HIV-1 infection. We summarizethe clinical data on the use of chemokine in-hibitors in treatment-naive and -experiencedpatients and discuss some of the challengesposed in the clinical development of these in-hibitors. We also review emerging concernsthat chemokine receptor blockade may in factcontribute to immune dysfunction. With thisbody of evidence in mind, we assess the po-tential role that chemokine inhibitors mayplay in future combination therapy againstHIV-1.

ENVELOPE-MEDIATED VIRALMEMBRANE FUSION

Entry of HIV-1 into target cells proceeds bythe fusion of viral and cellular membranes.This event involves viral and cellular pro-tein interactions that lead to conformationalchanges in critical protein structures. As ourunderstanding of the mechanism of HIV-1entry improves, novel targets for inhibitionor antagonism are recognized.

The mechanism of HIV-1 entry shares anumber of features in common with otherenveloped viruses. The HIV-1 external andtransmembrane subunits of the envelope gly-coprotein (gp120 and gp41, respectively) me-diate viral binding to and fusion with host tar-get cells. The env gene encodes the precursorpolyprotein gp160, which is cleaved by cellu-lar proteases to yield the mature gp120 andgp41 subunits. Both gp120 and gp41 traffic tothe viral membrane, where they remain non-covalently associated with a stoichiometry ofone molecule of gp120 to one molecule ofgp41. Three of these units aggregate on themembrane surface to form the gp120/gp41heterotrimer (1, 2, 2a). The association ofgp120 with gp41 in the trimer traps gp41 in aconformationally metastable state, the energyfrom which can later be exploited to acceleratethe rate of fusion (3).

Binding of gp120 to its primary recep-tor on the cell surface, CD4, is the firststep in membrane fusion. Although CD4-independent entry can occur in vitro, all pri-mary HIV-1 isolates require CD4 for viralentry (4). The CD4 binding site is not fullyformed in unliganded gp120 but is stabilizedand fixed by the approach of CD4 (5). Bind-ing to CD4 typically is followed by bind-ing to either the CCR5 or CXCR4 corecep-tor, which is required for fusion to proceed(6–10). Coreceptor recognition is defined byseveral structural elements of gp120 that in-clude the first and second hypervariable re-gions (V1-V2), the bridging sheet (an antipar-allel, four-stranded β sheet that connects theinner and outer domains of gp120), and most

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importantly, the V3 loop (11–14). The V1-V2 stem influences coreceptor usage throughits amino acid composition as well as by thedegree of N-linked glycosylation (15). Littlestructural variation of the bridging sheet isfound in human and primate lentiviruses, sug-gesting that this structure serves as a commondeterminant for recognition of either core-ceptor. The V3 loop, by contrast, is highlyvariable and is the principal determinant ofcoreceptor specificity (7, 16–19).

According to current models of HIV-1 en-try, sequential binding of gp120 to CD4 andthe CCR5 or CXCR4 coreceptor leads to therelease of gp41 from its metastable conforma-tion. The hydrophobic N-terminus, or fusiondomain, of the gp41 ectodomain is therebyfreed to insert into the target cell membrane(3, 20, 21). Two trimeric coiled-coil structuresin gp41, comprising heptad repeats 1 and 2(HR-1 and HR-2, respectively), rearrange inan antiparallel orientation to form a six-helixbundle that leads to the approximation of thetwo membranes and eventual fusion (3).

THE HIV-1 CORECEPTORS:CCR5 AND CXCR4

The chemokine receptors CCR5 andCXCR4 belong to the family of seven–transmembrane segment G protein–coupledreceptors (GPCRs). Significant redundancyexists in the chemokine network—somereceptors have multiple ligands, and someligands bind to multiple receptors. Forexample, CCR5 binds RANTES (regulatedon activation, normal T cell expressed andsecreted), MIP-1α (macrophage inflam-matory protein 1α), and MIP-1β (22).Conversely, MIP-1α can bind to CCR1or CCR5, and RANTES can bind CCR1,CCR3, or CCR5, although MIP-1β bindsonly to CCR5. By contrast, the only naturalligand for CXCR4 is stromal-derived factor1 (SDF-1), which interacts exclusively withCXCR4. A standardized nomenclature forthese ligands has been developed (Table 1),

Table 1 Chemokine nomenclature

Chemokines

Systematic names Common names Chemokine receptor(s)CCL2 MCP-1 CCR2CCL3 MIP-1α CCR1, CCR5CCL4 MIP-1β CCR5CCL5 RANTES CCR1, CCR3, CCR5CCL7 MCP-3 CCR1, CCR2, CCR3CCL8 MCP-2 CCR3CCL11 Eotaxin CCR3CXCL8 IL-8 CXCR1CXCL12 SDF-1 CXCR4

HR: heptad repeat

GPCR: Gprotein–coupledreceptor

RANTES:regulated onactivation, normal Tcell expressed andsecreted

MIP: macrophageinflammatoryprotein

SDF:stromal-derivedfactor

ECL: extracellularloop

SI:syncytium-inducing

but in this review we refer to the ligands bytheir more familiar common names.

