Background
The introduction of biological therapies, including infliximab (IFX), has drastically altered the treatment course of chronic autoimmune disorders affecting children [
1]. IFX is a chimeric IgG1 monoclonal antibody targeting tumor necrosis factor-alpha (TNFα), a major pro-inflammatory cytokine implicated in the pathogenesis of inflammatory autoimmune disorders [
2]. As the first monoclonal antibody approved for pediatric indications, IFX remains one of the most well studied biologic agents and is widely used, on and off-label, for the management of several chronic autoimmune disorders, including inflammatory bowel disease (IBD) [
3], juvenile idiopathic arthritis (JIA) [
4,
5], and uveitis [
6‐
8].
Although IFX has demonstrated efficacy in the treatment of these pediatric autoimmune disorders, clinical response is variable. Studies in IBD and rheumatoid arthritis (RA) indicate that 30% of patients fail to respond to IFX therapy and up to 50% of those who do respond lose response by 1 year of treatment [
9,
10]. Inadequate drug exposure, determined by measuring serum trough concentrations of IFX, has been identified as a major source of primary therapeutic non-response or secondary loss of response to IFX in IBD [
11]. Moreover, inadequate drug exposure resulting in ongoing disease activity in any condition can have long term and significant consequences (e.g., increased disease morbidity, decreased quality of life). IFX is currently not labeled for use in children with JIA, due in part to the drug not achieving the primary endpoint in a pivotal phase 3 placebo-controlled randomized-controlled trial, in which the dosing strategy, guided by adult dosing parameters, ultimately failed to result in similar drug exposures in children [
12,
13].
Immunogenicity, the development of anti-drug antibodies (ADAs), is another major contributing factor to the pharmacokinetics of IFX and is believed to result in accelerated systemic clearance of IFX through immune-complex formation, resulting in risk of response failure [
14]. In addition to immunogenicity, a number of other patient factors, including: gender, systemic inflammatory burden, serum albumin concentrations, concomitant therapy with immunomodulators, and body weight are thought to modulate IFX clearance and serum trough concentrations [
15,
16]. Previous study results suggest that the pharmacokinetic properties of IFX vary by age resulting in increased clearance in the pediatric population [
17,
18]. Similarly, population-based pharmacokinetic studies have found that the pharmacokinetics of IFX vary by diagnosis [
19]. Anecdotally, in our experience, the prevalence and clinical concern for immunogenicity appear higher in children with IBD compared to patients treated for rheumatologic disorders. Therefore, the objective of this study was to compare IFX pharmacokinetics in children receiving IFX for the management of inflammatory autoimmune disorders and evaluate relevant sources of interindividual variability in real-world clinical practice, as the first step toward implementing precision therapeutics for IFX treatment of immuno-inflammatory disorders in children.
Methods
Study design and patients
This was a prospective cross-sectional study that collected blood samples and clinical data from a cohort of pediatric patients (n = 97) receiving maintenance IFX infusions at the Children’s Mercy Kansas City Infusion Center. Blood samples were collected from patients on stable IFX dosing (e.g., no changes in dose or interval for ≥2 dosing cycles), immediately prior to their scheduled infusion (i.e., trough samples). Blood samples were processed immediately, and the resulting serum aliquots were stored at − 80 °C prior to batch analysis. Relevant clinical and demographic data were collected from review of the electronic medical record. Information collected and used in these analyses included patient age, gender, diagnosis, IFX dose, prescribed dosing interval, time since last IFX infusion, concomitant disease modifying anti-rheumatic drug (DMARD) treatment with methotrexate (MTX) or azathioprine (AZA), most recent laboratory measurements of erythrocyte sedimentation rate (ESR), serum albumin concentrations, and C-reactive protein (CRP) levels, 71-joint count clinical juvenile arthritis disease activity scores (cJADAS-71), and IBD physician global assessment of disease activity (PGA) within 30 days of study visit, if available. cJADAS-71 was calculated as the sum of the physician global assessment (visual analog scale range of 0-10), the parent/patient global assessment (visual analog scale range of 0-10), and the active joint count (simple count range of 0-71). Clinical and laboratory parameters of IBD were assessed using PGA, with disease activity assigned as quiescent, mild, moderate, or severe via agreement by two independent pediatric gastroenterologists. Due to small sample size, severe (n = 1) and moderate (n = 7) IBD were combined and treated as a single disease activity group (moderate/severe) in subsequent statistical analyses. IFX dose intensity (mg/kg/d) was calculated as the average daily dose equivalent of IFX by dividing the patient’s weight-based IFX dose (mg/kg) by the dosing interval, represented by the number of days since the last IFX dose. The study was approved under the Children’s Mercy Kansas City institutional review board. Written informed consent/assent was acquired prior to inclusion of subjects in the study and collection of patient data and samples.
