REVIEWS Successes and failures of chemokine-pathway targeting in rheumatoid arthritis Zoltán Szekanecz1 and Alisa E. Koch2

Abstract | Chemokines and chemokine receptors are involved in leukocyte recruitment and angiogenesis underlying the pathogenesis of rheumatoid arthritis (RA) and other inflammatory rheumatic diseases. Numerous chemokines, along with both conventional and atypical cell-surface chemokine receptors, are found in inflamed synovia. Preclinical studies carried out in animal models of arthritis involving agents targeting chemokines and chemokine receptors have yielded promising results. However, most human trials of treatment of RA with antibodies and synthetic compounds targeting chemokine signalling have failed to show clinical improvements. Chemokines can have overlapping actions, and their activities can be altered by chemical modification or proteolytic degradation. Effective targeting of chemokine pathways must take acount of these properties, and can also require high levels of receptor occupancy by therapeutic agents to prevent signalling. CCR1 is a promising target for chemokine-receptor blockade. Chemokines and chemokine receptors mediate leuko­ cyte extravasation during inflammatory processes, including rheumatoid arthritis1–4 (RA; FIG. 1); some are also involved in angiogenesis underlying inflammatory arthritis. More than 50 chemokines and 19 chemokine receptors have been identified1,5,6. In this Review, we summarize the involvement of chemokines and their receptors in the perpetuation or control of synovitis in RA. We discuss approaches for the therapeutic targeting of chemokines and chemokine receptors, and consider reasons for the failure of many of these strategies. Department of Rheumatology, Institute of Medicine, University of Debrecen Faculty of Medicine, Nagyerdei Str 98, Debrecen, H‑4004, Hungary. 2 University of Michigan Health System, Department of Internal Medicine, Division of Rheumatology, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109, USA. Correspondence to Z. S. (szekanecz.zoltan@ med.unideb.hu). 1

doi:10.1038/nrrheum.2015.157 Published online 26 Nov 2015

Chemokines and chemokine receptors Chemokines (chemotactic cytokines) are small (8–10 kDa) signalling proteins that can be classified according to the location of cysteine (C) residues near the N terminus of the primary sequence. These residues form intramolecular disulfide bridges with other C resi­ dues. The XC-chemokines contain a single N‑terminal cysteine, the CC-chemokines have two adjacent residues, CXC-chemokines have two cysteines separated by one other amino acid, and CX3C-chemokines have three amino acids between the C residues. Many chemokines also have nonsystematic names that are often still in use, but to reflect their designation as ‘chemokine ligands’, a systematic nomenclature has been developed for both chemokines and their receptors. Chemokine receptors are proteins with seven transmembrane domains that are

generally coupled to G proteins, and are expressed on the surface of target cells5 (FIG. 1). On the basis of relation­ ships between structure and function, chemo­kine recep­ tors have also been classified as ‘conventional’ receptors that mediate cell migration and ‘atypical’ receptors that influence chemokine bioavailability7,8.

Chemokines and chemokine receptors in RA In inflammatory conditions such as RA, the molecular mechanisms involved in chemoattraction of neutro­ phils, lymphocytes and monocytes into the synovium mediated by chemokines include leukocyte integrin expression and L‑selectin shedding, cytoskeletal reor­ ganization, neutrophil degranulation and phagocytosis, and the production of proteases and other inflamma­ tory mediators. Chemokines are also involved in endo­ thelial activation and angiogenesis, synovial fibroblast migration and proliferation, pseudoemperipolesis and the regulation of cartilage and bone metabolism1,2,6,9. Chemokines The CXC-chemokines CXCL1 (GROα), CXCL4 (PF4), CXCL5 (ENA78), CXCL6 (GCP2), CXCL7 (NAP2), CXCL8 (IL-8), CXCL9 (MIG), CXCL10 (IP10), CXCL12 (SDF1), CXCL13 (BCA1) and CXCL16 (SR-PSOX) have variously been detected in sera, synovial fluids and syno­ vial tissues of patients with RA1,2,4,10–20, and are mainly produced by synovial macro­phages12,17. The functions of

