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Aim To investigate the relationship between the serum concentration of infliximab, a chimeric monoclonal antibody to tumour necrosis factor-α (TNF-α), and its effectiveness in treating uveitis in Behçet disease.
Methods The authors studied 20 patients with Behçet disease who had been treated with infliximab (5 mg/kg). After informed consent was obtained, samples of the peripheral blood were taken. The authors began collecting blood samples after at least 4 months of infliximab infusions. The first sample was collected at 1 h after an infliximab infusion (Day 0), then after 4 weeks (Week 4), and finally at 1 h prior to the infliximab infusion 8 weeks later (Week 8). The clinical data on uveitis were collected from the clinical charts of the patients. The serum concentration of infliximab was measured by an enzyme-linked immunosorbent assay.
Results The mean serum concentration of infliximab in the 20 patients was 117.4±28.2 μg/ml on Day 0, 11.4±6.8 μg/ml on Week 4 and 6.3±4.8 μg/ml on Week 8. The serum concentration of infliximab in each patient was significantly correlated with its effectiveness in resolving the recurrent episodes of uveitis.
Conclusion Monitoring infliximab serum concentrations is useful in determining the effectiveness of infliximab treatments for uveitis in Behçet disease.
- Behçet disease
- anti-TNF-α antibody
- uveoretinitis
- serum concentration of infliximab
- Immunology
- inflammation
- treatment medical
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- Behçet disease
- anti-TNF-α antibody
- uveoretinitis
- serum concentration of infliximab
- Immunology
- inflammation
- treatment medical
Introduction
Behçet disease is a systemic inflammatory disorder of unknown aetiology.1 Patients with Behçet disease have recurrent episodes of uveoretinitis, which can cause irreversible damage to the neural retina and optic nerve, leading to vision decrease.2 The intense intraocular inflammation and recurrent uveitis are treated by both systemic corticosteroids and immunosuppressive agents such as ciclosporin.3 However, these conventional therapies fail to reduce the frequency of uveitis recurrence in many patients.
Infliximab, a chimeric monoclonal antibody to tumour necrosis factor-α (TNF-α), was developed and used to treat systemic inflammatory disorders such as rheumatoid arthritis4 5 and Crohn disease.6 In the eye, infliximab has been used to treat severe intraocular inflammatory diseases of non-infectious origin such as Behçet disease.7–15
Although the effectiveness of infliximab in treating Behçet disease has been reported in many studies,7–15 little is known about the relationship between the serum concentrations and their effectiveness. Thus, the purpose of this study was to determine the relationship between the serum concentrations of infliximab and its effectiveness in treating uveitis in patients with Behçet disease. In the current study, we examined the clinical outcomes of intravenous infliximab in four different groups of patients with Behçet disease.
Methods
A total of 20 patients with Behçet disease with refractory uveoretinitis were treated with infliximab at the Tokyo Medical and Dental University Hospital. Patients were given an initial infliximab intravenous infusion of 5 mg/kg, and then subsequent infusions at 2 weeks, at 6 weeks and then every 8 weeks after the initial infusion. Therefore, infliximab infusions were given at 0, 2, 6, 14, 22 and 30 weeks, and then every 8 weeks thereafter until discontinued.
After the patients had been receiving the infliximab infusion for at least 4 months, we began to collect blood samples with peripheral blood samples taken 1 h after the infliximab infusion (Day 0), at 4 weeks (Week 4) and at 8 weeks (Week 8) after the infusion. Blood was also collected just before the infusion of infliximab at 8 weeks. In addition, if the patients had uveitis attacks, a blood sample was collected at the time of the attack. Informed consent was obtained from each patient.
Based on a standard curve, serum levels of infliximab were measured by enzyme-linked immunosorbent assay (ELISA) using a mAbs specific for the TNF-α binding site on a chimeric monoclonal antitumour necrosis factor a (cA2). The captured cA2 was detected using a biotinylated mAbs specific for an epitope in the variable region of cA2, which does not inhibit the binding of TNF-α. The lowest level of infliximab that could be reliably detected was 0.10 μg/ml. The serum levels were measured in a commercial laboratory (Tanabe R&D Service, Tokyo, Japan) in accordance with a previously reported method.7 The coefficient of variation or relative error values for the intra- and interassays were within 20% or within ±25%, respectively.
The neutralising antibody titre to infliximab (ATI) was also measured by ELISA in accordance with a previously reported method.7 The ATI was only measured in patients who had undetectable serum concentrations of infliximab (<0.10 μg/ml).
