Introduction

Crohn’s disease and ulcerative colitis, two forms of inflammatory bowel disease (IBD) require lifelong treatment and patient monitoring. Current predictors of relapse and therapeutic success have limitations, therefore there is a high unmet need to identify novel biomarkers in IBD. Monitoring approaches for IBD are mostly invasive, time-consuming, and expensive. Therefore, a simple, rapid, and non-invasive test, with the ability to differentiate IBD from other gastrointestinal conditions and to monitor disease activity, will have important clinical implications. Our previous mucosal cytokine profiling identified that Plasminogen activator inhibitor type 1 (PAI-1) is expressed in IBD patients with active disease, but not in control.

Aims & Methods

Therefore, we aimed to assess the utility of PAI-1 in monitoring disease activity and therapeutic response of IBD patients in different biological samples.
Serum, biopsy, and faecal samples were collected from more than 160 IBD patients and controls without IBD. ELISA method was used to define the level of PAI-1 in the mucosa, serum, and faecal samples and the serum CRP levels were also determined. For the mucosal gene expression analysis qRT-PCR was used. The correlation and ROC analyses were applied to observe the relationship between the disease activity and PAI-1 level.

Results

The applied screening approach detected significantly higher expression of PAI-1 in active IBD compared to the control. We demonstrated that the serum, mucosal, and faecal PAI-1 concentration was significantly elevated in IBD patients showing clinical and endoscopic activity. Additionally, the initial PAI-1 level decreased significantly upon successful therapy in responder patients, whereas it remained unchanged in non-responders. qRT-PCR analysis of the mucosa showed same results of PAI-1 expression pattern as the ELISA. We also showed that faecal PAI-1 selectively increases in active IBD patients, but not in other organic gastrointestinal diseases. In addition, the stability analysis of faecal PAI-1 showed that the protein was detectable for up to one week at room temperature and 4ºC. The serum, mucosal, and faecal PAI-1 levels showed positive correlation to the endoscopic activity. ROC curves demonstrated that faecal PAI-1 showed a relatively high power to distinguish between active and control patients. However, the serum CRP level reflected to the disease activity, but no to the therapeutic response. Correlation between the serum CRP and faecal PAI-1 level was not observed.

Conclusion

Our results suggest that serum, mucosal and faecal PAI-1 expression reflects to the disease activity in IBD, which could be used to disease monitoring and possibly therapeutic response. The correlation between the faecal PAI-1 level and the endoscopic activity promotes that it could be used as a novel non-invasive disease specific faecal biomarker in the IBD diagnosis.

Disclosure

LDN-051 is owned by Ladon Therapeutics Ltd.

Introduction

Inflammatory Bowel Diseases (IBD) require lifelong treatment and patient monitoring. Current predictors of relapse and therapeutic success have limitations, mostly invasive, time-consuming, and expensive. The faecal biomarker can help to monitor the disease activity and therapeutic response by simple and non-invasively. Faecal Calprotectin (FC) is the gold-standard, but it has many disadvantages. We previously described that the correlation between the faecal Plasminogen activator inhibitor type 1 (PAI-1) (FP) level and the endoscopic activity and therapeutic response promote that it could be used as a novel non-invasive faecal biomarker in IBD diagnosis. Moreover, the faecal PAI-1 protein level is stable at room temperature and 4°C.

Aims & Methods

To observe the exact faecal biomarker potential, we aimed to define the FP level of IBD patients and subjects with other gastrointestinal (GI) diseases and compare it with the FC. Faecal samples were collected from 128 patients with IBD (CD-active: 18, CD-inactive: 10, UC-active: 20, UC-inactive: 10) and other GI diseases [colorectal cancer (CRC): 20, diverticulosis [DIV]: 21, irritable bowel syndrome [IBS]: 10, adenoma: 10) and 9 healthy subjects. ELISA method was applied to define FP level, and it was validated for stool samples by us and 0.5 ng/g PAI-1 level was defined as the cut-off value. For FC observation diagnostic FC ELISA kit (Orgentec) was used. 50 mg/g cut-off value was applied at FC. Confusion matrix analysis was utilized to define Youden-index, positive predictive value (PPV), negative predictive value (NPV), accuracy, specificity, and sensitivity of the measured values.