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REVIEWS Key points • Chemokines and chemokine receptors have been implicated in leukocyte recruitment and angiogenesis underlying rheumatoid arthritis (RA) and other inflammatory rheumatic diseases • Chemokines and chemokine receptors are abundant in the synovium and other inflammatory sites • Promising results from preclinical studies of agents targeting chemokines and chemokine receptors in animal models of arthritis have not been replicated in human trials of antibodies and synthetic compounds in RA • Possible reasons for the lack of positive results from human trials include pathway redundancy, incomplete antagonism and interspecies differences that limit translation of results from animal models • An alternative approach to the targeting of individual chemokines is chemokine-receptor blockade • The CCR1 receptor is a potential target, assuming that a high level of receptor occupancy can be maintained throughout treatment

CXC-chemokines in RA have been reviewed elsewhere3. CXCL1, CXCL5 and CXCL8 have neutrophil chemo­ attractant properties. CXCL5, CXCL7, CXCL8 and CXCL12 are angio­genic. CXCL4, CXCL9 and CXCL10 are angiostatic. Other functions of CXC-chemokines include induction of glycosamino­glycan synthesis and recruitment of B cells, T cells and monocytes3. Synovial CXC-chemokine expression can vary at dif­ ferent stages of RA. The synovial expression of CXCL4 and CXCL7 mRNA is more abundant in early RA than in long-standing disease21. These chemokines co-localize with blood vessels, platelets and synovial macrophages22. Furthermore, CXCL13 levels are pre­dictive of disease activity and therapeutic response in early RA, and high levels of CXCL13 might indicate a ‘window of opportunity’ for treatment23. The CC‑chemokines CCL2 (MCP1), CCL3 (MIP1α), CCL5 (RANTES), CCL7 (MCP3), CCL8 (MCP2), CCL13 (MCP4), CCL14 (HCC1), CCL15 (HCC2), CCL16 (HCC4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP3β), CCL20 (MIP3α), CCL21 (SLC) and CCL28 (MEC) are all expressed in sera and synovia in RA1,2,17,19,20,22,24–28. These chemokines exert chemotactic activity mainly for monocytes and lymphocytes1,5,20. XCL1 (lymphotactin), which is chemotactic for lymphocytes but not for monocytes or neutrophils, is involved in the accumulation of T cells and subchondral mesenchymal cells in joints affected by RA1,29. CX3CL1 (fractalkine) has also been detected in RA, and has a role in chemoattraction of synovial fibroblasts, and also in angiogenesis30,31. Protein citrullination (in which arginine is converted to citrulline) has been implicated in the patho­genesis of RA; antibodies that recognize citrullinated proteins can be detected up to 10 years before disease onset32. Chemokines are potential targets for citrullination, and citrullinated CXCL5 and CCL2 have been detected in synovial fluid of patients with RA21,33. CXCL5 and CCL2 are generally chemotactic for neutro­phils and mono­ nuclear cells, respectively, but citrullination reduces the chemotactic activity of CCL2 towards monocytes and converts CXCL5 to a mono­c yte chemoattractant21,33.

Alteration of the structure and activity of chemo­ kines might contribute to the failure of some strategies targeting chemokine signalling21.