CCR5 is expressed on memory T cells, ac-tivated T cells, gastric-associated lymphoidtissue, and macrophages. It demonstrates agreater affinity for soluble gp120 (Kd <

10 nM) than does CXCR4 (Kd 200–500 nM)(23). The tyrosine-sulfated N-terminus ofCCR5 forms an essential determinant of bind-ing to gp120 (24, 25). The current modelof gp120–CCR5 binding involves an ini-tial interaction between sulfated tyrosines inthe CCR5 N-terminus and gp120 followedby a second interaction between gp120 andthe CCR5 transmembrane domains. Stud-ies implicate extracellular loop 2 (ECL2) ofCCR5 and/or a segment at the transmem-brane 4/ECL2 junction as important for thesecond interaction and subsequent HIV-1 in-fection (25, 26).

CORECEPTOR UTILIZATION BYHIV-1

Soon after infection with HIV-1, most pa-tients harbor virus that grows relatively slowly,does not induce fusion of T cells (syncytiumformation) in vitro, and grows equally wellin monocytes and lymphocytes. Later in in-fection, a more highly cytopathic, syncytium-inducing (SI) variant can be isolated frommany patients (27, 28). These SI viruses tendto grow more rapidly in tissue culture and are

www.annualreviews.org • Chemokine Antagonists and HIV-1 447

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usually characterized by their ability to growin T cell lines (T cell tropism). Emergenceof SI variants is associated with an accelerateddecline in CD4+ lymphocyte counts (29). Sev-eral studies have found that individuals withSI isolates of HIV-1 progress more rapidly toAIDS than do those with non-SI isolates (30).Presence of an SI isolate is also a significant in-dependent risk factor for disease progressionand death (31).

SI strains are now known as X4 strains(because they use the CXCR4 coreceptor)and non-SI strains as R5 strains (becausethey use the CCR5 coreceptor). Occasionallyduotropic (R5/X4) viruses are isolated. Theseviruses can use both CCR5 and CXCR4coreceptors and can infect monocytes andCD4+ lymphocytes with similar efficiency. Itis thought that R5/X4 viruses represent inter-mediate forms in the evolution of SI virusesfrom non-SI strains.

Previous work demonstrated that most pa-tients from whom SI viruses were isolated hada mixture of SI and persistent non-SI viruses.This observation has now been confirmed us-ing contemporary assays that test the ability ofviruses to replicate on cells that express eitherthe CCR5 or CXCR4 coreceptor (32–34).Samples that contain only R5 viruses will showevidence of growth only on CCR5-expressingcells; conversely, samples that contain only X4viruses replicate only on CXCR4-expressingcells. Samples that contain a mixture of R5and X4 viruses, or those that contain virusesthat are dually tropic for both coreceptors,will show evidence of replication on both

cell lines. Distinguishing between these twopossibilities (mixed R5 and X4 versus dualtropism) requires clonal analysis; for thisreason, samples giving positive results onboth cell lines are said to have “dual/mixed”tropism.

Data from cross-sectional studies showthat the 70%–80% of patients with early-stagedisease harbor only R5 viruses; the remainderhave dual/mixed tropic viruses (Table 2). Bycontrast, nearly half of patients with advanceddisease have dual/mixed viruses; the other halfremain R5, with only a few percent showingpure X4 virus (33, 35). In these analyses, lowerCD4 count was an independent risk factor forpresence of dual/mixed or X4 virus.

The role of chemokine receptor tropismin determining the rate of disease progres-sion remains unresolved. Although the preva-lence of X4 variants increases with decreas-ing CD4+ cell count, X4 variants emerge inonly half of patients who progress to AIDS(28–32, 34). The possibility that emergenceof X4 variants is a consequence, rather thana cause, of advancing immunodeficiency re-mains a plausible alternative explanation forthe apparent association of X4 virus with dis-ease progression. Macaques infected with aSHIV (SIV/HIV chimera) that expresses anX4 HIV-1 envelope show rapid depletion ofCD4+ cells, suggesting a causal role of X4viruses in rapid disease progression (36, 37).On the other hand, the long interval betweeninfection and emergence of X4 viruses in mostpatients argues for strong selection against X4viruses early in the course of HIV disease. The

Table 2 Prevalence of R5 and X4 virus in different cohorts of HIV-1-infected subjects

Population Sample size R5 only R5/X4 X4 only ReferenceART-naive∗ 323 88% 12% 0% 84ART-naive 979 82% 18% 0.1% 32ART-naive 402 81% 19% N/A 34ART-experienced 117 67% 28% 5% 84ART-experienced 125 78% 22% N/A 34ART-experienced 724 50% 48% 2% 33

∗ART, antiretroviral therapy.


