Infliximab trough levels among patients with inflammatory bowel disease in correlation with infliximab treatment escalation: a cross-sectional study from a Greek tertiary center

Introduction

The introduction of biologic agents in the late 1990s transformed the management of inflammatory bowel disease (IBD), reshaping therapeutic strategies. Infliximab, a chimeric IgG1 monoclonal antibody targeting tumor necrosis factor-α (TNFα), was the first agent used, and to date it remains a cornerstone in providing effective induction and maintenance of disease remission in both Crohn’s disease (CD) and ulcerative colitis (UC) [1]. However, a substantial number of patients experience difficulties with the infliximab treatment. Approximately 30% of patients present no clinical improvement following the induction phase (primary non-response, PNR), and an additional 20-40% may develop a secondary loss of response (SLR) over time, with an annual risk estimated to be around 10-13% per patient year of treatment [2-4]. The SLR is commonly addressed through empirical approaches, such as dose or frequency escalation, the introduction of an immunomodulator, or transition to an alternative anti-TNF agent or to an agent targeting a different pathway of inflammation [5]. Pharmacokinetic and pharmacodynamic mechanisms both play a role in PNR and SLR to anti-TNFα therapy. Pharmacokinetic factors include low or undetectable drug concentrations, which may result from increased non-immune drug clearance, often related to a heightened inflammatory burden, or from immune-mediated clearance due to the development of immunogenicity. This is characterized by the production of antibodies to infliximab (ATI), also associated with immune-mediated reactions, such as infusion reactions. Pharmacodynamic factors are involved when therapeutic drug concentrations are achieved but the patient fails to respond, indicating an inflammatory pathway that is not primarily TNF-driven [6,7].

Several exposure-response relationship studies have shown a positive correlation between infliximab trough levels (TLs) and therapeutic outcomes, including clinical, biochemical, endoscopic and histological remission, in IBD, particularly during maintenance treatment, while certain IBD phenotypes, such as fistulizing CD, perianal CD and acute severe UC, may require even higher drug concentrations because of the increased inflammatory burden [8-14]. Moreover, infliximab TLs have been utilized to optimize treatment in patients with IBD [15-17]. Studies evaluating the drug concentration–effect relationship in IBD suggest a therapeutic window of infliximab at 3-7 μg/mL during maintenance therapy; however, others prefer higher infliximab trough concentrations at 5-10 μg/mL to reach better objective results, including mucosal healing, and to prevent suboptimal drug exposure and the development of ATI in the future [3,6,18,19]. Therapeutic drug monitoring, which involves the measurement of infliximab TLs and ATI, followed by dose titration to maintain levels within a therapeutic range, has become a valuable tool for diagnosing underexposure and guiding dose optimization [20].

Despite extensive recent research in this area, the optimal range of therapeutic infliximab concentrations required to achieve positive therapeutic outcomes remains largely uncertain. Moreover, few therapeutic drug monitoring studies of infliximab have been performed in a Greek population. This study aimed to assess the correlation between serum infliximab TLs and positive therapeutic outcomes in Greek patients with CD or UC, following intravenous infliximab maintenance therapy. In addition, the relationship between the infusion reactions and the presence of ATI was investigated.

Patients and methods

Results

Participants, infliximab treatment characteristics, infliximab TLs and ATI

A total of 77 patients (62.3% male, median age 43 years) were enrolled in this study. Twenty-six patients (33.8%) had UC, 48 (62.3%) had CD, and 3 (3.9%) unclassified colitis. The median IBD duration was 10 years, with a median age at diagnosis at 28 years. Of the 77 patients, 61 (79.2%) were biologic-naive prior to infliximab initiation. Among the remaining 16 patients (20.8%), 12 (15.6%) had been exposed to 1 biologic agent, 3 (3.9%) to 2 agents, and 1 (1.3%) to 3 agents. All 16 patients had been previously treated with anti-TNFα (7.8% with infliximab, 9.1% with adalimumab and 7.8% with golimumab). The baseline characteristics of the patients who were included in this study are presented in Supplementary Table 1.