Chemokine receptors Conventional chemokine receptors. The CXCchemokine receptors (CXCRs) CXCR1 and CXCR2 are the main neutrophil-associated receptors that have been implicated in RA in humans1,2,6,7. CXCR3 is important for the homing of leukocytes into inflammatory sites associated with type 1 T helper (TH1) cells, such as the synovium in RA1,34 (FIG. 1). CXCR4 mediates chemo­ taxis of lymphocytes into the synovium1,14. CXCR4, CXCR5, CXCR6 and CXCR8 are involved in physio­ logical lymphoid organization and synovial lymphoid neogenesis1,15,16,20,35,36. The CC‑chemokine receptors (CCRs) CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 and CCR10 are abundantly expressed in the synovium in RA1,2,7,20,28. Although CC‑chemokines are primarily involved in mononuclear-cell migration, CCR2 has been identi­ fied as a major neutrophil chemoattractant associated with disease activity and flares in RA37. CCR5 expres­ sion is characteristic of TH1 inflammatory infiltrates34. A single-nucleotide polymorphism variant responsible for the production of a nonfunctional truncated form of CCR5 is protective against RA, including extra-articular symptoms and joint erosions38. CCR6 is involved in the ingress of type 17 T helper (TH17) cells into rheumatoid joints18,39. CCR7 is associated with synovial lymphoid neogenesis40. CCR10 and its ligand CCL28 co-localize on synovial myeloid and endo­thelial cells, and they are involved in synovial angiogenesis28. XC-chemokine receptor 1 (XCR1) is expressed on synovial lympho­ cytes, macrophages and fibroblasts, whereas CX3CR1 is expressed on macrophages and dendritic cells, and is thought to be involved in the recruitment of TH1 cells1,41. Atypical chemokine receptors. Atypical chemokine receptors (ACKRs) are homologous to conventional chemokine receptors and bind chemokines, but do not signal through G proteins7,8. Rather than media­ ting intracellular signalling, binding of chemokines to ACKRs affects chemokine bioavailability. ACKR1 (also known as the Duffy antigen/chemokine recep­ tor (DARC)) has been detected in the synovium in RA, and acts as a receptor for a number of inflamma­ tory chemokines from both the CC-chemokine and the CXC-chemokine families42 (FIG. 2). ACKR2 (also known as chemokine-binding protein D6) binds several inflammatory CC‑chemokines, and can suppress TH17 cell responses to protein autoantigens43. ACKR3 (also known as CXCR7) is a receptor for CXCL11 (I-TAC) and CXCL12, and is implicated in inflammatory events as well as in angiogenesis associated with RA44. Synovial expression and activation of ACKR5 (a provisional designation for CCR-like 2 (CCRL2)) have also been described in RA45, and CCL18, the ligand for ACKR6 (a provisional designation for the membrane-associated phosphatidylinositol transfer protein 3 (PITPNM3)) has also been detected46.

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REVIEWS Bone marrow CXCR2 CXCR4 CXCR4 CXCR4

T cell

Neutrophil

B cell

Monocyte

NK cell

DC

Eosinophil

HSC

Thymus CCR2 CXCR4

TDP

TSP CCR4 CCR7

Synovium CCR7 TN

CCR1 CXCR1 CCR4 CCR2 CX3CR1 CX3CR1 CXCR2 CCR3 CXCR3 CCR6 CCR10 CCR5 CCR5 CCR2

?

TH1

TH17

TM

TEM

Blood TEM

CCR7 TN

CCR5 TM

TN CXCR5 CCR2

CCR7 CCR7 CXCR5 TCM CCR7

TCM TEFF CCR7 CCR7

T TEM CXCR5 Lymph node Spleen

Figure 1 | Leukocyte trafficking into the inflamed synovium. Various cell types and cell-surface chemokine receptors Nature Reviews | Rheumatology are involved in leukocyte extravasation and migration between the blood, haematopoietic compartments and the synovial tissue. CCR, CC‑chemokine receptor; CXCR, CXC-chemokine receptor; DC, dendritic cell; HSC, haematopoietic stem cell; NK cell, natural killer cell; TCM, central memory T cell; TDP, double-positive thymocyte; TEFF, effector T cell; TEM, effector memory T cell; TH1, type 1 T helper cell; TH17, type 17 T helper cell; TM, memory T‑cell; TN, naive T‑cell; TSP, single-positive thymocyte.

Chemokine-pathway targeting in RA Targeting of chemokines and chemokine receptors for therapeutic purposes in rheumatic disease can occur by both specific and nonspecific approaches1,2,23,47,48. These approaches have been tested in animal models and in human trials, and the largely negative results of these trials have called into question the existing strategies, leading to suggestions for new avenues of investigation.

