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Rheumatology Advance Access originally published online on October 27, 2007
Rheumatology 2007 46(12):1773-1778; doi:10.1093/rheumatology/kem222
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Blocking the receptor for C5a in patients with rheumatoid arthritis does not reduce synovial inflammation

C. E. Vergunst1, D. M. Gerlag1, H. Dinant1,2, L. Schulz3, M. Vinkenoog1, T. J. M. Smeets1, M. E. Sanders1, K. A. Reedquist1 and P. P. Tak1

1Division of Clinical Immunology and Rheumatology, Academic Medical Centre/University, of Amsterdam, The Netherlands, 2Jan van Breemen Institute, Amsterdam, The Netherlands and 3Promics Ltd., Brisbane, Australia.

Correspondence to: P. P. Tak, MD, PhD, Division of Clinical Immunology and Rheumatology, F4-218, Academic Medical Centre/University of Amsterdam, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. E-mail: p.p.tak{at}amc.uva.nl


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. All complement pathways lead to the formation of C5a, which is believed to contribute to the influx and activation of C5a-receptor (C5aR) bearing cells into the joints of patients with rheumatoid arthritis (RA). Studies in animal models of RA have suggested therapeutic potential of C5aR blockade. In this study, we examined the effects of the C5aR blockade on synovial inflammation in RA patients.

Methods. We performed a double-blind, placebo-controlled study using an orally administered C5aR-antagonist. Twenty-one patients with active RA were randomized 2:1 to treatment with a C5aR-antagonist AcF- (OpdChaWR) (PMX53) vs placebo for 28 days. Serum concentrations of PMX53 were determined. Synovial tissue was obtained at baseline and after 28 days of treatment for pharmacodynamic analysis using immunohistochemistry and digital image analysis.

Results. All patients completed the study. Areas under the curve (AUCs) of PMX53 in patients’ blood samples showed a mean of 40.8 nmol h/l. There was neither decrease in cell infiltration, nor changes in key biomarkers associated with clinical efficacy after active treatment. In addition, there was no trend towards clinical improvement in the C5aR-antagonist-treated group compared with placebo nor was there a correlation between the AUC and clinical response.

Conclusions. Treatment with PMX53 did not result in a reduction of synovial inflammation despite reaching serum levels of PMX53 that block C5aR-mediated cell activation in vitro. The data suggest that C5aR blockade does not result in reduced synovial inflammation in RA patients.

KEY WORDS: Rheumatoid arthritis, C5a, Complement, Clinical trial


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown aetiology affecting synovial tissue in multiple joints. The synovial inflammation can lead to invasion of cartilage and bone, which may result in radiological evidence of erosive damage within 2 yrs of onset of RA. Joint damage typically leads to permanent disability. Currently, there is no cure for RA, and available therapies are not satisfactory for all patients [1].

While healthy synovium is loosely structured and only contains a few scattered cells, the synovium of a joint affected by RA typically consists of a dense infiltrate of immune cells [2]. Although other pathogenic factors such as impaired apoptosis and inappropriate retention of cells may contribute to this infiltrate, chemokine-dependent recruitment of cells from the circulation into the tissue is required to explain the abundant presence of immune cells that maintain inflammation.

One of the chemotactic factors proposed to play a role in recruiting and activating cells in RA joints is C5a [3], a component of the complement cascade. All three pathways of complement activation (the classical, mannan-binding lectin and alternative pathway) lead to formation of C5 convertase, which cleaves C5 into C5a and C5b [4]. Whereas C5b is incorporated into the membrane-attack complex, C5a serves as a strong chemotactic factor for C5aR+ (CD88+) cells [5]. These cells are mainly members of the innate immune system, including monocytes and neutrophils [5], which are abundantly present in RA joints. C5a has been reported to have a mean concentration of 2.5 nmol/l in RA synovial fluid, which is sufficient to induce chemotaxis of C5aR+ cells [3]. In animal models of RA, a crucial role for C5a has been well documented [6, 7]. Most strikingly, C5aR–/– mice are completely protected from developing collagen-induced arthritis [8]. In addition, treatment with anti-C5 monoclonal antibodies may prevent collagen-induced arthritis [9]. Taken together, these data support the notion that C5a plays an important role in synovial inflammation.

Recently, a cyclic peptide AcF- (OpdChaWR) (PMX53) has been developed [10], which is a C5a mimetic that binds the C5aR with high affinity without causing agonist effects [10, 11] (Fig. 1). Because it is a small molecule, PMX53 has low immunogenecity and can be dosed orally for in vivo treatment purposes. PMX53 effectively inhibits C5a-induced neutropenia in the rat [10]. Moreover, rats treated with PMX53 showed a significant reduction of swelling, gait disturbance and severity of histopathology in the affected joints during experimental arthritis [12]. In vitro, PMX53 is able to inhibit chemotaxis and cytokine production in human neutrophils and macrophages [11].