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development of chemokine receptor antago-nists as a novel class of antiretroviral agentsmay provide new tools to address this impor-tant pathogenesis question.

CCR5 VARIANTS

The risk of disease progression is modulatedby a variety of host genetic factors. Amongthe most important are promoter polymor-phisms and allelic variants in the genes en-coding the chemokine coreceptors and theirligands. Several CCR5 promoter polymor-phisms have been described that either accel-erate or delay disease progression.

The best-known CCR5 allele, CCR5�32,results in a frameshift and truncation of thenormal CCR5 protein (38, 39). As a result,no CCR5 is expressed at the cell surface.This allele has a frequency of ∼10% in Cau-casian populations but is absent from nativeAfrican, American Indian, Tamil Indian, andEast Asian ethnic groups (39, 40). Individualshomozygous for the CCR5�32 allele (∼1%of Northern European Caucasians) resist in-fection with R5 virus (38). HIV-1-infectedpersons who are heterozygous for CCR5�32demonstrate slower rates of disease progres-sion than HIV-1-infected persons homozy-gous for wild-type CCR5 (41). The initialreports of CCR5�32 homozygotes demon-strated no additional phenotypes. This “ex-periment of nature” provides some measureof confidence that pharmacological blockadeof CCR5 will not lead to serious health con-sequences, and provides a strong rationale fordevelopment of CCR5 inhibitors.

Much speculation has surrounded the ori-gin and positive selection of CCR5�32. Al-though initial reports suggested that this dele-tion emerged during epidemics that spreadacross Europe in the thirteenth century, morerecent analyses based on improved and up-dated datasets suggest that CCR5�32 has beenpresent in the population for ∼5000 years andexhibits no positive selection over this timeperiod (42).

CCR5 ANTAGONISTS

Because coreceptor binding is essential forHIV-1 entry, antagonism of gp120 bindingto CCR5 presents an attractive target for ra-tional drug design. Early efforts to block thegp120/CCR5 interaction employed chemicalmodifications of the known CCR5 ligands,especially RANTES. These compounds suf-fered, however, from poor oral bioavailabil-ity and the induction of intracellular signalingcascades upon CCR5 binding. Several novelin vitro approaches have significantly reducedCCR5 cell surface expression and protectedcells from challenge with R5 HIV-1 virus.These include development of an intrabodythat retains bound CCR5 in the endoplasmicreticulum and construction of a lentiviral vec-tor that introduces siRNA into lymphocytesand inhibits CCR5 transcription (43, 44).

The most promising drugs in this class,however, are nonpeptidic, orally bioavailable,small-molecule antagonists of CCR5. Thesemolecules are noncompetitive allosteric an-tagonists of CCR5 with molecular weightsof 500–600 D. CCR5 antagonists have beengiven generic names with the suffix “-viroc,”an abbreviation for viral receptor occupancy.Several CCR5 antagonists have been evalu-ated for their therapeutic potential, includ-ing TAK-779, TAK-220, TAK-652, aplaviroc,maraviroc, AK602, SCH31125 (SCH-C), andvicriviroc (45–52). These compounds bindto the coreceptor in a cavity formed be-tween CCR5 transmembrane helices 1, 2, 3,and 7 (53, 54). Coadministration of multi-ple antagonists demonstrates that they bindto a common site on CCR5 (55). Three ofthese compounds—aplaviroc, maraviroc, andvicriviroc—have progressed to at least phaseIIb clinical trials. They exhibit potent inhibi-tion of HIV-1 replication in vitro and demon-strate antiviral activity against an array oflaboratory-adapted strains as well as primaryviral isolates. Anti-HIV-1 activity is evidentacross all clades of group M HIV-1. Theseagents do not appear to affect CCR5 cellsurface levels or intracellular signaling upon


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CCR5 binding. As we discuss below, a numberof unexpected events has complicated clinicaltrials with these drugs, and led to the termi-nation of aplaviroc’s development.