Indirect targeting of chemokine production Indirect targeting can include the use of immuno­ suppressive agents that, among other modes of action, have effects on chemokine production 2,48. Some NSAIDs and corticosteroids, as well as conventional and bio­logic DMARDs, can inhibit chemokine release and chemokine-receptor expression2. For example, early in vitro and animal model studies showed that NSAIDs and glucocorticoids block production of CXCL8 and

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REVIEWS Chemokine receptor

Chemokine receptor

CC-chemokines CCL1 (I-309)

CXC-chemokines

CCL2 (MCP1)

CXCL2 (GROβ)

CCL3 (MIP1α)

CXCL3 (GROγ)

CCL3L1 (LD78)

CXCL4 (PF4)

CCL4 (MIP1β)

CXCL5 (ENA78)

CCL5 (RANTES)

CXCL6 (GCP2)

Mouse CCL6 (C10)

CXCL7 (NAP2)

CCL7 (MCP3)

CXCL8 (IL-8)

CCL8 (MCP2)

CXCL9 (MIG)

CCL9 (MIP1γ) CCL11 (Eotaxin)

CXCL10 (IP10)

CCL12 (MCP5) CCL13 (MCP4)

CXCL12 (SDF1)

CCL14 (HCC1)

CXCL14 (BRAK)

CCL15 (HCC2)

Mouse CXCL15 (Lungkine)

ACKR1

ACKR3

CX3CR1

XCR1

Atypical CXCR8

CXCR6

CXCR5

CXCR3

CXCR2

CXCR1

ACKR4

ACKR2

ACKR1

CCR12

CCR10 CCR11

Chemokine ligand (alternative name)

CXCR4

Conventional

Atypical

CCR9

CCR8

CCR7

CCR6

CCR5

CCR4

CCR3

CCR2

Chemokine ligand (alternative name)

CCR1

Conventional

CXCL1 (GROα)

CXCL11 (I-TAC) CXCL13 (BCA1)

CCL16 (HCC4) CCL17 (TARC)

CXCL16 (SR-PSOX)

CCL18 (PARC)

CXCL17 (DMC)

CCL19 (MIP3β)

C-chemokines

CCL20 (MIP3α)

XCL1 (Lymphotactin)

CCL21 (SLC)

XCL2 (SCM1α) CX3C-chemokine

CCL22 (MDC)

CX3CL1 (Fractalkine)

CCL23 (MPIF1) CCL24 (Eotaxin 2) CCL25 (TECK) CCL26 (Eotaxin 3) CCL27 (CTACK) CCL28 (MEC)

Figure 2 | Known interactions between chemokines and their receptors. Some chemokines interact with a single Nature Reviews | Rheumatology receptor, others with multiple receptors. Likewise, some chemokine receptors only interact with a single chemokine, whereas others can bind multiple ligands. Before the introduction of standardized nomenclature, chemokines were known by a variety of different names. Chemokine receptors are classified as CC‑chemokine receptors (CCRs), CXC-chemokine receptors (CXCRs), C‑chemokine receptors (XCRs), CX3C-chemokine receptors (CX3CRs) and atypical chemokine receptors (ACKRs). Unlike the other receptor types, ACKRs are not coupled to G proteins, but can affect bioavailability of chemokines. Aliases for ACKRs include: DARC (ACKR1), D6 (ACKR2), CXCR7 (ACKR3) and CCRL1 (ACKR4). CXCR8 is a putative receptor; its classification is awaiting confirmation. The receptor targets of CXCL14 and CXCL15 have not been determined.

CCL2 (REFS 49,50).Conventional DMARDs (sulfasala­ zine, sulfapyridine, methotrexate.and leflunomide) inhibit the production of several chemokines, both in synovial cultures in vitro and in animal models of RA 2,49,51–53. Biologic DMARDs that target TNF can inhibit the release of multiple chemokines in RA2,54–56. Among other biologic DMARDs, tocilizumab57 and rituximab58 decrease CCL20 and CCL5 production, respectively. The Janus kinase inhibitor tofacitinib also suppresses expression of multiple chemokines, inclu­ ding CXCL10, CXCL13 and CCL2, in synovial tissue in RA59. Inhibition of chemokine expression by biologic DMARDs can also have implications for safety; the

use of anti-TNF agents increases the risk of develop­ing tuberculosis, and evidence from the treatment of RA and ankylosing spondylitis with infliximab suggests that this effect is the result of disruption of TNF-dependent gradients of chemokines such as CXCL8, CCL2 and CCL3 (REF. 60). Synthetic compounds and natural products that are not primarily treatments for arthritides can also affect chemokine secretion. Antioxidants, such as N‑acetyl-l‑cysteine and L‑2‑oxothiazolidine4‑carboxylate, and the bioflavonoid quercetin inhibit the stimulation of chemokine expression by TNF in cultured human RA synovial fibroblasts (RASFs)61,62.

