Crohn Disease (CD), IBD (Inflammatory Bowel Disease), Ulcerative Colitis (UC)
Conditions
Keywords
IBD, Ulcerative Colitis, Crohn's Disease, Combination Therapy, Ex Vivo Human Organ Culture
Brief summary
The purpose of this study is to evaluate whether simultaneously targeting three inflammatory pathways (TL1A, IL-23, and α4β7) produces a stronger anti-inflammatory effect in intestinal tissue from patients with Inflammatory Bowel Disease (IBD) compared with targeting individual pathways. The study aims to investigate whether this combined approach may help overcome limitations associated with single-pathway inhibition. This is a single-center, interventional ex vivo study conducted at IRCCS Ospedale San Raffaele. The study includes 30 participants divided into three cohorts: 10 patients with Ulcerative Colitis, 10 patients with Crohn's Disease, and 10 non-IBD control participants. Participant involvement is limited to a single day and is integrated into a clinically indicated, routinely scheduled colonoscopy and routine phlebotomy. During the scheduled colonoscopy, 8 additional mucosal biopsies are collected for research purposes, together with an additional 2 mL blood sample. No additional endoscopic procedure or separate study visit is required. No investigational drug or treatment is administered directly to participants. The collected intestinal biopsies are instead treated ex vivo in the laboratory with ATTO-1091, individual pathway inhibitors, or control conditions for 16 hours. Biological and molecular responses are evaluated using laboratory techniques including ELISA and RNA sequencing to assess inflammatory markers and gene expression. Laboratory personnel performing molecular and transcriptomic analyses are blinded to participants' clinical profiles until final data analysis.
Detailed description
The study is built on the hypothesis that the simultaneous blockade of TL1A, IL-23, and α4β7 pathways will exert a synergistic anti-inflammatory effect on the intestinal mucosa of IBD patients compared to single-agent inhibition, thereby potentially overcoming resistance to standard biological therapies. To test this, a monocentric, national, interventional ex vivo clinical study will be conducted at the IRCCS Ospedale San Raffaele using organotypic tissue cultures. The parallel-group design is based on three distinct clinical cohorts consisting of participants with Ulcerative Colitis, participants with Crohn's Disease, and non-IBD control subjects. To minimize bias and ensure scientific integrity, laboratory operators performing the molecular and transcriptomic analyses will be blinded to the clinical and response data of the source tissue until final statistical analysis. Participants will undergo a clinically indicated, routinely scheduled standard-of-care colonoscopy. During the procedure, 8 additional mucosal biopsies will be collected for research purposes according to a standardized sampling protocol, together with an additional 2 mL blood sample collected during routine phlebotomy. No investigational product or treatment will be administered directly to study participants. Instead, the active experimental phase will be conducted entirely ex vivo in the laboratory, where the collected intestinal tissue samples will be exposed to ATTO-1091, individual pathway inhibitors, or control conditions for a standardized culture period of 16 hours. Participant involvement will be completed within a single day and fully integrated into routinely scheduled clinical care, with no additional study-specific clinical examinations or post-treatment follow-up visits required. There are no sub-studies included in this protocol. Biological data will be generated via high-throughput laboratory assays, specifically organotypic tissue cultures, enzyme-linked immunosorbent assays (ELISA) for cytokine evaluation, and RNA sequencing (RNA-seq) to map gene expression. These molecular methods are selected because they provide comprehensive measurements of cellular inflammation and pathway inhibition and allow comparison of the biological effects of combined pathway inhibition with single-pathway inhibition and control conditions. Finally, any new future research or secondary use of the collected data and biological samples not explicitly described in this protocol will be submitted to the Ethics Committee for formal approval before initiation.
Interventions
PROCEDURECollection of 8 additional intestinal mucosal biopsies during standard-of-care endoscopy for subsequent ex vivo testing
Eight additional mucosal biopsies will be collected during a clinically indicated, scheduled standard-of-care colonoscopy. Biopsies will be obtained according to a standardized sampling protocol, with the anatomical site of each biopsy documented. The additional biopsies are collected for research purposes and subsequently processed for ex vivo laboratory analyses. No investigational product is administered to study participants.
PROCEDURECollection of an additional 2 mL blood sample
An additional 2 mL blood sample will be collected during routine phlebotomy performed in conjunction with the scheduled study visit. The additional blood collection is performed for research purposes and does not require a separate study visit.
Sponsors
PhoenixLAB srls
IRCCS San Raffaele
Study design
Allocation
NON_RANDOMIZED
Intervention model
PARALLEL
Primary purpose
BASIC_SCIENCE
Masking
NONE
Masking description
This is an open-label study. Experimental compounds and vehicle controls are applied strictly ex vivo to mucosal biopsy tissue, with no in vivo intervention or masking required for participants or care providers. Laboratory personnel performing molecular and transcriptomic analyses are blinded to participants' clinical profiles until final data analysis.