Figure 1
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  		FIG. 1. Structure of PMX53 [10].

 
Here we report the results of the first application of PMX53 in human disease. We performed a double-blind, placebo-controlled, phase Ib clinical trial with orally administered PMX53 in 21 patients with active RA. In addition to monitoring safety, we tested the hypothesis that treatment with PMX53 reduces synovial inflammation in RA.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 Acknowledgements
 References
 
Patients
Patients between the ages of 18 and 75 meeting the criteria of the American College of Rheumatology (ACR) for the diagnosis of RA [13] were included over a period of 8 months. Patients had to have been diagnosed with RA at least 6 months prior to inclusion in the study, and not before the age of 18. We included patients with active disease that was refractory to current treatment. Active disease was defined as having at least four tender and at least four swollen joints of 28 examined, including at least an actively inflamed knee joint, ankle joint or wrist. A second required criterion was an erythrocyte sedimentation rate (ESR) ≥28 mm/h, or C-reactive protein (CRP) ≥10 mg/l or morning stiffness of at least 45 min. Concomitant therapy with methotrexate was obligatory for at least 3 months and was kept stable (>5 and ≤30 mg weekly) for at least 28 days prior to screening. Use of prednisone in a dose of ≤10 mg daily was allowed if this dose had been stable for at least 1 month. All other disease-modifying anti-rheumatic drugs (DMARDs; including injections with corticosteroids) had to have been stopped at least 1 month prior to screening. Prior use of leflunomide, TNF-{alpha} antagonists or an investigational agent had to be ceased at least 3 months before screening. The use of one non-steroidal anti-inflammatory drug (NSAID) was allowed, if the dose had been stable for 1 month and did not exceed the recommended maximum dose. Patients with severe physical incapacity (ACR functional class IV) [14] were excluded. Other exclusion criteria included the presence of any other major inflammatory disease or significant concurrent medical condition.

Clinical study protocol
The Medical Ethics Committee of the Academic Medical Centre from the University of Amsterdam approved the study protocol. Patients gave written informed consent according to the Declaration of Helsinki before screening. All patients included were from this centre and in generally good health at screening as determined by medical history, physical examination, ECG, chest X-ray and laboratory tests. After the screening visit, eligible patients were enrolled in the study within 4 weeks. Clinical assessment for disease activity was repeated at baseline (before the arthroscopy) and on day 27. This included a 28 joint count for joint swelling and tenderness, doctor's and patient's global health assessment on a scale from 0 (very good) to 100 (very bad), pain assessed by a visual analogue scale from 0 (no pain) to 100 (severe pain), quality of life {Health Assessment Questionnaire (HAQ) [15]} from 0 (no disability) to 3 (severe disability), ESR and CRP. One assessor performed the clinical evaluation. Results of the clinical assessments were used to determine response according to European League against Rheumatism (EULAR) criteria using disease activity score (DAS) 28 [16], and ACR20 response criteria [17]. Monitoring for adverse events occurred daily during the study until 2 weeks after the study by interviews, physical examination, laboratory testing and ECGs.

Patients took study medication each day for 28 days, starting at day 0 until day 27. On day 0, patients were randomized in a double-blind fashion in a ratio of 2:1 to PMX53 or placebo. The medication was offered in red capsules for oral administration, each capsule containing 100 mg of PMX53 or 100 mg lactose. Depending on the patient's body weight, an amount of 4–7 capsules (8 mg/kg) once daily was prescribed, to be swallowed in whole with water. The dose of 8 mg/kg was chosen because PMX53 demonstrated activity in rat models for chronic inflammatory disease at dose levels ranging from ~3 to 10 mg/kg when dosed orally [12, 18]. When dosed orally in healthy volunteers at levels of up to 10 mg/kg, it has shown high levels of safety and tolerability, as well as pharmacokinetic profiles similar to those seen when dosed orally in rats (and other animal species tested).

Arthroscopy
To obtain synovial tissue samples, arthroscopy under local anaesthesia was performed in all patients at baseline 2 days before treatment and repeated at day 28, 1 day after the study-medication was discontinued. Arthroscopies were performed in an actively inflamed wrist, knee or ankle joint. Biopsies were taken twice from the same joint in each patient. Synovial tissue was snap-frozen immediately after biopsies were taken, then sectioned and slides were stored at –70°C until used for staining. Arthroscopies, tissue sampling and storage were done as described previously in detail [19].

