Introduction

Predicting and monitoring methotrexate (MTX) responses in patients with Crohn’s disease (CD) is presently not possible. Measurement of MTX-polyglutamates (MTX-PGs) in red blood cells (RBC) might enable therapeutic drug monitoring (TDM) as shown in other immune-mediated inflammatory diseases. Our aims were to assess the relation between MTX-PGs and treatment response, and to identify predictors of response in CD patients treated with MTX.

Aims & Methods

In a multicenter prospective cohort study, CD patients starting subcutaneously (s.c.) MTX without biologics were included and followed for one year. At baseline, clinical and biochemical parameters were recorded. 8,12, 24, and 52 weeks after start of therapy or when dropping out, blood samples were collected and individual MTX-PG levels (MTX-PG₁ - MTX-PG₅) were assessed in RBCs using mass spectometry. Primary outcome was either s.c. MTX discontinuation or initiation of step-up therapy due to disease activity or toxicity. MTX-PGs were analysed in an extended Cox model, and corrected for prednisone (at start) and budesonide. Secondary outcomes included biochemical disease activity, measured with fecal calprotectin (FCP).

Results

Eighty CD patients enrolled (mean age 55±13y, 35% male) with a median FCP of 268 µg/g (IQR 73-480). After one year 21 patients were still on MTX sc monotherapy. Twenty-one patients stopped MTX because of disease activity, 29 because of toxicity, and four because of a combination of both reasons (5 patients censored: ended study participation or stopped MTX because of undefined reasons).
MTX-PG₃ was the most abundant MTX-PG species with a median concentration of 51 nmol/L RBC (IQR 37-62) at week 12. A higher MTX-PG₃ concentration was associated with a higher rate of MTX drug survival (HR 0.86:for every 10 nmol/L increase in MTX-PG₃ the rate of MTX discontinuation decreased with 14%, 95%CI 0.75-0.99), lower FCP (β -3.7, SE 1.3) as well as biochemical response (FCP < 250, OR 1.1, 95% CI 1.0-1.3).
A higher HBI at baseline was associated with an increased rate of s.c. MTX monotherapy discontinuation (HR 1.08, 95% CI 1.02-1.16). Predictors of discontinuation due to disease activity (cause specific hazards) were male sex (3.83, 1.62-9.05), baseline eGFR (1.06, 1.02-1.09), baseline HBI (1.12, 1.02-1.23) and baseline plasma folate (0.94, 0.88-0.99). Sex and plasma folate were not correlated with HBI. No toxicity-specific predictors for stopping MTX could be identified.

Conclusion

RBC MTX-PG₃ concentrations are related to better MTX drug-survival and decreased biochemical disease activity. Therefore, the measurement of RBC MTX-PG₃ holds potential as a tool for TDM. Lower plasma folate at baseline and male sex are predictors for MTX-specific failure in the first year.

Introduction

Biologic treatment plays a crucial role in managing Crohn’s disease (CD) prior to considering surgical options. However, treatment failure and withdrawal are common and jeopardize the maintenance of remission in CD.

Aims & Methods

This study aims to analyse and compare drug survival – used as a surrogate marker for treatment efficacy – and reasons for withdrawal of the commonly used biologics in CD. This is a retrospective, monocentric study of CD patients treated at the University Hospital Heidelberg and registered in the IBD database from 2017 to April 2024. The primary endpoint was drug survival; the secondary endpoint was reason for treatment withdrawal. Drug survival was analysed using Kaplan-Meier estimators in SPSS. First-line treatments with Adalimumab (ADA) and Infliximab (IFX) were compared using log-rank tests following 1:2 Propensity Score Matching in R Studio.