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REVIEWS Epigallocatechin-3‑gallate, an ingredient of green tea, suppresses the production of multiple chemokines by RASFs63. The anti­arthritic effects of traditional Oriental medicines such as triptolide, lingzhi and curcumin might also result in part from the inhibition of chemokines and chemokine receptors64.

Specific targeting of chemokines and receptors Studies in animal models. In animal models of RA, administration of antibodies that bind to chemokines including CXCL1, CXCL5, CXCL8, CXCL12, CXCL13, CXCL16, CCL2, CCL3, CCL5, CCL24 (Eotaxin 2) and CX3CL1 has preventive and therapeutic effects with respect to synovitis2,15,65–68. In rats, treatment with a small-molecule inhibitor of endogenous CCL2 (p8A‑MCP‑1) has a positive effect on adjuvant-induced arthritis (AIA)69. Inhibition of either CXCL12 (REF. 70) or CXCL13 (REF. 68) has a therapeutic effect on synovitis in a collagen-induced arthritis (CIA) model. To increase the potential efficacy of the blockade, various antichemo­ kine strategies have been combined, with somewhat greater effects than inhibition of single chemokines71. Inhibitors of chemokine receptors have also been tested in animal models. An antibody directed against CXCR3 inhibits AIA in rats72. Synthetic oral antag­ onists of CXCRs inhibit arthritis in various rodent models 73,74. For example, SCH546738, a synthetic compound targeting CXCR3, attenuates CIA devel­ opment in mice 75 . Plerixafor, previously known as AMD3100, is a small-molecule inhibitor of the CXCL12 receptor CXCR4 that attenuates synovitis in rodent models of arthritis76 and that has been approved for bone marrow stem cell therapy. Another CXCR4 antagonist — T140 — also ameliorates CIA in mice77. Inhibition of CXCR7, the second receptor for CXCL12, reduces synovitis and angiogenesis in the CIA model78. CCR1, CCR2 and CCR5 each bind multiple CC‑chemokines (FIG. 2) that have important roles in the pathogenesis of RA2,23,47,79. Numerous antagonists to these receptors have been developed and tested in animal models2,47,79. The CCR1 antagonist J‑113863 has positive effects on murine CIA80, as do low doses of a monoclonal antibody directed against CCR2 (MC‑21)81, and development of CIA in the rhesus monkey is inhibi­ ted by the CCR5 antagonist SCH‑X82. Low doses of the MC‑21 monoclonal antibody lead to improvements in murine CIA, although high doses aggravate CIA and have proinflammatory effects81. Inhibition of the CCL25 (TECK) receptor CCR9 ameliorates CIA in mice83. MetRANTES is a dual antagonist of CCR1 and CCR5, and inhibits CIA in mice and AIA in rats84,85. Dual target­ ing of CCR2 and CCR5 has also been undertaken in preclinical studies86. Studies targeting chemokines in humans. Data from clinical trials targeting chemokines in human RA are limited, and not altogether positive. In a randomized, placebo-controlled trial87, 33 patients received ABN912, an antibody directed against CCL2, whereas 12 patients received placebo. Although ABN912 treatment was well-tolerated, no histological or clinical benefit was