Aplaviroc

Aplaviroc, formerly GSK 873,140, demon-strated antiviral activity with minimal toxi-cities during short-term monotherapy stud-ies. Although aplaviroc blocked MIP-1β

at nanomolar concentrations, RANTES-mediated signaling was less susceptible toaplaviroc inhibition (55). Aplaviroc in variousdosing regimens led to a 1–1.6 log10 reduc-tion in viral loads during 10 days of treatment(56). In phase IIb trials, however, 4 out of∼300 patients developed severe hepatotoxi-city, which on liver biopsy was found to beconsistent with drug-induced hepatitis. Thisfinding led to the termination of the trial (57).Similarly, 1 out of 26 patients participatingin a phase III trial of aplaviroc demonstratedelevation of alanine aminotransferase to 24times normal levels. This trial was also termi-nated, and clinical development of aplavirocwas halted. No deaths occurred, and the hep-atitis resolved with aplaviroc discontinuation.Additional analysis may shed light on the re-lationship of aplaviroc to the development ofhepatitis in those patients.

Maraviroc

Maraviroc, formerly UK 427,857, is aspirodiketopiperazine CCR5 antagonist withpotent in vitro and in vivo anti-HIV-1 activity.The molecule is a pure CCR5 antagonist thatblocks MIP-1α- and RANTES-mediated sig-naling at nanomolar concentrations (45). In a10-day monotherapy trial conducted in HIV-1-infected subjects with R5 virus, administra-tion of maraviroc at doses up to 600 mg dailyresulted in ≥1.6 log10 reductions of plasmaHIV-1 (46). Treatment-related adverse effectswere minor and included headache, dizziness,asthenia, gingivitis, and nausea. A shift in vi-ral tropism, however, was noted in two sub-

jects. One subject experienced transient emer-gence of a mixed tropic population on day11 of therapy that was not detectable on day40. The other subject developed a dual- ormixed-tropic virus by day 11 that remaineddetectable through day 433 of follow-up. Ad-ditional studies suggested that these virusesemerged from a pretreatment reservoir of X4viruses that was not detected on initial tropismtesting (58).

Ongoing trials are evaluating the activity ofmaraviroc as part of a combined antiretrovi-ral regimen in treatment-naive and treatment-experienced HIV-1-infected subjects. Inferiorefficacy compared with a standard efavirenz-based regimen led to discontinuation of the300-mg once-daily arm in the treatment-naive trial, but the 300-mg twice-daily armis continuing. One subject receiving maravi-roc at a dose of 300 mg daily, in combina-tion with zidovudine and lamivudine, devel-oped severe hepatotoxicity that necessitatedliver transplantation (59). The subject was in-fected with hepatitis C virus (HCV) withoutdetectable HCV RNA and had recently ini-tiated other potentially hepatotoxic medica-tions, including isoniazid and trimethoprim-sulfamethoxazole. This patient had elevatedtransaminases prior to receiving the first doseof maraviroc. The causal role of maraviroc inthis patient’s liver failure is therefore unclear,but careful monitoring of liver function in pa-tients receiving maraviroc is warranted untiladditional safety data are available.

Vicriviroc

Vicriviroc, formerly known as SCH417690or SCH-D, is an order of magnitudemore potent than the first-generation com-pound Schering C (SCH-C) (60). Like mar-aviroc, this molecule blocks signaling bythe C-C chemokines at nanomolar con-centrations (61). Clinical trials with SCH-C demonstrated unacceptable cardiovascularand central nervous system (CNS) side effects,presumably due to drug affinity for receptorsother than CCR5 (61). The QTc prolongation


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seen with SCH-C administration may resultfrom an interaction with the cardiac hERGK+ channel (human ether-a-go-go–relatedgene potassium channel), whereas the CNSside effects are partially attributable to theaffinity of SCH-C for muscarinic receptorsin brain. Vicriviroc improves receptor selec-tivity for CCR5 and decreases, but does noteliminate, binding to muscarinic receptors orthe hERG K+ channel (60).

Preclinical studies of vicriviroc in animalsshow a dose-limiting toxicity of seizures. Test-ing in healthy volunteers or HIV-1-infectedsubjects has not, to date, revealed QTc prolon-gation or CNS side effects. Vicriviroc is me-tabolized by CYP3A4 and thus can be boostedwith ritonavir. Data from a 14-day monother-apy trial demonstrated a reduction of plasmaHIV-1 RNA by ∼1.0–1.5 log10 copies/ml (51).A phase II trial of vicriviroc in treatment-naiveHIV-1-infected subjects was discontinued be-cause of increased rates of virologic failure inthe vicriviroc arms compared to the controlefavirenz arm [all subjects also received zi-dovudine plus lamivudine as a fixed-dose com-bination (ZDV/3TC)] (62). Subjects random-ized to the vicriviroc arms received an initial14-day period of vicriviroc monotherapy priorto addition of ZDV/3TC. Twenty-eight of68 subjects (38%) receiving vicriviroc expe-rienced virologic breakthrough. Genotypicanalysis demonstrated that virus from all sub-jects who experienced virologic breakthroughdeveloped the M184V mutation for 3TC re-sistance. This finding supports the concernthat vicriviroc may have been underdosed inthis study.