Results

Of 1020 IBD patients, 396 CD patients were eligible. The included cohort had a median age of 48 years (IQR: 35-60) with 44.9 % male and a median disease duration of 9 years (IQR: 2-20) until therapy initiation. 25.5 % reported to have perianal disease. Among their 1035 immunosuppressive treatments, 771 involved the biologics ADA, IFX, Vedolizumab (VDZ) and Ustekinumab (UST). ADA showed the highest median drug survival (27 months; 95 % CI: 19.8-34.2) which was significantly longer than that of IFX (14 months; 95 % CI: 10.6-17.4). Median drug survival of UST was 22 months (95 % CI: 13.4-30.6) and 15 months for VDZ (95 % CI: 7.2-22.8).
The most common reason for withdrawal for ADA (41.3 %), VDZ (76.8 %) and UST (61.8 %) was insufficient response which included persistent clinical symptoms, elevated inflammatory markers, or endoscopic activity. For IFX side effects (24.4 %), insufficient response (21.1 %) and immunogenicity (20.3 %) were almost equally present.
In first-line treatments (47 IFX vs. 94 ADA-cases), the log-rank test showed significantly longer drug survival for ADA compared to IFX (49.7 vs. 33.6 months; p=0.034).

Conclusion

Adalimumab showed greater overall and first-line drug survival than infliximab, hinting at a preference of adalimumab in clinical practice. Most biologic treatments were withdrawn due to insufficient response. More prospective, multicentric studies are encouraged to confirm these findings and to support optimised treatment decisions.

Disclosure

Annika Gauss received lecture fees and consulting fees from AbbVie, Johnson & Johnson, Takeda, Tillotts Pharma, Hexal, Pfizer and Sanofi.

Introduction

MTX polyglutamates (MTX-PGs) are generated during treatment with methotrexate (MTX), by the enzyme folylpolyglutamate synthetase (FPGS), which intracellularly catalyzes the additions of up to seven glutamate residues to MTX within 24 hours after administration. MTX-PG₃ in red blood cells (RBC) show a concentration-effect relationship in patients with Crohn’s disease (CD) and is therefore a potential tool for therapeutic drug monitoring. However, RBCs are neither effector cells in the pathophysiology of CD nor are they the target cells in the treatment of CD. The extent of variability of MTX-PGs in white blood cells (PBMCs), which are the effector cells, or in the intestinal mucosa, i.e. the target cells, have as of yet not been determined.

Aims & Methods

In a multicenter prospective cohort study, CD patients were followed for 12 months upon the initiation of subcutaneous (sc) MTX therapy. Eight, 12, 24, and 52 weeks after start of therapy or when MTX was stopped, blood samples were collected. Mucosal biopsies were obtained from non-inflamed rectum and/or inflamed mucosa (if present) during a ileocolonoscopy for clinical purposes or rectoscopy for study purposes after 24 weeks. Individual MTX-PGs (MTX-PG₁ - MTX-PG₆) were analyzed in intestinal mucosa, RBCs and PBMCs by mass spectrometry (UPLC-ESI-MS/MS) using stable-isotope-labelled internal standards. In PBMCs, only MTX-PG₁ to MTX-PG₄ were measured.

Results

We collected 24 mucosal, 6 PBMC and 193 RBC samples during the follow-up of eighty CD patients. We were able to measure MTX-PG_1-6­ in all mucosa samples and MTX-PG_1-4 in all PBMCs. MTX-PG₆ was not detectable in RBC samples. The PBMC MTX-PG concentrations were higher than in matched RBCs (MTX-PG₁ x18, PG₂ x12, PG₃ x3, PG₄ x1.5). The prevalence of the MTX-PGs differed between the matrices: mucosa = PG₁ (29%)> PG₅ > PG₄ > PG₃ > PG₂ > PG₆ , PBMC = PG₁ (48%) > PG₂ > PG₃ > PG₄ , and RBC = PG₃ (32%) > PG₁ > PG₄ > PG₂ > PG₅. The MTX-PG₃ concentration (median; first quartile - third quartile) was 60.9 (44.4 -136.8) fmol/mg protein in mucosa, 12.8 (7.1 - 34.9) fmol/10⁶ cells in PBMCs and 5.0 (3.5 - 6.2) fmol/10⁶ cells in RBCs.
We collected 3 mucosal, 3 PBMC and 19 RBC samples after MTX discontinuation (2-10 weeks). We found a rapid and deep decline of MTX-PGs in PBMCs, but not in mucosa and RBCs. There was a non-significant decrease of long-chain MTX-PGs in contrast to the fast and significant decrease of short-chain MTX-PGs after MTX discontinuation.

