Four patients with active disease each received an i.v. dose of 10 mg/kg canakinumab. Within 1 d, their urticarial rashes had disappeared, and a complete clinical response was achieved within 1 wk. CRP and SAA (), as well as plasma levels of IL-6 and IL-1Ra, returned to their normal ranges (), whereas levels of IL-1α and TNF-α did not change (). Sensitive markers for neutrophil (S100A12) and monocyte/macrophage activation (S100A8/9) (18
) showed a rapid decline () along with a normalization of neutrophil counts (). Levels of soluble IL-1RII in serum were within the normal range and showed no change after treatment (). Canakinumab induced long-lasting complete clinical response; the median time until redosing after relapse was 185 d (). Patients were retreated i.v. with 1 mg/kg canakinumab, resulting in clinical remissions for a median of 90.5 d. Three further patients were enrolled into the study, and all seven patients received a fixed 150-mg s.c. dose with repeat treatment on clinical disease flare. The median treatment duration in the study was 26.5 mo (range 13.5–28.5) and the median duration of clinical remission after each 150-mg s.c. dose was 127 d (range 55–230). Together, these data indicate that CAPS is solely driven by IL-1β.
Figure 1. Acute phase response after treatment with canakinumab. (A–E) Data for four patients treated i.v. with 10 mg/kg canakinumab as mean ± SEM. A, SAA and CRP; B, IL-6 and IL-1Ra; C, TNF-α and IL-1α; D, S100 A12 and A8/9; E, (more ...)
Time until redosing in days after different doses and routes of administration of canakinumab
It appears that time until relapse is a function of the duration of IL-1β neutralization. To study this hypothesis, a mathematical model was developed with the aim of gaining insights into the production of IL-1β in vivo and to predict the time until disease relapse. This was possible because IL-1β, which was undetectable in sera of patients at baseline (assay detection limit <0.1 pg/ml), could be detected by an assay that measured IL-1β complexed with antibody. The formation of these complexes resulted in the extension of IL-1β's normal half-life of ~3.5 h to that of the antibody (t1/2
30 d), thereby enabling the detection of IL-1β produced in vivo. Data, including concentrations of canakinumab, complexed IL-1β levels in blood, acute phase responses, and time until disease flares, were incorporated in the creation of the model ( and supplemental text
). The model included the diffusion exchange of canakinumab and IL-1β between the tissue and plasma compartments, plus elimination rates for free canakinumab, free IL-1β, and the canakinumab–IL-1β complexes from plasma. Because there is a direct, although nonlinear, relationship through the binding reaction between antibody, free IL-1β, and their complex, free IL-1β concentration can be calculated from measurement of canakinumab () and total IL-1β () levels. Canakinumab rapidly suppressed free central and interstitial IL-1β () to levels that were insufficient to stimulate downstream responses such as CRP, SAA, and clinical flare (). All these response events had similar kinetic profiles, which were ultimately dependent on the concentration of canakinumab and were well described by the model over multiple dosing cycles.
Figure 2. Structure of the PK-biomarker-symptom model. (A) canakinumab is injected into the plasma compartment and then permeates and distributes to a peripheral (tissue) compartment, where it can bind IL-1β. Unbound IL-1β in the tissue stimulates (more ...)
A posterior predictive check was performed on data from the three patients who had not been used in the creation of the model ( and Table S1
). Although drug levels were well predicted (), IL-1β production was predicted to be too high for patients 5 (, dashed lines) and 7 () and too low for patient 6 (). Consequently, the predictions for CRP (), SAA (), and time until relapse () were suboptimal. Correcting the model to fit the measured complexed IL-1β production allowed it to predict the remaining data correctly. Thus, the milder disease and longest remission time in patients 5 and 7, as well as the severe disease and short remission time in patient 6 (), were entirely driven by endogenously produced IL-1β. The slope of the IL-1β concentration–effect relationships for CRP and SAA were relatively shallow, with Hill coefficients of 0.8 and 0.9, reflecting the fact that flares occurred slowly over a time period of 2–4 wk. The estimates of the maximum effect (Emax
) of IL-1β on CRP and SAA were in the region of 79 mg/liter and 500 mg/liter, with half-maximum stimulatory concentrations (EC50
) of 0.8 and 2 nM, respectively. The real values of the three independent patients were 77, 21, and 288 mg/liter for CRP and 189, 85, and 1,060 mg/liter for SAA, whereas the median (25th–75th percentile) for the cohort of seven patients was 77 mg/liter (59–105 mg/liter) for CRP and 377 mg/liter (189–617 mg/liter) for SAA, indicating that the model predicts the maximum levels of these acute phase response parameters fairly well. Although seemingly complex, the model did not include the influence of membrane-bound or soluble IL-1 antagonists and still predicted the biological responses well, suggesting that natural inhibitors of the IL-1β do not substantially contribute to neutralization of IL-1β in this disease. The decoy IL-1RII, which is expressed on the cell surface or is produced as a soluble form, binds IL-1β with a high-affinity dissociation constant (Kd) of 1 nM. Its binding affinity is further enhanced by the IL-1R accessory protein (AcP) (19
). Serum levels of sIL-1RII in the studied CAPS patients were not different from those in healthy controls (range 6–21 ng/ml) and did not change after treatment with canakinumab (), indicating a basal, but not dynamic, contribution to IL-1β inhibition. Recombinant IL-1RII has been shown to dose-dependently compete with the binding of canakinumab to IL-1β, which suggests that IL-1β detected as a complex with canakinumab does not bind sIL-1RII. Thus, if there is endogenously produced IL-1β bound to and neutralized by the sIL-1RII, it cannot be detected by our assays. This makes it currently difficult to estimate the relative contribution of the sIL-1RII, as compared with canakinumab, to neutralization of endogenously produced IL-1β. The fact that canakinumab binds IL-1β in vitro with a Kd of 60 pM and its peak serum concentrations after a 150-mg s.c. dose are in the range of 15 µg/ml makes it likely that the vast majority of biological neutralization of IL-1β is mediated by canakinumab. However, given that there is a difference between the in vitro Kd and that estimated in vivo (healthy, 0.8 nM; CAPS, 0.39 nM; Table S1) may suggest that there is competition for binding between canakinumab and the sum total of other IL-1β binding entities in the system, whether they be soluble or membrane bound, in the plasma or in the tissues. Collectively, the amount of IL-1β production in vivo alone appears responsible for the severity of the disease and variable duration of remission after administration of canakinumab treatment.