Results

FP level was significantly higher in patients with active IBD compared to inactive and non-IBD subjects (0.64 vs 0.0 ng/g, p<0.0001). No significant differences were observed between CD-active and UC-active groups (0.46 vs 0.79 ng/g, p=0.65). In addition, FP concentrations were significantly increased in active IBD (0.76 pg/g) patients compared to inactive IBD (0.0 pg/g, p=0.0001), CRC (0.11 ng/g, p=0.02), DIV (0.05 ng/g, p=0.001), IBS (0.0 ng/g, p<0.0001), adenoma (0.0 ng/g, p<0.0001), and healthy (0.0 ng/g, p=0.0002) subjects. However, FC showed the same pattern as FP, except at the CRC, adenoma and DIV. In these groups, there were no significant differences compared to the active IBD (487.4 mg/g) subjects (vs CRC: 147.1 mg/g, p=0.09; p=; adenoma: 159.8, p=0.056. Confusion matrix analysis defined high specificity (83.5%) and moderate sensitivity (57.9%) values (TP: 22, FP: 14, FN: 17, TN: 71, PPV: 0.61, NPV: 0.816) at the FP measurements. Nevertheless, at FC levels 35.3% specificity and 94.7% sensitivity were measured (TP: 36, FP: 55, FN: 2, TN: 30, PPV: 0.396, NPV: 0.938). Calculated Youden-index was higher at the FP (0.41) compared to the FC (0.3). On the other hand, the accuracy of the FP is 75.6% and the FC is 53.6%. Based on the results of all FP levels measured (N=299) so far, the calculated values were specificity: 78%, sensitivity: 62%, PPV: 0.63, NPV: 0.78, Youden-index: 0.4, accuracy: 72.3%.

Conclusion

Our results suggest that the FP level selectively increased in the active IBD and discriminated more specifically from the other GI diseases compared to the FC. However, the sensitivity was lower, but it was improved by the increasing patient numbers at the pooled FP levels. Thus, the FP level could be useful in the diagnosis of IBD independently or combined with FC.

References

Jójárt B, Resál T, Kata D, Molnár T, Bacsur P, Szabó V, Varga Á, Szántó KJ, Pallagi P, Földesi I, Molnár T, Maléth J, Farkas K. Plasminogen Activator Inhibitor 1 Is a Novel Faecal Biomarker for Monitoring Disease Activity and Therapeutic Response in Inflammatory Bowel Diseases. J Crohns Colitis. 2024 Mar 1;18(3):392-405. doi: 10.1093/ecco-jcc/jjad160. PMID: 37751311; PMCID: PMC10906952.

Introduction

Crohn’s disease and ulcerative colitis, two forms of inflammatory bowel disease (IBD) require lifelong treatment and patient monitoring. Current predictors of relapse and therapeutic success have limitations, therefore there is a high unmet need to identify novel biomarkers in IBD. Cytokines may be utilized for this purpose in IBD therefore we aimed to assess and compare the inflammatory cytokine profile of the serum and mucosa of IBD patients and healthy controls in a Phase 2 clinical trial. Furthermore, the presence and predictive potential of the selected markers in fecal samples were assessed to identify Inflammatory bowel diseases (IBD) are defined by unregulated immune response leading to intestinal inflammation. Monitoring approaches for disease of the inflammation are mostly invasive, time-consuming and expensive. Therefore, a simple, rapid and non-invasive test, with ability to differentiate IBD from other gastrointestinal conditions and to monitor disease activity, will have important clinical implications. Our previous cytokine profiling identified that Plasminogen activator inhibitor type 1 (PAI-1) is expressed in IBD patients with active disease, but not in control.

Aims & Methods

Therefore, our aim was to assess the utility of PAI-1 in monitoring disease activity and therapeutic response of IBD patients in different biological samples. Serum, biopsy, and fecal samples were collected from 132 IBD patients and 30 controls without IBD. Inclusion criteria included change of therapy or initiation of new treatment for IBD patients. Organoid cultures (OC) were established from biopsies. Immunofluorescence (IF) staining was used to observe the presence and localization of cytokines. ELISA method was used to define the level of PAI-1 in the mucosa, serum and fecal samples. For the gene expression analysis qRT-PCR was used to observe the relative mRNA change of PAI-1 and other cytokines in the mucosa and OCs.