Conclusion

This is the first time that MTX-PGs are measured in intestinal mucosal samples and PBMCs of CD patients. We found a different pattern of the MTX-PG species across the matrices, possibly due to differences in FPGS activity, life span of the cells or ongoing absorption. MTX-PG₃ was not the most abundant species in the effector cell (PBMCs) and target cells (mucosa). However, the MTX-PG₃ concentration is three times higher in PBMCs than in RBCs. Whether MTX-PG_3, measured in PBMCs and mucosa, displays a clear concentration-effect relationship is presently unknown.

Introduction

Crohn’s disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Current therapies include monoclonal antibodies, such as adalimumab (ADA) and infliximab (IFX), that target the inflammatory cytokine tumor necrosis factor alpha, collectively known as anti-TNF. While it has been shown that anti-TNF is effective at inducing and maintaining remission in patients with CD, this treatment fails in up to 40% of CD patients^​. Ineffective treatment results in disease progression and complications, emphasizing the need to understand mechanisms behind non-response and to define prognostic biomarkers. We previously identified differences in the peripheral blood DNA methylome associated with response and non-response. However, it is unclear how differential methylation can affect the transcriptome.

Aims & Methods

In this study, we assembled a cohort of 58 adult CD patients that were scheduled to start ADA or IFX treatment. Response to treatment was assessed after 6-9 months based on strict combination of endoscopic (≥50% reduction SES-CD), biochemical (CRP reduction ≥50% or CRP≤5 mg/L and FCP reduction ≥50% or FCP ≤250 µg/g) and clinical criteria (≥3 point drop in HBI or HBI ≤4 without corticosteroids). Whole peripheral blood leukocytes (PBL) were collected from patients before baseline endoscopy or at first infusion/injection, and 6-9 months later. mRNA was extracted using QIAsymphony system, converted into cDNA and sequenced in a paired-end format on the Illumina NovaSeq at the Amsterdam UMC Core Facility Genomics. Differential expression (DE) analysis was carried out within the R statistical environment (v 4.3.1) using the Bioconductor package DESeq2 (v 1.40.2).

Results

Prior to the start of treatment, we identified 504 and 750 differentially expressed genes (DEGs) distinguishing responders from non-responders in ADA and IFX cohorts, respectively. While 13 genes presented response-associated differential expression for both ADA and IFX, no overall correlation was observed between response-associated differential expression profiles. However, based on canonical pathway analysis, we found that expression differed in both ADA and IFX responders and non-responders at the level of neutrophil degranulation, B-cell receptor regulation, and cell growth and differentiation.

Conclusion

Our data suggest that response to ADA and IFX can be associated to the PBL transcriptome prior to treatment. Furthermore, while the differences between responders and non-responders to ADA and IFX display limited overlap in DEGs, we observe that response-associated pathways are shared.

Introduction

Advances in therapeutic drug monitoring (TDM) have significantly improved the understanding of the relationship between anti-TNF-alpha drug concentrations and disease activity in patients with Crohn’s disease (CD). Early dose optimization of adalimumab (ADAL) and higher serum trough concentrations are associated with increased clinical, biochemical, and endoscopic remission in CD^1,2,3,4. This study aimed to evaluate whether early dose optimization of ADAL to a trough level >15 µg/mL improves clinical and biochemical remission rates at 12 weeks compared to standard dosing.

