Radiolabelled interleukin-1 receptor antagonist for detection of synovitis in patients with rheumatoid arthritis

P. Barrera, C. J. van der Laken1, O. C. Boerman1, W. J. G. Oyen1, M. T. P. van de Ven, P. L. E. M. van Lent, L. B. A. van de Putte and F. H. M. Corstens1

Departments of Rheumatology and
1 Nuclear Medicine, University Hospital, Nijmegen, The Netherlands


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To investigate the distribution of radiolabelled interleukin-1 receptor antagonist (IL-1ra) in patients with rheumatoid arthritis (RA) and to assess whether this cytokine is suitable for scintigraphic visualization of synovitis.

Methods. In patients with active RA, scintigraphy was performed after a single i.v. dose of [123I]IL-1ra. Clearance and organ distribution of radiolabelled IL-1ra were studied. To assess whether radiolabelled IL-1ra targets the synovial IL-1 receptors, the scintigraphic images obtained with IL-1ra were compared with those obtained by the use of a non-specific control agent. In addition, autoradiography was performed in mice with antigen-induced arthritis that were injected with either radiolabelled IL-1ra or a size-matched, non-receptor-binding protein.

Results. Radiolabelled IL-1ra allowed clear visualization of inflamed joints. Specificity in the detection of synovitis was high, whereas a number of painful and swollen joints were not visualized by scintigraphy. The procedure was well tolerated and [123I]IL-1ra was rapidly cleared from the circulation (t1/2{alpha} 11 min, t1/2ß 612 min) and excreted mainly in the urine. The definition of synovial contours by IL-1ra scintigraphy was not better than that observed with a non-specific agent. Although radiolabelled IL-1ra retained its affinity for IL-1 receptors, no binding to synovium was observed by autoradiography.

Conclusions. Radiolabelled IL-1ra allows the visualization of synovitis in patients with RA. However, neither the imaging nor the autoradiographic studies indicate that joint accumulation of radiolabelled IL-1ra is due to specific IL-1 receptor targeting. IL-1ra has proved its therapeutic value in RA, but with the dose schedule in this study it does not behave as a specific radiopharmaceutical that is suitable for scintigraphic detection of inflammation.


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a systemic disease characterized by chronic symmetrical polyarthritis. Although the aetiopathogenesis of RA is not completely understood, proinflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF) play a crucial role in synovial inflammation and joint destruction [13]. This has led to the introduction of therapeutic approaches aimed at inhibiting IL-1 and TNF activity in RA [47].

Specific blockade of IL-1 can be achieved using its natural inhibitor, the IL-1 receptor antagonist (IL-1ra). This molecule belongs to the IL-1 superfamily and acts as an endogenous IL-1 inhibitor by binding with high affinity to IL-1 receptors without initiating signal transduction [8].

The therapeutic potential of recombinant human IL-1ra has been studied in phase II/III trials in patients with active RA. These studies show that repeated administration of IL-1ra results in rapid clinical improvement and decreases radiological progression [4, 5]. This underscores the role of IL-1 as mediator of inflammation and cartilage damage in RA.

Most radiolabelled agents currently used to localize inflammation accumulate in inflammatory foci through non-specific mechanisms and there is a need for more specific agents [9]. In patients with RA and other inflammatory joint diseases, such agents could provide information about local disease activity and severity and help to optimize therapies.

In recent animal studies, we have shown that radiolabelled IL-1 and IL-1ra localize in inflammatory foci by binding to IL-1 receptors [10]. Since the latter are abundant in rheumatoid synovium [11], their targeting with radiolabelled IL-1 or IL-1ra could be feasible.

Administration of IL-1 is, however, hampered by undesirable side-effects even at doses as low as 0.1–0.3 µg/kg. In contrast, doses up to 10 mg/kg of IL-1ra are safe and well tolerated [4, 5], which makes this cytokine suitable for scintigraphic studies.

Herein, we report the in vivo behaviour, in terms of scintigraphic imaging, tissue distribution and pharmacokinetics, of systemically administered, radiolabelled IL-1ra in patients with active RA.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
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Patients
Patients with RA according to the American College of Rheumatology criteria [12], aged >= 18 yr and with active disease were studied. Active RA was defined by the presence of >= 10 swollen joints and at least three of the following criteria: >= 10 tender joints; severe or very severe disease according to the patient; severe or very severe disease according to the investigator; and C-reactive protein >15 mg/dl. Doses of non-steroidal anti-inflammatory drugs and/or oral corticosteroids (up to 10 mg/day prednisone) were kept constant for at least 4 weeks prior to the study. Exclusion criteria were those used in the clinical studies with IL-1ra [4, 5], plus systemic or intra-articular corticosteroid administration in the past 4 weeks and hyperthyroidism.