IFX and anti-IFX analysis
Serum samples were submitted for analysis to ARUP Laboratories (Salt Lake City, UT) and IFX and anti-IFX antibodies were detected using a NF-ƙβ luciferase gene-reporter assay (GRA) [
20]. The lower limit of IFX quantitation for the assay was 0.65 μg/mL and the upper limit of quantitation was 40 μg/mL. Serum IFX concentrations below the limits of quantitation were reported as 0 μg/mL and samples measuring above the limit of quantitation were reported as 40 μg/mL. Anti-IFX antibody detection was reported as positive or negative based on an infliximab neutralizing titer of 1:20 or greater. Anti-IFX antibodies in serum were additionally assessed using a commercial enzyme-linked immunosorbent assay (ELISA) in combination with an acid dissociation step following the manufacturer’s protocol (Eagle Biosciences, Amherst, NH). Anti-IFX antibody levels were reported in arbitrary units/mL. Samples with signal greater than two times background were deemed positive for anti-IFX antibodies.
IFX clearance estimation by population pharmacokinetic modeling
Estimates for pediatric pharmacokinetic parameters for IFX were obtained from a population pharmacokinetic model developed from 112 children from the phase 3 REACH study and were used to estimate IFX clearance (Cl) in our patients [
21]. Pharmacokinetic estimates for a typical child were as follows: clearance (Cl), 5.43 mL/kg/d; volume of distribution in the central compartment (V
1), 54.2 mL/kg; volume of distribution in the peripheral compartment (V
2), 29.2 mL/kg; intercompartmental clearance (Q), 3.52 mL/kg/d. A 2-compartment model with a 2-h intravenous infusion and first order elimination was used to estimate Cl of the IFX for each patient using a nonlinear mixed-effects approach in MONOLIX (Lixoft, Antony, France) [
22]. Interindividual variability of Cl was evaluated using an exponential random effects model. The intraindividual variability was described as an additive residual error model. Undetected IFX concentration in four patients was substituted to half of the lower limit of quantification (i.e. 0.325 μg/mL).
Covariate analysis
Variables associated with interindividual variability in Cl estimates were identified by population pharmacokinetic covariate analysis in MONOLIX. While IFX trough concentrations were measured in 97 patients, 15 patients were excluded due to missing covariates, and 82 patients were used to conduct covariate pharmacokinetic modeling. The covariates having significant influence were added in a stepwise manner with forward addition and backward elimination [
23]. The covariate model was evaluated by the difference in the objective function value (ΔOFV), such that ΔOFV greater than 3.84 (
p < 0.05, degree of freedom = 1) in forward addition and 7.88 (
p < 0.005, degree of freedom = 1) in backward elimination, was indicative of significance using the log likelihood ratio test.
Statistical analysis
Unpaired grouped analyses were conducted by Wilcoxon rank-sum testing. Spearman’s rank correlation analysis was used to evaluate correlations between continuous variables. Data analysis and statistical testing was conducted using JMP software v11 (SAS Institute Inc., Cary, NC). Statistical significance is considered for p < 0.05.
Discussion
Despite being a relatively well-studied biologic therapy, there remain significant gaps in knowledge regarding interindividual variability in IFX exposure, and the factors driving it in real-world practice. Even though IFX under-exposure is recognized as a major cause of primary drug non-response and secondary loss of response in pediatric patients, few pediatric studies have focused on understanding additional sources of interindividual variability in IFX exposure [
17,
18,
24]. Ours is the first study to directly compare factors contributing to variability in IFX exposure in a diverse pediatric population, across three different diagnoses, with incorporation of population pharmacokinetic modeling to control for observed variation in dosing practices in real-world patient care.