observed87. In a phase II randomized, placebo-controlled trial involving patients with active RA receiving metho­ trexate88, 35 patients also received 10 mg/kg of a fully human monoclonal antibody to CXCL10 (MDX‑1100) every other week. Relative to placebo, treatment with MDX‑1100 decreased levels of C‑reactive protein and disease activity, and improved physical function88. The ACR20 response rate (indicating a 20% improve­ ment in symptoms of RA) was significantly higher in patients treated with MDX‑1100 (54%) compared with those treated with placebo (17%) on day 85 (P = 0.0024), but ACR50 and ACR70 response rates did not differ between the two groups88. No serious adverse events were reported88. In rodent models of atherosclerosis, deletion of the gene encoding CXCL10 reduced athero­ sclerotic plaque formation and increased markers of reg­ ulatory T (TREG)-cell numbers and activity, suggesting CXCL10 blockade affects arthritis-related cardiovas­cular comorbidity89. However, information on the further development of MDX‑1100 is not yet available7 (TABLE 1). Studies targeting chemokine receptors in humans. The development of a number of oral CCR1 antagonists, including MLN3897, c‑4462, BMS‑817399, CCX354‑C and CP‑481,715, has been reviewed previously 7,79. CP‑481,715 is well-tolerated in doses up to 3,000 mg90 and, at a dose of 300 mg per 8 h over the 2 weeks of a phase I study, decreased the number of total and inti­ mal macrophages and CCR1‑positive cells in the syno­ vial tissue of patients with RA, compared with placebo treatment91. One-third of the 12 patients treated with CP‑481,715 also fulfilled ACR20 criteria91. In a phase IIa study involving patients with active disease who were receiving methotrexate, MLN3897 was well tolerated, but did not improve ACR20 compared with placebo, despite a high degree of CCR1 occupancy92. c‑4462 was developed for the treatment of RA93, but had no efficacy and further development was terminated. The results of a phase II trial of BMS‑817399 in RA are pending94,95. In a 12‑week, phase II trial96 involving 160 patients with active RA, CCX354-C was well tolerated. ACR20 response rates at week 12 were 43% and 52% in patients treated with CCX354-C (100 mg twice daily and 200 mg once daily, respectively) and 39% in the placebo group (no significant difference between treatment and pla­ cebo groups)96. A preclinical study with CCX354-C demonstrated that >90% receptor occupancy at all times is required for effective blockade of inflammatory-cell infiltration97,98. Overall, the evidence suggests that CCR1 is a potential target in the treatment of RA, but further development is required to determine its true value7,98. Several CCR2 inhibitors have been developed, but only a few have progressed from animal studies to clinical trials in relation to RA7,99 (TABLE 1). MK‑0812, a small-molecule inhibitor of CCR2, did not lead to improvement in a phase II trial in patients with RA7,100,101. In a phase IIa clinical trial involving 32 patients with active RA, treatment with the CCR2‑blocking antibody MLN1202 (three infusions of 0.5 mg/kg or 1.5 mg/kg over a 6‑week period) resulted in no clinical benefit compared with placebo102. In a phase I trial, the selective,

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REVIEWS Table 1 | Studies involving the targeting of chemokines and chemokine receptors in RA Molecular target

Drug

Outcome

Refs

Studies with results that demonstrated clinical improvement CXCL10

MDX‑1100 (mAb)

Significant ACR20 response (phase II)

88

CCR1

CCX354 (sm)

Effective and well tolerated (phase II)

96

Studies with results that did not demonstrate clinical improvement CCL2

ABN912 (mAb)

No clinical improvement (phase I)

87

CCR1

MLN3897 (sm)

No efficacy (phase Ib/IIa)

92

CCR1

CP‑481,715 (sm)

Well tolerated, no clinical improvement (phase I/Ib)

CCR1

c‑4462 (sm)

No efficacy (phase II)

93

CCR1

BMS‑817399 (sm)

Results unpublished (phase II)

94

CCR2

MLN1202 (mAb)

No clinical improvement (phase IIa)

102

CCR2

MK‑0812 (sm)

No efficacy (phase II)

101

CCR2

INCB3284 (sm)

No efficacy (phase II)

103

CCR5

SCH351125 (sm)

Adverse events, no efficacy (phase Ib)

105

CCR5

AZD5672 (sm)

No efficacy (phase IIb)

104

CCR5

UK‑427,857 (maraviroc; sm)

Well tolerated, no clinical improvement (phase IIa)

106

90,91

CCL, CC-chemokine ligand; CCR, CC-chemokine receptor; CXCL, CXC-chemokine ligand; mAb, monoclonal antibody; RA, rheumatoid arthritis; sm, small molecule.