Figure 3. A prediction check of the model. Simulations were performed for each of the three patients enrolled to the study who were treated only with 150 mg canakinumab injected s.c. Predictions from the model were then compared with the observed data, taking account (more ...)
IL-1β has been shown to stimulate its own production in vitro (20
). We looked for a similar positive-feedback loop in vivo by comparing the production of IL-1β in CAPS with healthy subjects and found that complexed IL-1β levels were much higher for CAPS patients over the first 8 wk after canakinumab treatment (); thereafter they were similar. This feedback mechanism was modeled by including a control loop whereby IL-1β stimulates its own production. Canakinumab, by suppressing free IL-1β, appears to disrupt this feedback mechanism, reducing IL-1β production to a normal and constitutive and, hence, IL-1β–independent rate (). This constitutive production of IL-1β was calculated to be 6 ng/d for both diseased and healthy subjects, whereas CAPS patients produced an additional 25 ng/d of IL-1β. Thus, the in vivo production of IL-1β in CAPS of 31 ng/d is about fivefold higher than in healthy subjects and is IL-1β dependent. This finding is consistent with the observations that anakinra treatment decreased in vitro–produced IL-1β in CAPS patients (16
) and that baseline IL-1β messenger RNA (mRNA) levels were higher in CAPS than in healthy subjects (). Furthermore, IL-1β mRNA expression in CAPS, but not in healthy subjects, declined within 24 h after canakinumab treatment () and increased at each relapse ( and Fig. S1
), paralleling the increase of free IL-1β. This confirms that the model correctly distinguished between IL-1β dependence and constitutive production of IL-1β. Interestingly, it took ~8 wk for IL-1β production to fully normalize in CAPS patients, despite the observed rapid clinical remission and normalization of CRP and SAA levels, suggesting that some level of inflammatory response was still ongoing. Although the mechanism is unclear, there was also complete normalization of S100A12 and S100A8/9 over a similar 8–10-wk period among CAPS patients who were treated with canakinumab, which had not occurred during long-term treatment with anakinra (H. Wittkowski, posters at The fifth International Congress on FMF and Systemic Autoinflammatory Diseases, Rome, 2008, and 15th Pediatric Rheumatology European Society Congress, London, UK, 2008). The similarities in the kinetics of S100A8/9 and IL-1β production suggest that S100A8/9 may serve as a sensitive marker for subclinical inflammation in CAPS (18
). Whether IL-1β stimulates the production of S100A8/9 or S100A8/9 stimulates the production of IL-1β has not yet been fully elucidated. Although IL-1β has been shown to induce S100A8/9 production in human monocytes in vitro (22
), engagement of TLR-4 by S100A8 stimulated the production of IL-1β (23
Figure 4. IL-1β production in CAPS patients as compared with healthy controls. (A) Total IL-1β levels in plasma for four CAPS patients (red) and six healthy volunteers (blue) after 10 mg/kg canakinumab i.v. (B) Model-derived estimation of IL-1β (more ...)
A simpler nonlinear mixed effect pharmacokinetic (PK) flare probability model was created (Table S2
) to identify a dosing regimen that should keep IL-1β production below the threshold associated with clinical evidence of CAPS. This model established an effective inhibition constant, Ki
, describing the concentration at which there is a 50:50 probability of a flare event, and a Hill coefficient. The Hill coefficient, through the logit transformation, approximates the inverse of the variance of a Gaussian distribution of canakinumab concentrations over which patients transition from a remission to a flare state (a Hill coefficient of 4.22 is, therefore, a variance of 0.24 and a standard deviation of 0.49, such that the 5–95% probability interval for canakinumab concentrations runs from 0.56 to 2.3 µg/ml, with 50:50 at 1.13 µg/ml, for a typical average patient). The model fitted the time of flare and the need for redosing very well. Across all observed events, patients were retreated at a mean of 69% probability of flare. To deduce a dose and regimen for preventing flare, Monte Carlo simulations were run using 1,000 replicates of the seven-patient database. The most practical posology was suggested to be 150 mg s.c. every 8 wk to keep patients flare free. This regimen has been applied to a randomized placebo-controlled study with a total of 31 patients. Data show that within a period of 24 wk, all patients randomized to canakinumab remained disease free, whereas 81% of patients on placebo flared (24
In summary, a targeted medicine approach using the anti–IL-1β antibody canakinumab in the rare monogenetic disease CAPS, along with data generated in healthy human subjects, has generated novel insights into in vivo IL-1β regulation. Modeling and simulation data indicated that the majority of elevated IL-1β in CAPS is produced by its own production and is completely restored after canakinumab treatment. Constitutive IL-1β in healthy subjects is IL-1β independent and not targeted by canakinumab. Further evolution of this mechanism-based concept should also allow new insights into more heterogeneous and complex chronic inflammatory diseases.