Immunohistochemical analysis
Staining was performed to detect fibroblast-like-synoviocytes (CD55), T-cells (CD3, CD4 and CD8), macrophages (CD68), subsets of macrophages (CD163, MRP14, MRP8 and MRP8/14), B cells (CD22), CD38+ plasma cells, CD15+ neutrophils, mast cells and the pro-inflammatory cytokines TNF-{alpha}, IL1-ß and IL-6. Additionally, cells bearing C5aR (CD88) were identified. Serial sections were stained with the following monoclonal antibodies: anti-CD3 (SK7, Becton-Dickinson, San Jose, CA, USA), anti-CD55 (clone 67, Serotec, Oxford, UK), anti-CD68 (EBM11, Dako, Glostrup, Denmark), anti-CD4 (SK3, Becton-Dickinson), anti-CD8 (DK25, Dako), anti-CD15 (cd3-1, Dako), anti-CD22 (CLB-B-Ly/1,6B11, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), anti-CD38 (HB-7, Becton-Dickinson), anti-CD163 (Ber-MAC3, Dako), anti-mast cell marker (AA1, Dako), anti-MRP8 (8-5C2, BMA Biomedicals, Augst, Switzerland), anti-MRP14 (S36.48, BMA Biomedicals), anti-MRP8/14 (2,70E+11, BMA Biomedicals), anti-TNF-{alpha} (52B83, Monosan, Uden, The Netherlands), anti-IL6 (polyclonal, Department of Nephrology, Leiden University Medical Centre, The Netherlands), anti-IL1-ß (2D8, ImmunoKontact, Stockholm, Sweden) and anti-CD88 (LS112, Dako). Sections with non-assessable tissue, defined by the absence of an intimal lining layer, were omitted before analysis. For control sections the primary antibody was omitted or irrelevant antibodies were applied. Staining was performed according to a three-step immunoperoxidase method as previously described in detail [20].

Digital image analysis
The slides were evaluated by quantitative computer-assisted image analysis. For the cytokine-stainings, integrated optical densities (IOD) were calculated for evaluation, from the cell marker stainings the numbers of cells were calculated (mean counts). The tissue sections stained for CD68 were digitally analysed both for total mean counts per mm2 and for mean counts in the synovial sublining per mm2. Both IOD and the numbers of cells positive for CD88 per mm2 were calculated in order to quantify both the expression levels of C5aR before and after treatment, and the number of C5aR+ cells in the synovium. All sections were coded and analysed as previously described in detail in random order by an experienced, independent observer (M.V.), who was unaware of the clinical data [21].

Pharmacokinetics
On the first (day 0) and the last day (day 27) of administering the study medication, serum samples were obtained to determine concentrations of PMX53 in peripheral blood of the patients at distinct time points: 30 min before administration of the drug and 15, 30, 60 and 90 min, 2, 4 and 6 h after administration. Compound concentrations were determined by mass spectrometry and the areas under the curve (AUC) were calculated.

In vitro inhibition of neutrophil activation by PMX53
Neutrophils were isolated with Lympholyte®-H (Cedarlane, Canada) using freshly drawn venous blood of a healthy donor. After isolation, the cells were incubated for 30 min at 37°C with medium alone (as a control), 1 nM, 10 nM or 100 nM PMX53. Cells were then stimulated for 30 min at 37°C with medium alone, or medium containing 1 nM C5a, 10 nM C5a, 100 nM C5a or 10 nM IL-8. Cells were incubated for 30 min with FITC-labelled anti-human CD11b (BD Pharmingen, San Diego, CA, USA) or isotype control mouse IgG2a (BD Pharmingen). The cells were washed and diluted in FACS-buffer and analysed by FACS. The experiments were performed three times using blood from different donors. The absolute mean fluorescence intensity of CD11b was normalized compared to cells stimulated with medium alone.

Statistical analysis
Patient characteristics were compared between the two treatment groups and clinical responders vs non-responders to the study treatment using {chi}2-tests and t-tests. Results before and after treatment and changes from baseline were compared by paired t-tests. A two-sample t-test was used to compare the changes from baseline between the two treatment groups.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 Acknowledgements
 References
 
Patients groups were comparable at baseline
Twenty-one patients were included in the study over an 8-month period. Fig. 2 shows the inclusion and disposition of patients in the study. Patient baseline characteristics are summarized in Table 1. In the PMX53-treated group erosive disease, RF and anti-CCP antibodies tended to be more prevalent. In contrast, tender joint count, swollen joint count and DAS28 tended to be slightly higher in the placebo group. With the exception of anti-CCP antibody positivity, which was observed at a significantly higher frequency in the patient group treated with PMX53 at baseline, no statistically significant differences were observed between patient groups treated with placebo and PMX53. No differences were observed between clinical responders vs non-responders to the study treatment (data not shown).