The median duration of infliximab treatment until the measurement of infliximab TLs and ATI was 2.9 years. Infliximab treatment was escalated according to the physician’s decision in 47 patients (61%). All of them had a decrease in dose intervals; 35 (45.5%) had an infusion every 4 weeks and 12 (15.6%) every 6 weeks. Seven patients (9.1%) had a further dose escalation, among whom 6 (7.8%) received infliximab 7.5 mg/kg and 1 (1.3%) infliximab 10 mg/kg. Concomitant therapy combined with infliximab at the time of TLs and ATI sampling was given to 13 patients (16.8%); 4 patients (5.2%) received azathioprine as an immunosuppressant, while 9 patients (11.7%) received corticosteroid therapy (Table 1).

Table 1 Characteristics of infliximab treatment in patients with inflammatory bowel disease (n=77)

Infliximab mean TLs were 7.2±4.9 μg/mL. Mean TLs were only correlated with treatment escalation (9.7 vs. 3.6 μg/mL, P<0.001), but not with sex, BMI, smoking status, IBD type (UC vs. CD), CRP levels, albumin levels or the combined treatment with azathioprine or corticosteroids (Supplementary Table 2). Twelve patients (15.6%) had undetectable infliximab TLs, 9 (11.7%) had TLs that were detectable but <3 μg/mL, 14 (18.2%) had TLs in the range of 3-7 μg/mL and 42 patients (54.5%) had levels >7 μg/mL.

Of the 20 patients with infliximab TLs <1 μg/mL, only 10 patients were positive for ATI.

Correlation of TLs with remission in patients with or without treatment escalation

A subgroup analysis indicated that, among the 30 patients without infliximab treatment escalation, 27 (90%) had clinical remission, while steroid-free clinical remission was observed in 25 of them (83.3%). Biochemical remission was observed in 22/30 patients (73.3%). Combined remission was observed in 19/30 patients (63.3%). Mean infliximab TLs were significantly greater among patients with biochemical (4.2 vs. 0.8 μg/mL, P=0.020) and combined (4.3 vs. 1.5 μg/mL, P=0.035) remission (Table 2, Fig. 1). According to the ROC curve analysis, TLs could accurately predict (AUC 0.768, 95%Cl 0.584-0.952, P=0.016) those patients with combined remission. The optimal TL cutoff was set at 3.4 μg/mL (sensitivity 63.2%, specificity 91%, PPV 92.3%, NPV 58.8%; Fig. 2).

Table 2 Subgroup analysis of correlations between infliximab trough levels and clinical remission, steroid-free clinical remission, biochemical remission and combined (steroid-free and biochemical) remission in patients with or without infliximab treatment escalation

Figure 1 Comparison of infliximab trough levels (μg/mL) among patients without treatment escalation with or without combined remission

Figure 2 Receiver operating characteristic curve analysis for infliximab trough levels among patients without treatment escalation with combined remission

Among 47 patients with infliximab treatment escalation, clinical remission was observed in 41 of them (87.2%), while steroid-free clinical remission was observed in 37 patients (78.7%). Biochemical remission was observed in 26/47 patients (55.3%) and combined remission was observed in 24/47 patients (51.1%). Mean TLs for each therapeutic outcome are shown in Table 2 and Fig. 3. According to the ROC curve analysis, TLs could predict with fair accuracy (AUC 0.653, 95%CI 0.527-0.755, P=0.018) patients with combined remission. The optimal TL cutoff was set at 11 μg/mL (sensitivity 66.7%, specificity 69.6%, PPV 69.6%, NPV 66.7%; Fig. 4).