Aims & Methods

This study aimed to evaluate whether early dose optimization of ADAL to a trough level >15 µg/mL improves clinical and biochemical remission rates at 12 weeks compared to standard dosing. This was a randomized, multicenter, open-label trial evaluating TDM-guided ADAL dose optimization versus standard dosing in biologic-naïve and experienced patients with active CD. In the dose optimization arm , after standard induction doses at Weeks 0 (160 mg) and 2 (80 mg), patients with trough levels ≤15 µg/mL at week 4 received an additional 80 mg at Week 6 followed by 40 mg every week. Patients in the optimization arm who had trough levels >15 µg/mL at week 4 and all patients in the standard care arm remained on 40 mg every 2 weeks. The primary endpoint was a composite of clinical and biochemical remission at Week 12, defined as Harvey-Bradshaw Index (HBI) <5 and C-reactive protein (CRP) <5 mg/L or fecal calprotectin (FCal) <250 µg/g.

Results

A total of 77 patients were enrolled in the trial (39 in optimization arm vs 38 in standard care arm). At week 4, 15/39 patients in the intervention arm were eligible for dose optimization, with twelve going on to receive the accelerated regimen. At week 12, 6/12 (50%) of the optimized patients achieved an ADAL trough level >15 µg/mL. A numerically higher proportion of patients in the dose optimization arm achieved the primary endpoint compared to the standard care arm, however this difference was not statistically significant (17 (43.6%) vs 12 (31.6%), p = 0.394). In total, eleven patients in the optimization arm (28.2%) and seven patients in the standard care arm (18.4%) achieved an ADAL trough >15 µg/mL, with mean trough levels being numerically higher in the optimization group (18.1 vs 12.8 µg/mL, p = 0.160). ROC analysis of the predictive value of Week 12 trough levels on the primary outcome determined an optimal threshold level of 18.2 µg/mL (AUC 0.68, sensitivity 88.9%, specificity 39.3%). When analyzing the entire cohort, patients who achieved an ADAL trough >15 µg/mL (whether they were dose-optimized or not) at Weeks 8 or 12 were significantly more likely to achieve combined clinical and biochemical remission (19/27 patients (70.4%) vs 10/50 patients (20.0%), p < 0.001). Taken separately, both clinical and biochemical remission occurred more frequently in patients who achieved an ADAL trough >15 µg/mL (clinical: 22/27 (81.5%) vs 16/50 (32.0%), p <0.001; biochemical: 22/27 (81.5%) vs 14/50 (28.0%), p < 0.001). No difference in adverse effects was observed between trial arms. The major limitation of the trial was the small sample size and patient withdrawal due to the COVID 19 pandemic.

Conclusion

Although proactive dose optimization did not significantly increase remission rates compared to standard dosing, achieving an ADAL trough level >15 µg/mL at Week 8 or 12 was strongly associated with higher remission rates. These findings support early TDM-driven dose escalation and suggest that maintaining ADAL >15 µg/mL is a key target for achieving biochemical remission.

References

1. Little RD, Swaine A, Reynolds R, et al. Adalimumab Drug Levels at Secondary Loss of Response Do Not Predict Response to Dose-intensification in Crohn’s Disease: A Retrospective, International Multicenter Study. Inflamm Bowel Dis. 2024;30(10):1714-1723. doi:10.1093/ibd/izad248
2. Assa A, Matar M, Turner D, et al. Proactive Monitoring of Adalimumab Trough Concentration Associated With Increased Clinical Remission in Children With Crohn’s Disease Compared With Reactive Monitoring. Gastroenterology. 2019;157(4):985-996.e2. doi:10.1053/j.gastro.2019.06.003
3. Chaparro M, Guerra I, Iborra M, et al. 538 Correlation Between Adalimumab Serum Levels and Remission After the Induction Phase in Crohn’s Disease Patients. Gastroenterology. 2015;148(4):S-107-S-108. doi:10.1016/S0016-5085(15)30370-X
4. Kim MJ, Kim E, Kang B, Choe YH. Therapeutic Drug Monitoring of Adalimumab During Long‐term Follow‐up in Paediatric Patients With Crohn Disease. J Pediatr Gastroenterol Nutr. 2021;72(6):870-876. doi:10.1097/MPG.0000000000003070

Introduction

In Crohn’s disease (CD), an exposure-response relationship is suggested for ustekinumab, with higher serum levels associated with better outcomes. Pharmacokinetic data are scarce, with suggested target maintenance trough level associated with remission at 0.8-1.4 µg/mL. ^{1, 2} The REScUE study (NCT04245215) investigated the effect of two different re-induction regimens with ustekinumab in patients with CD. No significant difference was observed on clinical, endoscopic and biological outcomes.³ Here we present the pharmacokinetic and pharmacodynamics results of the REScUE study.