Vicriviroc is currently being evaluated ina phase IIb trial in antiretroviral-experiencedpatients by the AIDS Clinical Trial Group(protocol number A5211). That study is com-paring the safety and efficacy of three differentvicriviroc dosages in combination with an op-timized background regimen (OBR) to OBRalone. Interim results raised concern whenfive cases of malignancy occurred in patientsreceiving vicriviroc: one case of recurrentHodgkin’s disease; a case of non-Hodgkin’s

disease in a subject with previously treatedHodgkin’s; two de novo cases of lymphoma(one Hodgkin’s and one non-Hodgkin’s); andone case of gastric adenocarcinoma (63). Nocancers have been reported to date in the con-trol arm. Because of the small sample size(n = 118) and the participants’ advanced stageof HIV disease, the significance of these re-sults is unclear, and a causal link with vicrivi-roc has yet to be established. Vicriviroc has notpreviously been shown to be carcinogenic ormutagenic, and preclinical toxicology studiesin animals showed no increased malignancyrisk. Severe hepatitis has not been observedto date in clinical trials of vicriviroc.

OTHER CCR5 INHIBITORS

In addition to small-molecule CCR5 antago-nists, several monoclonal antibodies directedagainst CCR5 are being developed, includingPRO140 (Progenics) and CCR5mAb004(Human Genome Sciences). Given theoral bioavailability and advanced stage ofclinical development of the small-moleculeinhibitors, it is unclear what role CCR5monoclonal antibodies may play in futureantiretroviral therapy regimens. CCR5monoclonal antibodies could have a distinctmechanism of action, presumably stericallyhindering gp120-CCR5 binding through themuch larger size of the antibody structure.Compelling clinical data are not yet availablefor this class of entry inhibitor compounds.

HIV-1 RESISTANCE TO CCR5INHIBITORS

The high error rate of HIV-1 reverse tran-scriptase and rapid turnover of the viral pop-ulation facilitate the emergence of drug-resistant mutants under conditions of partialdrug efficacy. In the case of CCR5 in-hibitors the drug target is a host cell pro-tein, which is unlikely to undergo mutationin response to drug pressure. Viral adaptationto CCR5 inhibitors could, however, involvechanges in the viral envelope protein that alter


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dependence on CCR5. Of greatest theoreticalconcern is the potential selection of CXCR4-tropic virus in response to CCR5 blockade.Whether or not emergence of X4 viruses inthis setting would accelerate disease progres-sion is the subject of much speculation (23,64). Data from two macaques infected withR5/X4 virus demonstrated a transient increasein X4 viral load, followed by a rapid declineback to baseline during therapy with a CCR5antagonist (65).

Concerns over the emergence of X4 virushave been somewhat allayed by experience todate. Serial in vitro passage of HIV-1 in thepresence of various CCR5 inhibitors does notselect for X4 virus. Rather, resistance appearsto be mediated by changes in HIV-1 gp120that allow the protein to interact with CCR5in the drug-bound conformation. For exam-ple, selection experiments performed withthe investigational CCR5 inhibitor AD101resulted in high-level (>20,000-fold) resis-tance after 19 passages and conferred cross-resistance to SCH-C (66). The mutant viruscontinued to use CCR5 and not CXCR4for entry. Complete resistance to AD101 re-sulted from four amino acid substitutions—K305R, H308P, A316V, and G321E—in theV3 loop of gp120 (67). Individually, these mu-tations conferred partial resistance to AD101,suggesting that the development of high-level resistance to CCR5 antagonists requiresthe step-wise accumulation of mutations ingp120. The degree of resistance conferred bythese mutations depended on the envelopebackbone into which they were introduced.