Across the study population (n = 97), IFX dose intensity (i.e., dose and dosing interval) correlated strongly and positively with IFX trough levels (p < 0.0001). Only patients with undetectable IFX trough levels had ADAs detected by GRA and may reflect the lack of tolerance of this assay to detect ADA in the presence of excess free infliximab. Six additional patients were identified to have ADAs by immunoassay, highlighting differences in sensitivity across assay types used in clinical practice. All ADA positive patients (9% of study population, all with IBD) had significantly lower IFX trough levels than patients without ADAs, putting them at increased risk for therapeutic failure and increased risk of infusion reactions to IFX, which represents one of the only two biological agents approved for pediatric IBD.
Overall, IFX trough levels were significantly lower in pediatric patients with IBD, compared to JIA or uveitis (Fig.
2b;
p ≤ 0.001); however, this is likely due to the lower dose intensity used in IBD, represented by both lower doses and longer dosing intervals. Population pharmacokinetic modeling confirmed that once we accounted for variability in dosing practices, IFX clearance rates were comparable across the three pediatric diagnoses investigated (Fig.
4). Nevertheless, the observed variability in dosing practices across diagnoses is important and offers an opportunity to compare and optimize practices across subspecialties. Looking at current dosing practices, 21 % of children with IBD were found to have IFX trough concentrations below the recommended trough levels associated with mucosal healing of IBD based on previous studies (i.e., < 5 μg/mL) [
24]. Furthermore, only patients with IBD had detectable ADAs in our cohort, begging the question whether the real-world dosing practices observed in our IBD cohort (median [IQR] of 7.7 [6.2,9.4] mg/kg every 6 [4,8] weeks) are adequate to prevent loss of treatment response to IFX. Two other recent analyses have similarly questioned the adequacy of standard 5 mg/kg IFX dosing every 8 weeks for pediatric IBD, advocating for treat-to-target approaches for IFX [
25,
26]. No patients with uveitis or JIA had ADAs or trough levels < 5 μg/mL, however, unlike IBD, there are no established IFX therapeutic trough targets currently utilized for JIA or uveitis. One may speculate that IFX troughs for rheumatologic conditions may need to be higher, based on evidence to suggest that systemic exposure may be inadequate to achieve acceptable drug concentrations at the target tissue of interest (e.g. the joint), which has resulted in trials utilizing direct intra-articular injections with IFX, or other biologic agents [
27,
28]. Failure to observe ADAs in patients with JIA or uveitis in our study may indicate a reduced propensity for ADA formation in this patient population, or is more likely to be related to the maintenance of higher trough concentrations [
29]. An alternative explanation may be that higher cumulative IFX dosing in JIA and uveitis may have resulted in higher IFX trough concentrations that potentially masked ADA detection by GRA or immunoassay in these patients [
30]. Even though the immunoassay we used included an acid-dissociation step that significantly increases the drug tolerance of the assay, drug tolerance is one inherent limitation of studies examining ADAs [
31]. An additional explanation for the lower incidence of IFX ADAs in patients with JIA and uveitis may be related to the higher rate of concomitant immunomodulator therapy use in this population (75% in JIA/uveitis vs. 30% in IBD); although, in our current cohort, concomitant medications did not appear to influence IFX clearance or the development of ADAs.