Figure 2
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  		FIG. 2. Patient inclusion and disposition. The flow diagram template was obtained from www.consort-statement.org [46].

 

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  		TABLE 1. Patient characteristics at baseline

 
Adequate PMX53 serum levels were achieved in the majority of patients
Serum samples were obtained for pharmacokinetic analysis from all patients at day 0. On day 27, analyses could not be performed on two patients because they mistakenly took their study medication at home before coming to our trial unit. The mean of AUCs of day 0 was 40.8 nmol h/l (range of 1.8–130.7) (Table 2). In all but two patients, PMX53 AUCs were higher than 10 nmol h/l. There was no correlation between AUCs and clinical response.


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  		TABLE 2. AUCs of individual patients treated with PMX53

 
PMX53 inhibits neutrophil activation by C5a in vitro
Experiments were performed to confirm that levels of PMX53 reached in the patients could effectively inhibit C5aR+ cell activation. Stimulation of freshly isolated neutrophils with increasing concentrations of C5a resulted in an increase in CD11b surface expression (Fig. 3). Pre-treatment of neutrophils with PMX53 at concentrations that reflect the AUC-values observed in patients inhibited C5a, but not IL-8-induced CD11b up-regulation in a dose-dependent manner.


Figure 3
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  		FIG. 3. Neutrophil activation by C5a is reduced by PMX53 in a dose-dependent manner. Freshly isolated neutrophils were pre-incubated with different concentrations of PMX53, then stimulated with different concentrations of C5a, medium or IL8 (x-axis). CD11b expression was measured by FACS analysis (y-axis). N = 3 different donors. Error bars represent SDs.

 
PMX53 treatment is generally well tolerated
All patients completed the study. No serious adverse events were reported. Nine patients (65%) receiving PMX53 and seven patients (100%) in the placebo group reported at least one adverse event during the study. Of a total of 47 reported adverse events (25 in the placebo group), six events were possibly related to the treatment: two were reported in placebo patients, one patient in the PMX53 group once reported nausea and one other patient reported ‘feeling abnormal’ once after taking the medication. No major abnormalities in haematological, serum chemical or urinary findings occurred during or after the study (data not shown).

PMX53 treatment does not ameliorate rheumatoid arthritis
Patients treated with the C5aR antagonist PMX53 did not show clinical improvement when compared with patients who received placebo (Table 3). The placebo-treated patient who showed a moderate DAS28 response is not the same patient who had an ACR20 response.


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  		TABLE 3. Clinical response to study medication

 
The synovial cell infiltrate and pro-inflammatory cytokine expressions are not affected by PMX53 treatment
Fig. 4 shows representative pictures of infiltration by CD68+ macrophages and C5aR+ cells in serial synovial biopsies. Table 4 shows the mean (non-significant) changes in cells per mm2 of the various inflammatory cell types and Table 5 the (non-significant) changes in IOD per mm2 of the inflammatory cytokines. Paired analysis of the changes following treatment showed no significant differences from baseline for any of the markers examined, including key biomarkers associated with active treatment (Fig. 5) [22, 23]. In addition, comparison of the mean changes from baseline did not show (trends towards) differences between the patient groups.


Figure 4
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  		FIG. 4. Representative synovial expression of CD68+ macrophages and CD88 (C5aR)+ cells before and after treatment of a placebo patient (A, B) or a patient treated with PMX53 (C, D). The original magnification 200x.

 

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  		TABLE 4. Data on synovial tissue analysis: changes in the presence of various immune cells

 

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  		TABLE 5. Data on synovial tissue analysis: changes in pro-inflammatory cytokine expression

 

Figure 5
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  		FIG. 5. Treatment with PMX53 did not reduce the number of macrophages (A) and C5aR+ cells (B) in the synovial tissue. Different lines represent paired values before and after treatment in individual patients. (A) Expression of CD68+ cells in the synovial sublining in serial synovial biopsy specimens after either treatment with PMX53 or placebo for the individual patients. Paired analysis showed no significant change in numbers of macrophages in patients treated with PMX53 (I) (P = 0.5) or placebo (II) (P = 0.1). (B) Expression of CD88 (C5aR)+ cells in serial synovial biopsy specimens after treatment with either PMX53 or placebo for the individual patients. Paired analysis showed no significant change in number of macrophages in patients treated with PMX53 (I) (P = 0.3) or placebo (II) (P = 0.4).