Maraviroc-resistant HIV-1 variants havebeen generated by serial passage in vitro (68).Two mutations in the V3 loop, T316A andV323I, were associated with maraviroc resis-tance; a third V3 loop mutation, A319S, wasnot consistently observed. Standard drug sus-ceptibility testing showed a decrease in thepercent maximal inhibition achievable withmaraviroc, without an appreciable shift inthe 50% inhibitory concentration (IC50) (68).This pattern has been ascribed to the non-competitive nature of maraviroc inhibition of

gp120 binding to CCR5, and it suggests thatresistant viruses have developed the capacityto interact with CCR5 in the drug-bound con-formation. Maraviroc-resistant recombinantsretained sensitivity to both aplaviroc and en-fuvirtide. Aplaviroc-resistant viruses also havebeen isolated after serial in vitro passage. Allviruses retained CCR5 tropism, although thecells used for passage had either no or low lev-els of CXCR4. In contrast to maraviroc resis-tance, aplaviroc resistance was characterizedby a rightward shift in the IC50 and no changeor plateau in the percent maximal inhibition(69). Mutations that conferred decreased sen-sitivity to aplaviroc did not cluster in the V3loop and were found in the C1–5, V1, and V3regions of gp120 and within gp41 as well. Inaddition, the phenotypic effects of these mu-tations depended on the env context in whichthey were expressed. That is, mutations thatemerge in a given env backbone do not con-fer resistance when engineered into env froma different viral strain.

Clonal analysis of V3 loop sequences ob-tained from subjects experiencing virologicfailure on vicriviroc showed evidence of ge-netic selection of the V3 loop sequences,although no common pattern of mutationscould be identified across samples from differ-ent subjects (70). As in the case of maravirocresistance, the V3 loop changes that emergedwith vicriviroc treatment were associated witha decrease in the percent maximal inhibitionachievable without a significant shift in IC50.The contribution to vicriviroc susceptibilityof other envelope regions outside the V3 loopremains to be determined.

In summary, the available data suggestthat unlike resistance to reverse transcrip-tase and protease inhibitors, resistance tosmall-molecule CCR5 antagonists may notresult in the selection of stereotypical muta-tions. Rather, the selected mutations may leadto env-specific structural changes that allowgp120 to adapt to an inhibitor-bound con-formation of CCR5. The multiple conforma-tions of CCR5 that exist in vivo, as for anyallosteric protein, may also contribute to the


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variety of CCR5-inhibitor resistance mu-tations observed to date. That is, small-molecule antagonists may bind to severaldifferent conformations of CCR5. The emer-gence of a variety of seemingly unrelated mu-tations could, in the context of a particularenvelope molecule, give rise to commonstructural changes that improve the efficiencyof HIV-1 fusion and viral entry into targetcells.

CONSEQUENCES OF CCR5BLOCKADE

Although GPCRs have been targets of phar-macological inhibition in the past, chemokineinhibitors are the first antiretroviral drugsthat target host proteins. The apparent ab-sence of any significant immunological deficitamong individuals with naturally occurringloss-of-function mutations (i.e., CCR5-�32homozygotes) provides some reassurance thatpharmacological blockade of CCR5 will berelatively benign (71). Presumably, redun-dancy in the chemokine network allows otherchemokine receptors to subsume the functionof CCR5. However, pharmacological block-ade of a receptor in mature individuals mayhave different consequences than congeni-tal absence of the receptor. Thus, the long-term safety of CCR5 blockade remains to beproven.

Several studies of CCR5 knockouts in miceas well as epidemiological data from humanCCR5-�32 homozygotes suggest this dele-tion may have previously unrecognized con-sequences. For example, although no overtpathological changes were noted in a mouseCCR5 knockout, alterations of macrophagefunction and increased susceptibility to cryp-tococcal infections of brain have been re-ported (72, 73). Moreover, increased mor-tality from West Nile virus encephalitis wasseen in CCR5−/− mice and was linked to de-creased leukocyte trafficking into the brain(74). CCR5-deficient mice also have an ab-normal immune response to ocular infec-tion with herpes simplex virus type 1 (75).

In a mouse model of T cell–mediated hep-atitis, CCR5 deficiency increased mortalityand liver injury (76). The lack of signalingdue to the absence of CCR5 was thought toprevent downregulation of the natural killerT cell response, resulting in fulminant hep-atitis. The relevance of this finding to thecases of aplaviroc-induced hepatotoxicity isunclear.

The murine studies have been corrob-orated by studies in humans showing thatCCR5�32 heterozygotes have a sixfold in-creased risk for severe West Nile virus infec-tion and a fivefold increased risk of mortal-ity (77). The immunological importance offunctional CCR5 is underscored by severalstudies demonstrating improved outcomes inpatients with immunologically mediated con-ditions such as rheumatoid arthritis and organtransplantation, among others, in CCR5�32homozygotes (77a, 77b).