Consistent with previous studies, our data support findings of increased IFX clearance in patients with lower serum albumin concentrations, elevated ESRs, and patients positive for IFX ADAs [
16,
17]. However, we failed to demonstrate a significant relationship between IFX clearance and other previously investigated covariates, such as diagnosis, age, weight, and DMARD use. By covariate analysis using population pharmacokinetic modeling, we were able to develop a model to describe the relationship between IFX clearance for individuals and clinical covariates including ADA positivity by GRA, serum albumin levels and ESR. In contrast to previous work, weight and DMARD use were not shown as significant covariates for IFX clearance in our data [
21]. We did find that IFX troughs were significantly higher in patients receiving combination therapy with methotrexate than monotherapy with IFX (21.2 [12.3,39.5] vs 13.1 [5.9,25.2] μg/mL,
p = 0.01), however, patients receiving combination therapy were also receiving higher cumulative doses of IFX. Once dosing differences were accounted for in the pharmacokinetic modeling, no detectable effect of MTX on IFX pharmacokinetics was observed. This is contrary to observations of decreased IFX clearance with combination therapy in a post hoc pooled population analysis of the REACH trial of IFX in pediatric IBD [
21]. This discrepancy may reflect the significantly higher IFX dose (median [IQR]: 8.2 [6.4,9.7] mg/kg) and shorter dosing interval (median [IQR]: 6 [4,7] weeks) used in our study compared to the REACH trial (5 mg/kg every 8-12 weeks). In particular, early studies investigating the impact of MTX on reducing IFX clearance demonstrated the greatest impact at extremely low doses of IFX (i.e. 1 mg/kg), with significantly reduced effects as IFX doses were increased [
32]. Other studies have commented on observations of higher IFX troughs in patients receiving combination therapy with azathioprine or 6-mercaptopurine [
33]. No significant differences were observed in our study; however, this may be due to the small number of patients receiving concomitant therapy with azathioprine (
n = 3).
In a separate analysis we evaluated whether clinical disease activity in IBD and JIA were associated with increased IFX clearance. Such an analysis could not be performed for uveitis, as standardized disease activity scoring data were lacking for these patients. Although we had a limited number of patients with JIA with corresponding clinical disease activity scores, a significant positive correlation between cJADAS-71 and IFX clearance was observed. Specifically, when stratified by a low disease activity cJADAS-71 cut-off score of 2.5 that could be applied to both oligoarthritis and polyarthritis, patients with active disease had estimated IFX clearance values 49% higher than patients with low disease activity. This suggests that these patients would require higher IFX doses to achieve a similar level of exposure, indicating that these patients may be hypermetabolic. A similar trend was observed in our IBD cohort, with IFX clearance 50% higher in children with moderate/severe vs. quiescent disease, and 47% higher in moderate/severe vs. mild disease; however this trend did not reach statistical significance (p ≤ 0.08), likely due to limitations in sample size for moderate/severe IBD (n = 8). There may also be a potential limitation in the clinical assessment used for disease activity in the IBD population. In our study, we used the readily and consistently available PGA score, a compilation of clinically meaningful signs and symptoms in addition to objective laboratory values, rather than endoscopy, which is invasive and not always clinically indicated or performed to assess treatment response or mucosal healing. Nevertheless, there remained an association of increased IFX clearance with elevated ESR and low albumin levels, both physiologic markers of inflammatory burden. Together, these data support a potential relationship between increased disease burden and enhanced IFX clearance and suggest that IFX dosing intensity may need to be increased in the presence of active disease and continued elevations in inflammatory markers.
Employing therapeutic drug monitoring (TDM) as an individualized treatment strategy to guide IFX dosing has been shown to optimize efficacy, safety and cost effectiveness of biologic agents such as IFX in IBD [
34‐
36], and a trial is currently underway to determine the effectiveness of standardized TDM in IFX management across different diagnoses in adults [
37]. Our data suggest that dose individualization for children may need to go well beyond standard pediatric dosing practices in IBD (e.g., 5 mg/kg every 8 weeks), and perhaps clinical features such as markers of disease activity could judiciously guide higher dosing in children with rheumatic disease. However, future prospective longitudinal studies will be necessary to further delineate the relationship between disease activity and dose intensity, as the risk for under dosing children with IFX is unquestionable when disease activity is inadequately treated and long-term morbidity is at stake. Future studies will also need to further delineate the relationship between drug toxicity and dose intensity to begin to weigh the risks of increased or excessive exposure to IFX. Identification of potential upper thresholds of exposure may be able to minimize IFX related toxicities, such as excessive immunosuppression and risk for infection, or simply identify dosing that exceeds additional benefit to maximize cost effectiveness. In the realm of pediatric rheumatic disease, there are significant gains to be made in this domain.
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