Results

The applied screening approach detected the presence of plasminogen activator inhibitor 1 (PAI-1) in active IBD. We demonstrated that the serum, mucosal and fecal PAI-1 concentration is elevated in IBD patients showing clinical and endoscopic activity. Additionally, in responder patients the initial PAI-1 level decreased significantly upon successful therapy, whereas it remained unchanged in non-responders. OCs derived from active IBD patients produced higher concentration of PAI-1 compared to the controls suggesting that epithelial cells could be a major source of PAI-1. We also showed that faecal PAI-1 selectively increases in active IBD patients, but not in other organic gastrointestinal diseases.

Conclusion

We successfully demonstrated in a Phase 2 clinical trial that serum, mucosal and fecal PAI-1 expression reflects to the disease activity in IBD, which could be used to disease monitoring and possibly therapeutic response. The correlation between the fecal PAI-1 level and the endoscopic activity promotes that it could be used as a novel non-invasive disease specific fecal biomarker in the IBD diagnosis.

Disclosure

LDN-051 is owned by Ladon Therapeutics Ltd.

Introduction

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders characterized by alterations of acute relapses and remission. The past decades have brought substantial advances in the pharmacological management of IBD by a wide range of medicines. However, the sustained efficacy of the currently available anti-inflammatory therapies is still far from optimal. Therefore, the development of novel therapies represents a major unmet clinical need in this field. Recently, the enrichment of the coagulation pathway genes and among these, Plasminogen activator inhibitor 1 (PAI-1) was described in IBD, whereas PAI-1 emerged as a link between the epithelium and inflammation. Moreover, the genetic deletion of PAI-1 prevented the severity of experimental colitis in mice.

Aims & Methods

Based on these, we aimed to develop and test novel PAI-1 inhibitors for the treatment of IBD.
The novel, amino acid-based PAI-1 inhibitor, LDN-071 was synthesized by the A³SMO^® platform. The inhibitory potential and cytotoxicity effects of the peptide were determined by in vitro assays. Acute and chronic colitis was induced in mice with DSS and 10 mg/bwkg LDN-071 was administered per os. The changes of body weight, spleen weight, and colon length were measured. Haematoxylin eosin (HE) and Periodic acid-Schiff (PAS) staining were performed for histology. ELISA and qRT-PCR measurements were applied to analyze the cytokine expressions. Hydroxyproline assay was used to quantify the mucosal collagen deposition. In vivo, the toxicity of LDN-071 was determined in mice. Human colon organoid and fibroblast (FB) cell cultures were generated from IBD patients and the effects of LDN-071 were observed.

Results

LDN-071 significantly inhibited PAI-1 in vitro without decreasing the cell viability in a concentration range of 1µM-1mM. In the acute mice model, the administration of 10 mg/bwkg LDN-071 in the DSS-treated mice prevented the body weight loss, the development of bloody diarrhea, the decrease of colon length, and the increase of the spleen weight, which were displayed by the DSS+vehicle-treated animals. The histological analysis of the colon revealed that DSS treatment induced severe transmural inflammation, ulceration, loss of crypt structures, and goblet cell depletion. Importantly, all these parameters were significantly ameliorated by the administration of 10 mg/bwkg LDN-071 in the acute and chronic models as well. Additionally, mucosal gene and protein expressions of TNF-α, IL-1β, and IL-6 were significantly decreased by PAI-1 inhibition in the acute phase. Besides, the LDN-071 treatment significantly reduced the inflammation, epithelial erosion, and fibrosis caused by chronic administration of DSS. However, the collagen deposition was significantly lower in the PAI-1-inhibited DSS mice. Importantly, no signs of toxicity were observed in mice exposed to 100 mg/bwkg LDN-071 for 7 days. In our in vitro models, the PAI-1 inhibition reduced the protein level and gene expression of pro-inflammatory cytokines in human colon organoid of IBD patient. Moreover, the gene expression of fibrosis-related genes significantly decreased after the LDN-071 treatment in IBD patients-derived human FB cell culture.