oral CCR2 antagonist INCB3284 exhibited a pharma­ cokinetic profile suitable for once‑daily dosing103, but the drug showed no clinical efficacy in a phase II trial in RA7,23. The results of these studies suggest that CCR2 is not an optimal target in arthritis therapy. In a phase IIb trial104, 371 patients with active RA received 20 mg, 50 mg, 100 mg or 150 mg of the oral small-molecule CCR5 inhibitor AZD5672 once daily, oral placebo once daily or 50 mg subcuta­neous etaner­ cept once weekly. Although AZD5672 was well-tolerated, no difference was seen in ACR20 response rates between AZD5672 treatment and placebo, and etanercept was more efficacious than AZD5672 or placebo104. In a phase Ib trial105 involving patients with active RA, 20 patients received the oral, small-molecule CCR5 inhib­ itor SCH351125 and 12 received placebo. No improve­ ment was observed with active treatment compared with placebo 105. Maraviroc (UK‑427,857), a CCR5 antagonist used in the treatment of HIV, has been inves­ tigated in a phase IIa trial106 for its effect in RA. In the pharmacokinetic component of the trial, maraviroc was well-tolerated in the presence of metho­trexate106. In the proof‑of‑concept component, 110 patients receiving methotrexate were randomly assigned in a 2:1 ratio to receive 300 mg maraviroc or placebo twice daily for 12 weeks106. This component of the study was termi­ nated after the planned interim ana­lysis because of lack of efficacy of mar­aviroc106. Overall, the evidence suggests that CCR5 is not an appropriate target for the treatment of RA. Although several CXCR inhibitors have been devel­ oped for the treatment of HIV infection or cancer107, to our knowledge no clinical trial results obtained with any CXCR antagonists in relation to RA have been published. In addition, no results from clinical trials investigating targeting of ACKRs have yet been published7.

Prospects for chemokine-pathway targeting. Targeting chemokines and their receptors has yielded disappoint­ ing results in the treatment of RA, and the question remains of whether this approach is worthwhile. Several factors could interfere with these targeted strategies2,108 (BOX 1). For example, redundancy among chemokines and chemokine receptors might be important. Some receptors bind multiple ligands, and some ligands bind multi­ple receptors. Targeting one specific ligand or receptor might, therefore, be ineffective71,86,109. However, although redundancy in binding is clearly possible in vitro, spatial and temporal localization of ligands and receptors, as well as differences in binding affinities, might limit its influence in vivo. Moreover, binding of different ligands to a particular receptor might not have the same downstream effects108. The use of animal models to determine appropri­ ate inhibitory compounds could create problems, as the affinity of a compound for a rodent chemokine receptor can differ substantially from its affinity for the human equivalent. For example, the murine CCR2 and CCR5 molecules differ considerably from their human homologues, which could explain why anti‑CCR2 and anti‑CCR5 therapies that were successful in rodents failed in human RA98,102,104,105,108,110. Even when an appro­ priate inhibitor is identified, the most effective dosage and timing can be difficult to determine, so negative results might, at least in part, result from suboptimal administration81. Modification of the molecular structure of chemokines — for example by citrullination — can alter the char­ acteristics of their binding to receptors. Chemokinereceptor blockade that effectively blocks the binding of unmodified chemokines might not block the mod­ ified forms33. Similarly, proteolytic enzymes, such as matrix metalloproteinases, can cleave chemokines,

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REVIEWS Box 1 | Potential difficulties with targeting of the chemokine pathway2,108 Redundancy of chemokines and chemokine receptors • Multiple ligands can exist for one receptor, as well as multiple receptors for one chemokine, so blocking a specific chemokine or receptor might not be effective; however, the importance of redundancy has been challenged71,86,108,109 Cross-species target prediction • A chemokine-receptor inhibitor can have different affinity for the rodent and human forms of the targeted receptor. For example, both CCR2 and CCR5 show considerable species-specific variation98,102,104,105,108,110 Structure modification • Citrullination of chemokines can alter their receptor-binding characteristics, rendering blocking agents ineffective33 Cleavage of chemokines by proteases • Enzymes such as matrix metalloproteinases can cleave chemokines, potentially altering receptor targeting111,112 Choice of dosage and timing • Doses of agents and timing of delivery chosen for studies might not result in therapeutically optimal levels in vivo81,108 Undesired inhibition of anti-inflammatory cells • In addition to the blockade of inflammatory cells, chemokine-pathway targeting can simultaneously affect anti-inflammatory cells, such as regulatory T cells113,114 Interference with homeostatic function • In addition to inflammation, several chemokines (including CXCL12, CXCL13, CXCL16, CCL19 and CCL21) affect homeostatic functions, such as lymphoid development and physiological homing. Chemokine blockade might interfere with these physiological processes1,2,15,115,116 Insufficiency of receptor occupancy • Continuous, high levels of receptor occupancy might be required throughout the period of treatment, to prevent chemokine signalling96–98 CCL, CC-chemokine ligand; CCR, CC-chemokine receptor; CXCL, CXC-chemokine ligand.