CXCR4 ANTAGONISTS

The clinical development of CXCR4 in-hibitors has proceeded more slowly than thatof CCR5 inhibitors. Unlike CCR5 deficiency,CXCR4 deficiency has no naturally occurringmodels. SDF-1α, the natural CXCR4 ligand,is a primordial chemokine involved in a mul-titude of cellular processes, including organo-genesis, stem cell localization, and T cell traf-ficking (78–80). Knockout of either CXCR4or SDF-1 is lethal in mouse embryos, causingmarked hematopoietic, cardiac, and cerebel-lar defects. Nevertheless, small-molecule in-hibitors of CXCR4 have been developed overthe past decade that bind CXCR4 with highaffinity and inhibit replication of X4 virusesin vitro.

Short-term administration of AMD3100demonstrated anti-HIV-1 activity in a subjectwith mixed R5/X4 infection, resulting in a de-crease of the X4 population. Unfortunately,cardiac toxicities precluded further develop-ment of this compound. A backup compound,AMD11070, is currently in phase I–II stud-ies. Of note, CXCR4 blockade was found to


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cause the release of hematopoietic stem cellsfrom the marrow into the peripheral circu-lation. As a consequence, AMD3100 is nowbeing developed as an effective agent for stemcell mobilization prior to harvest in stem celltransplant patients.

CHEMOKINE INHIBITORS ASTOPICAL MICROBICIDES

Considerable interest surrounds the devel-opment of topical microbicides to preventsexual transmission of HIV-1. Multiple cellpopulations susceptible to HIV-1 infection,including Langerhans cells, dendritic cells,macrophages, and T cells, are present ingenital and rectal subepithelial tissues (81).Because these cells express CCR5 and/orCXCR4, topical preparations of chemokineinhibitors might protect against vaginal andrectal transmission of HIV-1. Prevention ofvaginal SHIV transmission in a macaquemodel was demonstrated with high doses ofPSC-RANTES, a modified analog of thechemokine RANTES (82). Combinations ofmicrobicides have also been shown to pro-tect macaques from vaginal SHIV challenge.The entry inhibitors BMS-378,806 [a smallmolecule that prevents exposure of the HR-1after CD4 binding (82a)], CMPD167 (a small-molecule CCR5 inhibitor), and C52L (a pep-tide inhibitor of gp41-mediated fusion) weretested alone or in two- and three-drug com-binations. Sixteen of 20 macaques were pro-tected by the two-drug combinations, and 3of 3 were protected by the application of thethree compounds together (83). Clinical trials

of microbicides are ongoing in Africa, with re-sults expected by late 2007. Combination mi-crobicidal prevention trials are set to begin in2007.

CONCLUSIONS

Viral entry provides a rich array of targetsfor pharmacological inhibition. However, de-spite a large number of candidate moleculesdirected against various steps in the entrypathway, progress has been slow. As of thiswriting, enfuvirtide remains the only entryinhibitor approved by the U.S. Food andDrug Administration. At present, most en-try inhibitors are being evaluated for usein treatment-experienced patients who har-bor drug-resistant HIV-1. Given the potency,safety, and established efficacy of availableregimens for the treatment of antiretroviral-naive patients, legitimate concerns have beenraised about testing novel agents in treatment-naive populations. However, the approach tofirst-line therapy is unlikely to remain staticbecause of the rising prevalence of trans-mitted drug resistance and possible long-term toxicity of currently available regimens.As additional entry inhibitors enter clinicaldevelopment, the possibility of synergisticcombinations will be explored, including thecombined use of enfuvirtide with a CCR5inhibitor or coadministration of CCR5 andCXCR4 inhibitors. Ongoing and future in-vestigations will elucidate and maximize thepotential of entry inhibitors to expand therepertoire of antiretroviral medications avail-able for HIV-1-infected patients.

DISCLOSURE STATEMENT

DRK has served as a consultant to, and received honoraria and/or research support from, thefollowing companies whose drugs and/or diagnostic tests are discussed in this review: Anormed,Bristol Meyers-Squibb, GlaxoSmithKline, Human Genome Sciences, Monogram Biosciences,Pfizer, Roche/Trimeris, Schering-Plough.

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Contents ARI 30 November 2006 12:12

Annual Review ofMedicine

Volume 58, 2007

Contents

The Drug Development Crisis: Efficiency and SafetyC. Thomas Caskey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Idiosyncratic Toxicity: A Convergence of Risk FactorsRoger G. Ulrich � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �17

Rethinking Electronic Health Records to Better Achieve Quality andSafety GoalsWilliam W. Stead � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �35

Schizophrenia: New Pathological Insights and TherapiesL. Fredrik Jarskog, Seiya Miyamoto, and Jeffrey A. Lieberman � � � � � � � � � � � � � � � � � � � � � � � � � � �49

Cardiac Resynchronization Treatment of Heart FailureAyesha Hasan and William T. Abraham � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �63