Conclusion

Our results showed that LDN-071, a novel amino acid-based inhibitor of PAI-1, significantly ameliorated the severity of acute and chronic colitis in mice. However, the LDN-071 reduced the expression of inflammation- and fibrosis-related genes and proteins in vitro human models. We propose that the inhibition of PAI-1 could be a potential novel therapeutic strategy in IBD.

Disclosure

LDN-071 PAI-1 inhibitor is owned by Ladon Therapeutics Ltd.

Introduction

Anti-TNF therapy has revolutionised the treatment of patients with Crohn’s disease (CD) and ulcerative colitis (UC). However, primary resistance or loss of efficacy are serious clinical challenges. Therapeutic drug monitoring (TDM) could help to manage these unfavourable therapeutic outcomes. The measurement of anti-TNF drug levels (DL) with point-of-care tests (POCT) is fast, simple tool to guide the therapy. TDM can be performed reactively in patients with active disease and proactively in asymptomatic patients. Nevertheless, the optimal utilization of TDM is still debated.

Aims & Methods

Our aim was to evaluate the impact of proactive and reactive TDM on the medium-term course of inflammatory bowel disease. We enrolled patients with UC and CD who were treated at our department with infliximab or adalimumab and underwent reactive or proactive TDM with POCT method between October 2021 and September 2023. We examined the types of therapeutical decision (dose intensification, switch within or out of class) were performed knowing DL and how the changes have affected the half-yearly outcome. Laboratory findings such as C-reactive protein (CRP), iron, albumin, haemoglobin, platelet, stool calprotectin level and calculated disease activity indices, Crohn’s disease activity index (CDAI), Harvey-Bradshaw Index (HBI), partial Mayo score (pMayo) and Simple clinical colitis activity index (SCCAI) were analysed at baseline and after 6 months. Statistical analysis was carried out with Graphpad Prism and SPSS.

Results

175 patients were enrolled, 80 men, 95 women, median age was 38 (IQR 30-50 years). 81 UC and 94 CD patients. In 79 cases proactive, in 96 cases reactive TDM was performed. In the reactive group the CRP (5.9 vs 12.2 mg/l, p=0.010), platelet count (278.3 vs 314.5 G/l, p=0.024) and the four activity indices (CDAI p=0.004, HBI p=0.0004, SCCAI p<0.0001, pMayo p<0.0001) were significantly higher, the albumin level (46.5 vs 45 g/l, p=0.019) was significantly lower compared to the proactive group at baseline. In the reactive group CRP (12.2 vs 5.7 mg/l, p=0.0083) and albumin level (46.5 vs 45 g/l p=0.0186) and all examined activity indices (CDAI p<0.0001, HBI p=0.0003, SCCAI p<0.0001, pMayo p=0.0002) changed significantly after 6 month from baseline. In the proactive group the lowering of SCCAI (p=0.0232), pMayo (p=0.0077) and CDAI (p=0.0471) were significant comparing baseline and 6-month parameters. Between the proactive and reactive groups no significant difference was found at month 6. In 41 (52%) cases in the proactive group subtherapeutical DL was found. Within these cases in CD patients CDAI (p=0.035, OR:1.02, 95%CI: 1.0-1.05) had a significant predictive value for the half-yearly outcome, in UC platelet count appeared to be nearly significant (p=0.097, OR: 1.07, 95%CI: 0.99-1.15) as a predictive factor. In 41 (52%) of the proactively measured cases no intervention was done, 27 (34%) dose-intesification, 5 (6%) de-escalation, 5 (6%) stop and 2 (3%) switch was carried out. In the reactively measured group 55 patients (57%) had subtherapeutical DL. In the reactive group no therapeutical change happened in 25 cases (26%), 42 (44%) underwent dose-intensification, 29 (30%) had switch (13 within class, 16 out of class).

Conclusion

We found no difference between the proactive and reactive drug level monitoring methods with POCT. Both affected the decision-making process and presented beneficial effect for medium-term and helped to achieve stride therapy goals.