Aims & Methods

This Belgian multicentre prospective double-blind randomized placebo-controlled trial included adult patients with CD treated with ustekinumab who presented with secondary loss of response to ustekinumab after documented primary response. All patients received a single IV re-induction with ustekinumab (≈6mg/kg) and were then randomized 1:1 to blinded maintenance ustekinumab 90 mg SC Q4W (intervention) or 90 mg SC Q8W (control) for 48 weeks. PK samples were obtained at baseline, week 4-8-12-20-28-36-48.

Results

In total, 108 patients were included: 67% female, median [IQR] age 41 [32-54] year, median disease duration 14 [7-22] years. Prior anti-TNF exposure was reported in 92% of the patients. Baseline ustekinumab trough levels at moment of loss of response were similar in both groups and at the suggested target level of 0.8-1.4 µg/mL ¹: median (IQR) q4 week 1.39 (0.84-2.74) µg/mL and q8 week 1.31 (0.91-2.34) µg/mL (p>0.9). Eight weeks after induction, there was a statistically significant difference in ustekinumab trough levels between both groups 10.8 (8.0-15.9) µg/mL versus 8.2 (4.9-11.1) µg/mL for q4weeks vs q8weeks respectively (p=0.002). After 48 weeks of treatment the statistically significant difference persisted with 6.0 (4.8-9.6) µg/mL versus 2.1 (1.6-2.9) µg/mL (p<0.001). No significant association between baseline/week 8/week 48 ustekinumab trough levels and (steroid free) clinical remisison [PRO2 (AP ≤ 1 AND SF ≤ 3) and FCP <250µg/g and no steroids for 90 days prior to week 48] or endoscopic remission [SES-CD <3 at week 48] was seen. However, there was a significant association between baseline/week 8/week 48 ustekinumab trough levels and biomarker remission [CRP <5 mg/L and FCP <250 µg/g at week 48]; OR (95% CI) 2.98 (1.85, 5.23), 1.12 (1.06, 1.19) and 1.35 (1.13, 1.66) (all p<0.01) respectively. As per quartile analysis of the ustekinumab trough levels at week 48, the lowest quartile (ustekinumab <0.9 µg/mL) had the lowest steroid free clinical remission in each treatment group.

Conclusion

In patients with CD experiencing secondary loss of response to ustekinumab, baseline ustekinumab levels are low. Dose optimization with a single IV re-induction followed by intensified maintenance dosing (90 mg SC q4W) leads to higher ustekinumab levels compared to IV re-induction followed by conventional maintenance dosing (90 mg SC q8W). No association was found between ustekinumab levels and different outcomes except for biomarker remission. This suggests that treatment with ustekinumab should be personalized on a patient level, guided by drug-clearance rather than guided by target trough levels.

References

¹ Adedokun O. et al Gastroenterology 2018;154:1660–1671
² Schreiber S. et al ECCO 2023 Copenhagen; P436
³ Bossuyt P. et al ECCO 2025 Berlin; OP35

Disclosure

PB: Financial support for research: AbbVie, EG. •Lecture fees: AbbVie, AMC ICP, Amgen, Bristol Myers Squibb, Celltrion, Dr Falk Benelux, EG, Galapagos, Globalport, Lilly, Medtalks, Materia Prima, Pentax, Springer Media. •Advisory board: AbbVie, Bristol Meyers Squibb, CIRC, Galapagos, Janssen, Lilly, Pentax, PSI-CRO, Roche, Takeda, Tetrameros.