New Approaches in the Therapy of Cardiomyopathy in MuscularDystrophyElizabeth M. McNally � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �75

Acute Ischemic Stroke: Overview of Recent TherapeuticDevelopmentsNijasri Suwanwela and Walter J. Koroshetz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

Use of Stents to Treat Intracranial Cerebrovascular DiseasePhilip M. Meyers, H. Christian Schumacher, Kurenai Tanji,

Randall T. Higashida, and Louis R. Caplan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 107

Kidney Disease and Cardiovascular RiskMarcello Tonelli and Marc A. Pfeffer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 123

Cardiovascular Risks of Antiretroviral TherapiesKristin Mondy and Pablo Tebas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 141

Airway Surface Dehydration in Cystic Fibrosis: Pathogenesisand TherapyRichard C. Boucher � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

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Toward a Comprehensive Set of Asthma Susceptibility GenesYohan Bosse and Thomas J. Hudson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 171

Does Anti-IgE Therapy Help in Asthma? Efficacy and ControversiesPedro C. Avila � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 185

Advances in the Treatment of Prostate CancerMark Pomerantz and Philip Kantoff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 205

Immunotoxin Treatment of CancerIra Pastan, Raffit Hassan, David J. FitzGerald, and Robert J. Kreitman � � � � � � � � � � � � � � � 221

NSAIDs and Cancer Prevention: Targets Downstream of COX-2Yong I. Cha and Raymond N. DuBois � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 239

The Multiple Endocrine Neoplasia SyndromesVipul T. Lakhani, Y. Nancy You, and Samuel A. Wells � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

Cancer Stem Cells: Models and ConceptsPiero Dalerba, Robert W. Cho, and Michael F. Clarke � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 267

Stem Cells and Chronic Lung DiseaseBrigitte N. Gomperts and Robert M. Strieter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 285

Progress and Potential for Regenerative MedicineGeoffrey C. Gurtner, Matthew J. Callaghan, and Michael T. Longaker � � � � � � � � � � � � � � � � � 299

The Leading Edge of Stem Cell TherapeuticsIlyas Singec, Rahul Jandial, Andrew Crain, Guido Nikkhah, and Evan Y. Snyder � � � � � 313

New Reagents on the Horizon for Immune ToleranceE. William St. Clair, Larry A. Turka, Andrew Saxon, Jeffrey B. Matthews,

Mohamed H. Sayegh, George S. Eisenbarth, and Jeffrey Bluestone � � � � � � � � � � � � � � � � � � � � 329

T Cell Costimulation: A Rational Target in the TherapeuticArmamentarium for Autoimmune Diseases and TransplantationFlavio Vincenti and Michael Luggen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 347

A Human Monoclonal Antibody Cocktail as a Novel Component ofRabies Postexposure ProphylaxisJohn de Kruif, Alexander B.H. Bakker, Wilfred E. Marissen, R. Arjen Kramer,

Mark Throsby, Charles E. Rupprecht, and Jaap Goudsmit � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 359

Why Hasn’t Eliminating Acute Rejection Improved Graft Survival?JogiRaju Tantravahi, Karl L. Womer, and Bruce Kaplan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 369

End-Stage Renal Disease Measures of QualityJonathan Himmelfarb and Alan S. Kliger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

Inflammatory Bowel Disease Genetics: Nod2Judy H. Cho and Clara Abraham � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 401

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New Therapeutic Approaches for Multiple SclerosisPhilip L. De Jager and David A. Hafler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 417

Cytokine Therapy for Crohns Disease: Advances in TranslationalResearchTheresa T. Pizarro and Fabio Cominelli � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 433

Chemokine Antagonists as Therapeutics: Focus on HIV-1Athe M.N. Tsibris and Daniel R. Kuritzkes � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 445

Current Concepts in AIDS Pathogenesis: Insights from theSIV/Macaque ModelAndrew A. Lackner and Ronald S. Veazey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 461

Macular DegenerationEdwin M. Stone � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 477

Targeting VEGF-A to Treat Cancer and Age-Related MacularDegenerationNapoleone Ferrara, Robert D. Mass, Claudio Campa, and Robert Kim � � � � � � � � � � � � � � � � � � 491

Indexes

Cumulative Index of Contributing Authors, Volumes 54–58 � � � � � � � � � � � � � � � � � � � � � � � � � � � 505

Cumulative Index of Chapter Titles, Volumes 54–58 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 509

Errata

An online log of corrections to Annual Review of Medicine chapters (if any, 1997 to thepresent) may be found at http://med.annualreviews.org/errata.shtml

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Documents

Chemokine Antagonists as Therapeutics: Focus on HIV-1