Clinical joint pain and swelling were assessed and the disease activity score [13] was calculated immediately before scintigraphy. Thyroid uptake of iodide was blocked with oral potassium iodide and potassium perchlorate. Patients received a single i.v. dose of 74–185 MBq (0.5 mg) [123I]IL-1ra. Total body scintigraphic images and spot views of inflamed joints were obtained with a MultiSpect2 gamma camera connected to a Scintiview image processor and ICON computer system (Siemens, Hoffmann Estates, IL, USA) 5 min and 1, 4, 8 and 24 h post-injection (p.i.). The scintigraphic images, collected in digital format, were scored for joint uptake (0 = no uptake, 1 = uptake) without knowledge of the clinical scores. The sensitivity and specificity of scintigraphy for the detection of joint pain and swelling were determined over the following regions: shoulders; elbows; wrists; metacarpophalangeal and proximal interphalangeal joints (as a group); knees; ankles; and midtarsal and metatarsophalangeal joints (as a group). Whole-body clearance was calculated. Ratios of uptake in regions of interest (drawn over positive joints and, if present, over the contralateral negative joint and/or periarticular normal tissue) were normalized for the area of the region.

Radioactivity measured in blood and urine samples, obtained between 0 and 24 h p.i., was expressed as percentage of the injected dose after correction for radioactive decay.

In two patients, scintigraphy with a non-specific radiopharmaceutical, [123I]human serum albumin ([123I]HSA) or 99technetium-methylene diphosphonate ([99Tc-MDP]), was performed as a control.

The study was approved by the ethics review board at our centre.

Autoradiography studies
Antigen-induced arthritis (AIA) was induced in male C57 Bl6 mice by intra-articular injection of 60 µg methylated bovine serum albumin (mBSA) in of 6 µl phosphate-buffered saline into the knee joint 21 days after immunization with mBSA in complete Freund's adjuvant (CFA). On day 7, a single i.v. injection of [125I]IL-1ra (n = 2, total dose 500 µCi) was administered and 24 h later knee joints were dissected. Control mice received a single injection of [125I]myoglobulin (n = 2) or [125I]BSA (n = 2) instead of [125I]IL-1ra. Dry, deparaffinized whole-knee tissue sections were exposed to a photographic emulsion and processed for histology and autoradiography.

Radiolabelling and characterization of [123I]IL-1ra
Human recombinant IL-1ra (166.7 µg IL-1ra, 40 mg/ml; gift of Bill Rich, Amgen, Boulder, CO, USA) was labelled with 370 MBq 123I using the Bolton-Hunter method [14] shortly before i.v. administration.

Radiolabelled IL-1ra retained its receptor-binding capacity (receptor-binding fraction >85% for conditions of infinite receptor excess in the EL-4–6–1 thymoma cell line) [15]. The specific activity of [123I]IL-1ra was 4–10 MBq/mg and its radiopharmaceutical purity was >98% after gel filtration.


    Results
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 Patients and methods
 Results
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[123I]IL-1ra scintigraphy and distribution in RA patients
Clinical and demographic data for four patients enrolled in this study are summarized in Table 1Go. In three of the four patients, [123I]IL-1ra localized in inflamed joints within 5 min after injection. Joints were clearly visualized from 1 to 8 h p.i. The uptake of [123I]IL-1ra in inflamed joints (Fig. 1AGo) was at least 1.5-fold higher than in periarticular normal tissue and reached peak values of around 2.5-fold. In patients with asymmetrical arthritis, the ratio of inflamed to contralateral unaffected joints was approximately 1.5. Uptake of radiolabelled IL-1ra was also noticed in the kidneys, liver, spleen, bladder and, from 8 h p.i. onwards, also in the bowel.


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TABLE 1. Characteristics of the patients

 


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FIG. 1. (A) Anterior spot views of the knees, hands and feet 4 h after injection of [123I]IL-1ra. (B) Autoradiography of a knee section in a mouse with AIA. Original magnification x400, showing no preferential binding of [125I]IL-1ra to synovial tissue but local accumulation in fibrin present in the inflammatory exudate.

 
[123I]IL-1ra rapidly cleared from the blood, with t1/2{alpha} 11 ± 3 min and t1/2ß 612 ± 39 min. The excretion was predominantly renal (approximately 60 and 80% of the injected radioactivity was recovered in urine 8 and 24 h p.i., respectively), although a minor amount of radiopharmaceutical was also cleared via the hepatobiliary route.

In one patient with less severe arthritis (patient 1), no articular uptake of IL-1ra was observed despite mild tenderness and/or swelling in several joints. Two-phase conventional scintigraphy with 99Tc-MDP performed 2 days later again yielded no joint uptake. This patient had skin vasculitis at the time of the study and was later diagnosed with type-II cryoglobulinaemia.