Figure 3 Comparison of infliximab trough levels (μg/mL) among patients with treatment escalation with or without combined remission

Figure 4 Receiver operating characteristic curve analysis for infliximab trough levels among patients with treatment escalation with combined remission

Discussion

Our study investigated the correlation between infliximab TLs and positive therapeutic outcomes (clinical, steroid-free, biochemical and combined remission) in Greek patients with IBD. We found a significant correlation between infliximab TLs and treatment escalation (P<0.001). Subgroup analysis revealed that patients without treatment escalation had significantly higher TLs associated with biochemical and combined remission (P=0.020 and P=0.035, respectively). Furthermore, ROC curve analysis showed that TLs could significantly predict the combined remission for both non-escalated and escalated patients (P=0.016 and P=0.018, respectively). The optimal infliximab TL cutoff for achieving combined remission was 3.4 μg/mL for non-escalated patients (sensitivity 63.2%, specificity 91%, PPV 92.3%, NPV 58.8%) and 11 μg/mL for escalated patients (sensitivity 66.7%, specificity 69.6%, PPV 69.6%, NPV 66.7%). These findings show that higher infliximab TLs were associated with clinical and biochemical remission, particularly in patients requiring treatment escalation.

Numerous studies have shown that higher infliximab TLs result in better therapeutic outcomes in terms of clinical, biochemical, endoscopic and histological remission, in patients with IBD [8-14]. It has also been suggested that patients with specific IBD phenotypes, such as fistulizing or perianal CD, extensive disease or acute severe UC, often require higher drug serum concentrations to attain remission, because of the greater burden of inflammation [10,13,14,22,23].

Interestingly, when we divided patients into 2 groups, based on treatment escalation or not, we found a markedly significant difference in the TLs of patients on a standard regimen with biochemical and combined remission. It should also be noted that CRP as a biomarker, although not specific or highly sensitive for intestinal inflammation, has a positive correlation with clinical and endoscopic activity and therefore, it could predict a relapse of the disease [24,25]. In addition, normalization of CRP between 8 and 14 weeks post-treatment has been shown to predict a sustained long-term response to anti-TNF agents [26-28]. According to STRIDE-II, achieving clinical remission and normalizing CRP levels should be considered as intermediate (medium-term) treatment goals in both CD and UC [21]. Regarding the relationship between infliximab TLs and CRP, there are studies suggesting that CRP levels may serve as an indicator of serum infliximab TLs, in terms of predicting a loss of response. In fact, it was shown that a decline in infliximab TLs <1 μg/mL occurred before the loss of response to the drug, which could also be easily identified by detecting an elevation in CRP [29]. In a study of patients with worsening symptoms while receiving infliximab, CRP levels above 12 mg/L were highly specific in identifying patients with infliximab TLs <3 μg/mL [30]. Roblin et al [31], demonstrated that a loss of response to infliximab can be accurately predicted by considering a combination of CRP, infliximab TLs and stable ATI. A previous Greek study showed a decrease in infliximab TLs over time, which was associated with a concurrent increase in CRP levels [32].

To our knowledge, no previous studies have demonstrated that higher infliximab TLs can predict positive therapeutic outcomes specifically in patients requiring treatment escalation, compared to those on standard dosing. In our cohort, patients who had previously undergone treatment escalation empirically due to SLR showed an initial favorable response and remained in infliximab treatment. This suggests that pharmacokinetic, rather than pharmacodynamic factors, may be contributing to the current cases of non-remission that are more frequently associated with SLR [33,34]. This observation likely reflects a higher inflammatory burden in patients who achieved and maintained clinical and biochemical remission after dose escalation. Recent studies advocate for targeting a broader therapeutic range for infliximab (3-10 μg/mL) [3,20,35-37]. This wider range could encompass both patients who have undergone treatment escalation and those on a standard dose regimen.