Uveitis attacks in each patient were determined by reviewing the clinical charts of the patients. Any type of acute episodes of intraocular inflammation was recorded as an uveitis attack. The interval between the uveitis attack and the infliximab infusion prior to the uveitis attack was also collected.
Diagnosis of Behçet disease was made according to the criteria of the Behçet disease Research Committee of Japan, which is part of the Ministry of Health and Welfare of Japan.16 17 Statistical analyses were performed using the Mann–Whitney U test. p Values <0.05 were considered to be statistically significant.
This study followed the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Tokyo Medical and Dental University.
Results
The mean±SD of the serum concentration of infliximab at 1 h (Day 0) after the infliximab infusion was 117.4±28.2 with a range from 86.2 to 176.3 μg/ml (table 1). The mean concentration of infliximab at Week 4 was 11.4±6.8 μg/ml, and this was significantly lower than that on Day 0 (p<0.0001; Mann–Whitney U test). In all patients except Patient 15, the serum infliximab concentration at Week 4 was >1.0 μg/ml. At Week 8 just prior to the infliximab infusion, the mean serum concentration was 6.3±4.8 μg/ml, which was significantly lower than that observed at Week 0 (p<0.0001) and Week 4 (p<0.05). In addition, in Patients 15 and 16, the infliximab levels were below the detectable level, and in Patients 17 and 18, the levels were <1.0 μg/ml (table 1).
Patient profiles and infliximab (IFX) serum levels during the treatment
In the 15 patients who did not develop any uveitis attacks, the mean serum concentration of infliximab at Week 8 was 7.3±4.6 μg/ml, while the mean concentration was 3.4±4.7 μg/ml in the five patients who had uveitis attacks (table 1). However, the difference in the serum concentrations of infliximab between these two groups was not significant (p >0.05).
Therefore, we divided the patients into four groups based on the relationship between the infliximab serum concentration and its effectiveness in treating the uveitis. The first group included 14 patients who did not develop any ocular inflammatory attacks after the infliximab during all the following test periods (Patients 1–14 in table 1). The infliximab serum concentration in these patients was high, even at Week 8, ranging from 1.8 to 15.6 μg/ml with a mean of 7.8±4.3 μg/ml. Representative results for Patients 1 and 2 can be seen in figure 1A. The second group consisted of three patients (Patients 16–18) who developed ocular inflammatory attacks between the 5th and 8th week after the infliximab infusion (table 1 figure 1B). The serum concentration levels of infliximab were far below 1.0 μg/ml. The third group included the two patients (Patients 19 and 20) who had repeated severe uveitis attacks after their initial infliximab infusion. The infliximab concentrations in these patients were high at each of the three times the serum was collected (figure 1C), and the uveitis attacks were considered to be irrelevant to the level of infliximab. Patient 15 (figure 1D) was the only patient in the fourth group, and she did not develop any uveitis attacks, even though her serum concentrations were below detectable levels at Weeks 4 and 8. Similar to Patients 19 and 20, the uveitis attacks in Patient 15 were considered not to be associated with the infliximab level.
Infliximab serum concentrations in patients with Behçet disease with refractory uveoretinitis were measured by ELISA at three time points after infliximab infusion; 1 h (Day 0), 4 weeks (Week 4) and 8 weeks (Week 8) after the infusion of infliximab. In some patients (Patients 16, 18, 19 and 20), serum concentration was also examined when there was recurrence in the patient while being treated (active uveitis attacks, bold numbers in the graph, eg, W3, W5, W6, W7). (A) First group, consisting of patients who maintained high serum concentrations and had a complete remission of the uveitis attacks during the infliximab therapy (Group 1; results for Patients 1 and 2 are shown). (B) Second group, consisting of patients who had low serum concentrations at Week 8 followed by uveitis attacks during weeks 5–8 prior to the next infusion (Group 2; results for Patients 16 and 18 are shown). (C) Third group, consisting of patients who had repeated uveitis attacks despite high serum concentrations similar to those seen in Group 1 (Group 3; results for Patients 19 and 20 are shown). (D) Serum concentrations were undetectable at Weeks 4 and 8, even though there were no uveitis attacks (Group 4; results for Patient 15 are shown). The y-axis indicates the infliximab serum concentrations (μg/ml).
As the presence of infliximab in ELISA test samples affects the neutralising antibody-to-infliximab (ATI) measurements, we were only able to measure ATI when the serum infliximab was below detectable levels. Therefore, we measured ATI in the serum of Patient 15 at Weeks 4 and 8 and in Patient 16 at Week 8. ATI measurements in these patients were 1:320 at Week 4, 1:80 at Week 8 for Patient 15 and 1:20 for Patient 16 at Week 8.