No uptake of [123I]IL-1ra was observed in clinically unaffected joints of any of the patients examined (specificity for painful and swollen joints was 100%). Conversely, not all clinically affected joints showed [123I]IL-1ra uptake (Table 1Go). Excluding patient 1, the average sensitivity of the IL-1ra scintigraphy was 60% for painful joints and 72% for swollen joints. Administration of [123I]IL-1ra was well tolerated and no adverse events were observed.

One patient underwent a control scintigraphy with [123I]HSA shortly after [123I]IL-1ra administration. Imaging with [123I]HSA yielded a similar joint uptake pattern, and the delineation of synovial contours was as sharp as or better than imaging with [123I]IL-1ra.

[123I]IL-1ra autoradiography in the AIA model of arthritis
All knee sections of mice on day 7 after induction of AIA showed an inflammatory infiltrate in the synovial layer consisting of polymorphonuclear cells, mononuclear cells and lymphocytes and an abundant polymorphonuclear cell exudate in the joint cavity. Autoradiography of knee joint sections after a single injection of [125I]IL-1ra showed no preferential accumulation of the label in tissue structures. In contrast, the fibrin present in the inflammatory exudate showed more intense staining than the surrounding inflamed synovium (Fig. 1BGo). Control mice injected with non-receptor binding proteins showed a staining pattern similar to that observed with radiolabelled IL-1ra.


    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The present study shows that scintigraphic detection of synovitis can be achieved by systemic administration of radiolabelled IL-1ra in patients with RA. However, our results do not support the hypothesis that this is due to a specific interaction of the exogenous IL-1ra with IL-1 receptors in the joint.

In all but one of the patients studied, radiolabelled IL-1ra localized in arthritic joints within 1 h after injection, resulting in clear visualization of these joints for at least 8 h. This articular uptake seemed to be specific initially, since it was not observed in any of the clinically unaffected joints. The patient with less severe RA had negative IL-1ra scintigraphy, which was confirmed by two-phase conventional bone scintigraphy with 99Tc-MDP. The latter is a non-specific radiopharmaceutical known to accumulate in inflammatory lesions, and the lack of joint uptake suggests that the disease activity might have been overestimated by concurrent pathology or that the latter could have interfered with the scintigraphy itself.

When this patient was excluded, 40 painful and 30% swollen articular regions failed to show uptake of radiolabelled IL-1ra, and we hypothesized that these false-negative joints could have been due to low or absent local expression of IL-1 receptors. We then assessed whether IL-1ra joint uptake was explained by specific binding of this cytokine to IL-1 receptors at the local level or was the result of non-specific mechanisms such as increased vascular permeability. Therefore, we compared scintigraphic results after[123I]IL-1ra administration with those after [123I]albumin administration in one patient, and we conducted autoradiography studies in mice with AIA. Receptor-blocking studies were considered but not carried out since adequate neutralization of IL-1 requires repeated or continuous and prolonged administration of large doses IL-1ra [1, 4, 5, 8].

Scintigraphy with albumin labelled under the same conditions as [123I]IL-1ra yielded a similar uptake pattern in affected joints, with similar or more clear synovial delineation. This suggested that the localization of radiolabelled IL-1ra could be due to non-specific uptake rather than to binding to the IL-1 receptors present in the synovial lining. That localization was due to non-specific uptake was confirmed by autoradiographic studies in mice.

It should be pointed out that the lack of binding of IL-1ra to murine synovial tissue cannot be explained by the radiolabelling or by species specificity: radiolabelled IL-1ra retained high affinity for mice thymoma cells, and human IL-1ra ameliorates this and other murine models of arthritis [1, 2, 16]. The accumulation of IL-1ra in fibrin present in the joint exudate rather suggests binding to extracellular proteins. Binding to fibrin and extracellular matrix proteins is a non-receptor-specific phenomenon involved in the homing of currently used radiopharmaceuticals, such as human IgG, on inflammatory foci [17].

It should be stressed that the therapeutic efficacy of IL-1ra in RA requires repeated administration of this cytokine at 1000-fold higher doses than those used in our study. It is therefore possible that, in conditions of receptor saturation, the doses used in our study were too low to displace endogenous IL-1 or IL-1ra from their synovial receptors. Our results do not allow us to draw conclusions about the binding site of IL-1ra responsible for its therapeutic effects at higher doses.

Neither the imaging characteristics nor the autoradiographic study, taken together with the dose used in this study, suggest that systemically administered, radiolabelled IL-1ra binds to IL-1 receptors in inflammatory foci. The joint uptake observed may rather be explained by non-specific mechanisms. Administration of higher doses of radiolabelled IL-1ra with a similar specific activity would require an unacceptable radiation burden for humans. Our results do not favour the future use of this cytokine as a specific radiopharmaceutical.


    Notes
 
Correspondence to: P. Barrera, Department of Rheumatology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Back


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 27 July 1999; revised version accepted 28 January 2000.