altering their structures and receptor-binding characteristics111,112. The same chemokine receptors can be expressed by both inflammatory and anti-inflammatory T REG cells, so that chemokine-receptor blockade can inhibit both cell types at the same time, resulting in conflict­ ing effects113,114. Likewise, some chemokines, including CXCL12, CXCL13, CXCL16, CCL19 and CCL21, are involved not only in inflammation, but also in physio­ logical, homeostatic processes, such as lymphoid tissue development and lymphocyte homing. Interference with

Szekanecz, Z., Vegvari, A., Szabo, Z. & Koch, A. E. Chemokines and chemokine receptors in arthritis. Front. Biosci. (Schol. Ed.) 2, 153–167 (2010). 2. Szekanecz, Z., Koch, A. E. & Tak, P. P. Chemokine and chemokine receptor blockade in arthritis, a prototype of immune-mediated inflammatory diseases. Neth. J. Med. 69, 356–366 (2011). 3. Koch, A. E. Chemokines and their receptors in rheumatoid arthritis: future targets? Arthritis Rheum. 52, 710–721 (2005). 4. Vergunst, C. E. & Tak, P. P. Chemokines: their role in rheumatoid arthritis. Curr. Rheumatol. Rep. 7, 382–388 (2005). 5. Zlotnik, A. & Yoshie, O. Chemokines: a new classification system and their role in immunity. Immunity 12, 121–127 (2000). 6. Szekanecz, Z. & Koch, A. E. Chemokines and angiogenesis. Curr. Opin. Rheumatol. 13, 202–208 (2001). 7. Bachelerie, F. et al. International Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. Update 1.

these homeostatic functions can result in unwanted adverse effects1,2,15,115,116. Evidence suggests that a high level of receptor occu­ pancy is required at all times in order to inhibit mono­ cyte migration into the synovium and to achieve a good clinical response to an antagonist92,97. The failure of some chemokine-receptor antagonists to give clinical benefit in the treatment of RA might, at least in part, result from insufficient receptor coverage2,92,97,98. Although some clinical efficacy has been seen with agents targeting CCR1, therapeutic strategies involving CCR2 and CCR5 have been less successful98. Although antibodies directed against each of these receptors inhibi­ ted monocyte chemotaxis induced by specific human chemokines in vitro, only CCR1 blockade resulted in the inhibition of monocyte migration induced by syno­ vial fluid from patients with RA98. Monocyte recruit­ ment in the synovium might be dependent on CCR1 but independent of CCR2 and CCR5 (REF. 98). Indeed, antibody blockade of CCR2 and CCR5 has failed to pro­ duce improvements in synovitis in clinical trials98,102,105. Although CCR1 blockade has also failed in some clinical trials, CCR1 might still be a good target for therapeu­ tic intervention in RA, subject to optimization of the inhibitory strategy23,91,92,96–98.

Conclusions Chemotactic signalling is thought to be important in the pathogenesis of RA, and numerous chemok­ ines, along with their respective receptors, have been implicated in leukocyte ingress into inflamed synovia. Chemokine signalling is, therefore, a target for thera­ peutic inter­vention in the treatment of RA. However, promising results in preclinical studies with antibodies and small-molecule inhibitors directed against chemok­ ines and chemokine receptors have generally led to dis­appointment when translated into cinical trials. Evidence gained from studies of CCR1 inhibitors sug­ gests that the development of strategies for the optimiza­ tion of inhibition requires consideration of factors such as continuous receptor occupancy. This insight could provide new opportunities for clinical trials involving existing inhibitors, and could contribute to the devel­ opment of more-effective inhibitors for the treatment of RA as well as other inflammatory conditions.

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Author contributions

Both authors contributed to researching data and writing the article, and to reviewing and editing the manuscript before submission.

Competing interests statement

The authors declare no competing interests.

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