Discussion
Our results demonstrated that the effectiveness of infliximab on the uveitis attacks in Behçet disease correlates with the infliximab serum concentrations. However, in some patients, the uveitis attacks were not related to the infliximab levels. At Day 0, the serum concentration was close to 100 μg/ml or higher in all patients, and the standard deviation was relatively small. As the infliximab serum concentration decreased with time after the infliximab infusion, this indicated that the infliximab serum concentration measurements were done correctly.
Our results indicated that the patients could be divided into four groups. The first group consisted of 14 patients (Patients 1–14 in table 1) who maintained high serum concentrations (>1.0 μg/ml) and had complete remission of their uveitis attacks during the infliximab therapy. In these patients, as the infliximab therapy was effective, it was reasonable to continue these treatments. The second group consisted of three patients (Patients 16–18) who had low serum concentrations at Week 8 followed by uveitis attacks during weeks 5–8 prior to the next infliximab infusion. In these patients, we suggest that the recurrence of uveitis occurred because of low infliximab concentrations. In fact, the serum concentration of infliximab was also low when these patients had active uveitis attacks. These patients experienced recurrences at 5–6 months after the infliximab treatments, which suggests these patients were responsive at the beginning of the treatments. We then shortened the interval between the infliximab infusion from 8 weeks to every 6 or 7 weeks, and after this, none of the patients had any further inflammatory attacks. However, one patient (Patient 18) had uveitis attacks on the 5th week after infusion. Thus, in these types of patients, treatment intervals need to be further shortened to 4 weeks.
The third group consisted of two patients (Patients 19 and 20) who had repeated uveitis attacks, despite having high serum infliximab concentrations at all times. In fact, these patients exhibited their first uveitis attacks immediately after the first infusion. Moreover, serum concentrations were also found to be high in the one patient who had active ocular inflammation. As infliximab was considered to be ineffective in these patients, we added ciclosporin or prednisolone to their treatments. In these patients, it was assumed that other inflammatory cytokines were related to their disease activity. The infliximab concentration in the last patient (fourth group; Patient 15) was undetectable at Weeks 4 and 8. However, this patient did not develop a uveitis attack, even though there were such low serum concentrations. Since we assumed that the infliximab did not have any effect on the ocular inflammation, we discontinued the treatment in this patient.
The serum concentrations of infliximab in the patients who did not develop recurrence of the uveitis (good responders) were not significantly different from those patients who developed a uveitis attack during their infliximab therapy (poor responders). However, this does not mean that the infliximab serum concentrations were not significantly related to its effectiveness on uveitis attacks in Behçet disease. There were two subgroups within the good responder group, one with high and one with very low infliximab serum concentrations. Similarly, there were also two subgroups in the poor responder group, one with high and one with very low serum concentrations. As such, there were no significant differences noted between the infliximab serum concentrations between the two groups.
At the present time, the optimal serum infliximab concentration levels in Behçet disease remain unknown. A previous study reported that the mouse/human chimeric monoclonal antibody neutralised TNF in vitro and in vivo at concentrations of 5–6 μg/ml.18 Furthermore, a study in rheumatoid arthritis reported that serum concentrations of infliximab above 1.0 μg/ml were required in order to be able to maintain good therapeutic effects in rheumatoid arthritis patients.19 Our results indicated that an infliximab serum concentration above 1.0 μg/ml is necessary to control ocular inflammations in Behçet disease.
It is well known that production of neutralising ATI during infliximab treatment in some patients can abolish the effectiveness of the infliximab treatment.7 20 Therefore, even though serum ATI measurements are considered to be very important, these measurements can be affected during ELISA if infliximab is present in the serum.20 When we measured ATI in our study, these antibodies were present in the two patients (Patients 15 and 16) whose infliximab concentrations in the serum were below detectable levels. However, we could not determine whether ATI was also present in other patients who had recurrences of ocular inflammation but who also had high concentrations of infliximab (Patients 19 and 20). We speculate that ATI might have been present and associated with the poor effectiveness seen for infliximab in these two patients.
In conclusion, the measurements of infliximab serum concentrations may provide useful information with regard to treatment efficacy in Behçet disease. Serum concentrations higher than 1.0 μg/ml are considered to be optimal in controlling ocular inflammation in Behçet disease. In some patients, however, shortening of the infliximab infusion interval might be helpful in maintaining the serum concentrations of infliximab and its efficacy.
References
Footnotes
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided by the Ethics Committee of Tokyo Medical and Dental University.
Provenance and peer review Not commissioned; externally peer reviewed.