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.

Clinical Case Summary

Cryptosporidiosis is a gastrointestinal infection caused by the protozoan Cryptosporidium spp. It is typically self-limited in immunocompetent patients. However, in immunocompromised individuals, it can lead to moderate to severe diarrhea and significant morbidity and mortality.¹
We present the case of a 31-year-old woman with a history of Crohn’s disease in remission, who presented with a one-week history of diarrhea (up to 10 bowel movements daily), abdominal pain, and fever (38°C). Laboratory tests revealed an elevated C-reactive protein (CRP), and intestinal ultrasound showed bowel wall thickening, hyperemia and wall disruption, suggestive of an active flare. However, routine stool culture isolated Cryptosporidium, and based on her clinical background, oral paromomycin and azithromycin were initiated, resulting in complete clinical remission.
Cryptosporidiosis is a common parasitic cause of diarrhea, particularly affecting immunocompromised patients, such as those with HIV, cancer or solid organ transplants.²
Although few cases have been described in inflammatory bowel disease (IBD), the immunosuppressive state of these patients may increase the risk of infection. Diagnosing cryptosporidiosis in this population can be challenging, as stool tests detect only about 30% of cases, and the clinical presentation often mimics an IBD flare.³
Treatment is generally supportive and reserved for severe or refractory cases. Nitazoxanide is the drug of choice, and in non-responders, it can be combined with fluoroquinolones or azithromycin. In IBD patients, the decision to initiate specific antiparasitic treatment depends on the degree of immunosuppression and the clinical scenario.⁴
A clear association between IBD and cryptosporidiosis has not been established, but it is crucial to rule out opportunistic gastrointestinal infections in suspected flare-ups before starting specific therapies.
image "Bowel wall thickening, hyperemia, and disruption of the normal layer pattern."

References

(1) Janssen B, Snowden J. Cryptosporidiosis. 2023 Jul 10. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan–. PMID: 28846353.
(2) Liu B, Schnider A, DeArmond M, Banach DB, Haubrich BA. Cryptosporidiosis in individuals with inflammatory bowel disease: a scoping review protocol. BMJ Open. 2024 Oct 15;14(10):e086529. doi: 10.1136/bmjopen-2024-086529. PMID: 39414295; PMCID: PMC11481120.
(3) Ferreira J, Baden R, Magge S, Cheifetz A. Cryptosporidiosis masquerading as a Crohn's flare. Inflamm Bowel Dis. 2011 Oct;17(10):E133-4. doi: 10.1002/ibd.21811. Epub 2011 Jul 7. PMID: 21739536.
(4) Manthey MW, Ross AB, Soergel KH. Cryptosporidiosis and inflammatory bowel disease. Dig Dis Sci 1997;42:1580–6.

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

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

Current treatments for Crohn’s disease (CD) and ulcerative colitis (UC) are associated with loss of treatment response. However, studies assessing the real-world burden of non-response in Canada are scarce and the clinical and humanistic burden remains unknown. We aimed to describe the burden of active disease on symptom prevalence and work impairment by describing patients with CD and UC according to remission status.

Aims & Methods

Data were drawn from the Adelphi Real World Inflammatory Bowel Disease Specific Programme™, a real-world, cross-sectional survey of gastroenterologists (GEs) and their consulting patients with CD and UC in Canada from June 2023 to January 2024. Data included physician-reported patient demographics and clinical characteristics. Descriptive analyses were conducted.

Results

19 physicians reported data for 144 patients (73 with CD and 71 with UC). Physicians reported 20% of patients not to be in clinical or endoscopic remission (non-remitters), 31% to be in clinical remission only and 49% in both clinical and endoscopic remission. Mean (standard deviation) age was 44.5 (20.5) years among non-remitting patients, 33.5 (13.3) years among those in clinical remission only and 40.4 (17.4) years among patients in those with clinical and endoscopic remission. Among non-remitting patients, 38% were an ex-smoker, versus 14% of clinical remission and 18% of clinical and endoscopic remission patients. The most reported comorbid conditions included anxiety (24% non-remitters, 14% clinical remitters, 18% clinical and endoscopic remitters), depression (14% non-remitters, 0% in clinical remitters, and 14% in clinical and endoscopic remitters) and hypertension (14% non-remitters, 9% clinical remitters, and 14% clinical and endoscopic remitters). Top three current symptoms included fatigue (52% non-remitters, 32% clinical remitters, 15% clinical and endoscopic remitters), abdominal pain (69% non-remitters, 14% clinical remitters, 7% clinical and endoscopic remitters), and non-bloody diarrhea (48% non-remitters, 7% clinical remitters, 6% clinical and endoscopic remitters). Regarding employment, 38% of non-remitters were working full-time versus 48% of clinical remitters, and 58% of clinical and endoscopic remitters.

Conclusion

Patients with CD/UC that had not achieved or maintained remission experienced more symptom burden, comorbid conditions, and were often not in full-time employment than those in disease remission. Notably, non-remitters experienced a high symptom burden, including fatigue, abdominal pain, and diarrhea, which impacted daily living. Patients with both clinical and endoscopic remission experienced fewer symptoms and full-time employment. This highlights the importance of, and significant unmet need for, effective treatment in IBD to manage disease symptoms and decrease inflammation.

Disclosure

Janssen Global Services funded the research and I am an employee of Janssen