Several previous studies have explored the association between infliximab TLs and treatment escalation after SLR [38-42]. It has been previously shown that responders exhibited a greater increase in serum infliximab TLs compared to non-responders during treatment intensification (8.8 vs. 3.0 μg/mL, P=0.035 using a reporter gene assay; 9.9 vs. 4.7 μg/mL, P=0.04 using a homogeneous mobility shift binding assay), indicating inadequate drug levels in a subgroup of patients [38]. Furthermore, infliximab TLs ≥1 μg/mL, low circulating interleukin 6 and adequate albumin levels before infliximab dose escalation in patients with loss of response, were significantly correlated with remission 40 weeks after escalation [39]. However, an earlier study found no difference in infliximab TLs at baseline between patients, who responded to treatment intensification and those who did not, suggesting that clinical improvement might occur after infliximab intensification, regardless of TLs [40]. In a retrospective study, infliximab TLs over 3.8 μg/mL identified with 90% specificity non-responders to either dose increase or switch to another anti-TNF agent [41]. Another study of patients with infliximab TLs >3 μg/mL and negative ATI found that TLs were significantly lower among patients in clinical remission compared to those with active symptoms, at both 6 and 12 months following treatment escalation, with an optimal cutoff at 4.8 μg/mL [42]. Nevertheless, Papamichael et al [20], suggested that, in cases of active disease during maintenance therapy, discontinuation of the infliximab treatment should be typically avoided unless drug concentrations exceed 10 μg/mL. In our study, there were no available data on infliximab concentrations prior to treatment escalation for comparison. Furthermore, the present study was not conducted under conditions of SLR but presented data only from patients routinely followed in our hospital center.

In our study, ATI were measured in 20 patients using a drug-sensitive assay, capable of identifying ATI only at low infliximab concentrations (<1 μg/mL). Consequently, the presence of ATI may have been underestimated. Recent advances in drug-tolerant assays have addressed this limitation by enabling ATI measurement even in the presence of infliximab, to a certain extent [43]. Nevertheless, the clinical benefit of employing a drug-tolerant assay in the presence of adequate infliximab TLs remains a subject of debate [20,43,44]. Among the 20 patients, 11 did not achieve combined remission, with 6 testing positive for ATI. This suggests a potential correlation between treatment failure and the development of immunogenicity, as indicated by the presence of ATI. However, because of the lack of serial ATI and infliximab TL measurements in our study, we cannot definitively establish this correlation. Intriguingly, among the remaining 9 patients, who achieved combined remission, 4 tested positive for ATI. This occurrence could be linked to a transient appearance of ATI.

A total of 8 patients reported reactions attributed to infliximab infusion. Notably, there was a tendency indicating a correlation between infusion reactions and the presence of ATI. Specifically, among the 5 patients who reported infusion reactions and had ATI measured, 4 tested positive for ATI. Similarly, 4 of 10 patients with positive ATI reported infusion reactions, in contrast to 1 patient with negative antibody status. While the limited sample size prevents definitive conclusions, these findings align with previous research that linked ATI development to a higher risk of infusion reactions to infliximab [45]. Immunogenicity against infliximab can be prevented and suppressed by the concomitant use of an immunomodulator [46].

Our study demonstrates a pioneering effort in identifying varying infliximab concentrations in relation to treatment escalation. Additionally, all patients were consistently followed at the same hospital center throughout the study period, where clinicians adhered to similar empirical methods. Moreover, all samples were stored under the same conditions and tested concurrently for infliximab TLs and ATI using the same ELISA plates in the same laboratory setting. It is important, though, that we also acknowledge certain limitations of our study. Firstly, the patient sample size was relatively modest, comprising solely individuals on infliximab maintenance therapy at a single hospital center. Moreover, our study lacked data on fecal calprotectin as a biomarker and endoscopic findings. Furthermore, as this was a cross-sectional study, each patient underwent only a single measurement of infliximab TLs, which could fluctuate over time, along with ATI, which might be transient if detected as positive. Another limitation is the utilization of a drug-sensitive assay, even though the debate over the clinical advantage of employing a drug-tolerant assay in the presence of adequate infliximab TLs remains ongoing.

In conclusion, our study demonstrated that infliximab TLs are associated with treatment escalation. Higher infliximab TLs are significantly linked to achieving biochemical and combined remission in patients without treatment escalation, highlighting the importance of maintaining adequate TLs for optimal therapeutic outcomes. Based on our results, targeting higher infliximab TLs and adopting a broader therapeutic range, as previous research has suggested, could include both patients undergoing treatment escalation and those on a standard dose regimen to achieve combined remission. These findings indicate that personalized infliximab dosing strategies, based on regular monitoring of TLs, could improve clinical and biochemical remission rates in IBD patients, particularly those requiring treatment adjustments. Large prospective studies are needed to confirm these associations and determine the optimal infliximab TL thresholds for patients with treatment escalation.