Introduction

High-quality endoscopy in IBD is associated with better clinical outcomes, while quality measurement supports effective quality control. The European Society of Gastrointestinal Endoscopy recommends using nine performance measures, depending on the indication for endoscopic examination in IBD.

Aims & Methods

Our study aimed to compare colonoscopy quality between tertiary and secondary referral centers for IBD patients, based on endoscopic reports. In this cross-sectional study, colonoscopy reports of IBD patients were analyzed. At our tertiary center, we consecutively enrolled patients over 18 years old with established IBD between July and October 2024, using their available colonoscopy reports. The secondary care group included patients referred to our tertiary center, with the last existing report from secondary care used for analysis, while the tertiary care group consisted of our patients whose most recent report was similarly used. Nine key performance measures (indication, bowel preparation, photo documentation, ileal intubation, biopsy, endoscopic activity score, high-definition and chromoendoscopy use, and neoplasia detection) were evaluated based on colonoscopy reports. Patients were stratified by procedure indication (diagnostic, activity assessment, or surveillance) as well. Descriptive statistics, Welch’s, and Fisher’s exact tests were used to compare groups, with p-values <0.05 considered significant.

Results

Overall, 207 colonoscopy reports were assessed, of which 68/207 were performed at secondary care level. A total of 78/207 patients were diagnosed with Crohn’s disease, while unclassified diagnoses were noted only at secondary centers. Activity control (88% vs. 38%, p<0.001) and surveillance (8% vs. 0%, p=0.02) examinations were more common at the tertiary level. Bowel preparation was better at tertiary centers (Boston Bowel Preparation Scale [BBPS] 8.3±2.3 vs. 7.2±1.2; p=0.003), and use of BBPS was more frequent at higher levels of care (p<0.001). Disease activity scores were more commonly used at the tertiary level during activity control examinations (51.2% vs. 19.2%, p=0.004), while ileal intubation was also more frequent at the tertiary level (53% vs. 28%; p<0.001). Procedure and withdrawal times did not differ between levels in diagnostic or activity control examinations. Neoplasia detection was low, and biopsy rates were similar.

Conclusion

Quality indicator targets were met in neither secondary nor tertiary centers; however, wider investigation is needed to verify these results. Broad education can help to improve the quality of colonoscopy in IBD care.

Introduction

Inflammatory bowel diseases (IBD), comprising Crohn´s disease (CD) and ulcerative colitis (UC), are characterised by chronic, relapsing and remitting inflammation of the gastrointestinal tract. Despite significant efforts to understand the pathogenetic mechanisms of IBD, the elucidation of its etiopathology and progression remains incomplete. This study aimed to clarify IBD pathogenesis using a multiomic approach in serum, urine, intestinal tissue and stool samples.

Aims & Methods

We performed a multiomic analysis (transcriptomic, proteomic, metabolomic and metagenomic) in a discovery cohort of 53 patients with active CD, 50 patients with active UC, and 33 healthy controls (HC). Transcriptomic and proteomic analysis of intestinal tissue was performed by RNA-seq and liquid chromatography-mass spectrometry, respectively. Serum and urine samples were used for metabolome study using nuclear magnetic resonance, and metagenomic analysis of stool samples was performed by 16rRNA gene sequencing.

Results

Gene expression profiles showed a detailed transcriptome landscape of intestinal tissue in patients with CD and UC. A total of 4,105 proteins were identified in mucosal biopsies, of which 2,128 were differentially expressed (DE) between CD and HC, 2,715 proteins DE in UC compared with HC, and 1,653 proteins DE between CD and UC. Metabolomic analyses revealed metabolites and pathways related to ketone, amino sugar and methyl histidine metabolism, Warburg effect, and ammonia recycling deregulated in IBD patients. Moreover, metagenomic results indicated that CD and UC differ in their microbial populations according to beta diversity. In comparison to HC, the microbial population in CD was different, and its alpha diversity was significantly lower.

Conclusion

The results showed that some of the identified analytes could play essential roles in the pathogenesis of CD and UC. New potential key mechanisms that could help to understand the information of omics layers that underlies the pathogenesis of IBD have been outlined.

Disclosure

None