 

   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
Acknowledgements
 References
 
In this double-blind, placebo-controlled, phase Ib trial, we have shown that treatment with the orally administered C5aR antagonist PMX53 in patients with RA did not alter synovial infiltration by immune cells, including C5aR+ cells, nor did treatment influence synovial expression of inflammatory cytokines. Of particular interest, no changes were observed in the number of CD68+ macrophages in the synovial sublining, a cell population which may serve as a highly sensitive biomarker of clinical response in RA [22, 23].

It has been postulated previously that the complement factor C5a may be a key regulator of inflammation in RA. Autoantibodies present in inflamed synovial tissue, including rheumatoid factor, form complexes that may activate the complement system. Resultant generation of C5a might then contribute to local inflammation by attracting and activating circulating cells expressing C5aR, particularly monocytes and neutrophils [24]. Consistent with this notion, production of C5a and the presence of C5aR+ cells have been detected in the RA synovial compartment [3, 25–28].

Additionally, C5aR+ mast cells have been proposed to serve as a critical link between autoantibodies and effector inflammatory cells in arthritis [29, 30]. This notion is supported by the observation that C5aR+ mast cells, isolated from peripheral blood from healthy individuals, can migrate towards C5a in vitro [31]. Local production of complement factors and regulators can be observed in RA synovial tissue, suggesting that with appropriate stimuli, complement activation via both classical and alternative pathways can take place in RA synovium [32, 33]. Finally, in diverse animal models of arthritis, C5a has generally been reported to play a key role in synovial inflammation [7, 8, 12, 34].

Despite this body of evidence, our trial suggests that blockade of C5a–C5aR interactions does not result in improvement in RA. Our analyses indicate that the failure of PMX53 to influence RA is not due to the trivial possibility that insufficient levels of the compound were reached in the patients to mediate a potential biological effect. First, there was no relationship between patient response and serum levels of PMX53; there was no response in patients with high serum levels of PMX53. Second, in vitro experiments demonstrated that PMX53 could effectively inhibit C5a-induced CD11b upregulation in vitro at concentrations comparable to those observed in our patients. Our findings are consistent with the results of a clinical trial in RA with eculizumab, an anti-C5 antibody [35]. Eculizumab strongly inhibits C5 in vivo, and thereby the formation of both C5b-9 and C5a, leading to the clinical efficacy observed in paroxysmal nocturnal haemoglobinuria (PNH), but not in RA [36, 37].

The trial design used in this study has been tested in a variety of studies of comparable size and treatment duration on a variety of (biological) DMARDs evaluating effective drugs like methotrexate, leflunomide, gold, corticosteroids, infliximab, anakinra and rituximab, and ineffective drugs like anti-CD4 antibodies, IFN-ß, IL-10 and anti-MCP1 antibodies [22, 23, 38–40]. Together, we detected high sensitivity to detect relevant changes in the synovium after effective treatment in relatively small groups of patients [23]. We have shown for a variety of anti-rheumatic drugs that patient groups of 10–12 per active arm are sufficient to detect changes in synovial biomarkers associated with changes in DAS28 with a standardized response means, as a measure for sensitivity to change, of at least 0.8, which indicates good sensitivity to change [22, 23, 38, 40]; in the present study we had 14 patients in the active treatment group. Importantly, it has previously been shown that the synovial features within patients who do not receive active treatment are on average stable [23, 41, 42]. Examples include anti-CD4 treatment, IFN-ß and IL-10 treatment, as well as anti-MCP1 treatment [20, 39, 41, 43].

In summary, biologically relevant levels of the C5a receptor antagonist PMX53 were reached in patients with active RA, but did not result in a decrease in synovial inflammation. Thus, although blockade of cell migration could be an attractive strategy to treat immune-mediated inflammatory disease [44, 45], C5aR blockade does not appear to be a promising approach to reduce the synovial infiltrate in RA.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
References
 
We wish to thank Dr Koen Vos, Dr Niek de Vries, Dr Dirkjan van Schaardenburg and Dr Arno van Kuijk for referral of patients, Margot Colombijn for invaluable logistic support, and Desiree Pots for excellent technical assistance with immunohistochemistry.

Funding: D.M.G. and T.J.M.S. were supported by the Dutch Arthritis Association (‘Reumafonds’), P.P.T. by the European Community's FP6 funding. This publication reflects only the author's views; the European Community is not liable for any use that may be made of the information herein. The clinical study was supported by Promics Ltd, Brisbane, Australia.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 17 April 2007; Accepted 18 June 2007


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