Intervention model description
This is an interventional ex vivo study using organotypic mucosal tissue cultures. Participants are enrolled into three parallel diagnostic cohorts based on their pre-existing clinical diagnosis: Ulcerative Colitis (UC, n=10), Crohn's Disease (CD, n=10), and non-IBD controls (n=10). During a scheduled standard-of-care colonoscopy, 8 additional mucosal biopsies are collected from each participant, together with an additional 2 mL blood sample during routine phlebotomy. Collected mucosal tissue is cultured ex vivo for 16 hours and exposed under standardized laboratory conditions to the experimental compound ATTO-1091, which simultaneously inhibits the TL1A, IL-23, and α4β7 pathways, individual pathway inhibitors (tulisokibart, risankizumab, and vedolizumab), or control conditions. No investigational product is administered directly to participants; all pharmacological exposures occur exclusively ex vivo on the collected tissue.
Eligibility
Sex/Gender
ALL
Age
18 Years to No maximum
Healthy volunteers
Yes
Inclusion criteria
* Patients of at least 18 years of age * Able to comply with the study procedures and to sign an informed consent form * Established diagnosis of UC or CD, with indication to start any biological or small molecule agents as per standard of care: UC patients with a clinical indication to start biologics or small molecules as per standard of care, with a Total Mayo Score of 6-12 (moderate-severe disease) and an endoscopic subscore ≥ 2; CD patients with a clinical indication to start biologics or small molecules as per standard of care, with a clinical CDAI score of 220-600 (moderate-severe disease) and endoscopic evidence of active mucosal inflammation/ulceration upon baseline examination; \- Patients with IBS or individuals undergoing colonoscopy for CRC prevention or routine surveillance, with no prior diagnosis of inflammatory bowel disease and no endoscopic evidence of intestinal mucosal inflammation.
Exclusion criteria
* Absolute contraindications to colonoscopy procedures * UC or CD patients in endoscopic remission * IBS or patients undergoing CRC prevention surveillance with inflamed mucosa during the endoscopy * Patients of at least 18 years of age unable to comply with the study procedures and to sign an informed consent form * Pregnancy or breastfeeding
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Concentration of pro-inflammatory cytokines in intestinal biopsy culture supernatants following ex vivo treatment with ATTO-1091 | 16 hours after ex vivo exposure | Pro-inflammatory cytokine concentrations will be quantified by enzyme-linked immunosorbent assay (ELISA) in culture supernatants collected after 16 hours of ex vivo incubation of intestinal biopsies with ATTO-1091, individual pathway inhibitors, or the corresponding control condition. Cytokine concentrations following ATTO-1091 exposure will be compared with those measured in control cultures and in cultures exposed to individual pathway inhibitors to quantify the anti-inflammatory effect of simultaneous TL1A, IL-23, and α4β7 inhibition. Each selected cytokine will be analyzed separately and reported as its concentration in the culture supernatant using the concentration unit specified for the corresponding ELISA assay. Comparisons between experimental conditions will be performed using Student's t-test or analysis of variance (ANOVA), as appropriate, with correction for multiple comparisons. |
| Change in gene expression following ex vivo treatment with ATTO-1091, expressed as log2 fold change | After 16 hours of ex vivo incubation | Gene expression profiles will be assessed by RNA sequencing (RNA-seq) in intestinal biopsies following 16 hours of ex vivo incubation with ATTO-1091 or the corresponding comparator conditions. Sequencing will be performed using paired-end reads (2 × 150 bp), targeting approximately 30 million read pairs per sample. After quality control, trimming, alignment to the human reference genome, and generation of gene-level counts, differential gene expression will be analyzed using DESeq2. Gene expression profiles following ATTO-1091 exposure will be compared with the corresponding control and single-pathway inhibitor conditions to identify genes significantly modulated by simultaneous TL1A, IL-23, and α4β7 inhibition. Changes in gene expression between experimental conditions will be expressed as log2 fold change (log2FC), with statistical significance assessed using adjusted P values. |
| Enrichment of disease-associated inflammatory biological pathways following ex vivo treatment with ATTO-1091 | After 16 hours of ex vivo incubation | Functional and pathway enrichment analyses will be performed using RNA-seq-derived differential gene expression data from intestinal mucosal tissue following 16 hours of ex vivo exposure to ATTO-1091, individual pathway inhibitors, or control conditions. Pathway enrichment will be assessed from differential gene expression data to identify disease-associated inflammatory biological processes and pathways modulated by simultaneous TL1A, IL-23, and α4β7 inhibition. Pathway enrichment following ATTO-1091 exposure will be compared with that observed under control conditions and following individual pathway inhibition. Biological processes and pathways with an adjusted P value ≤0.05 will be considered significantly enriched. The enrichment measure is an analytical output of pathway enrichment analysis and is not a clinical scale with predefined minimum or maximum values. |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Correlation between ex vivo gene expression response to ATTO-1091 and disease duration in participants with IBD | Through study completion, up to 12 months | Gene expression response to ATTO-1091 will be measured by RNA sequencing (RNA-seq) in intestinal mucosal biopsies following 16 hours of ex vivo exposure and analyzed using DESeq2. For each analyzed gene, gene expression response will be expressed as log2 fold change (log2FC) following ATTO-1091 exposure compared with the corresponding control condition and with each individual pathway inhibitor (tulisokibart, risankizumab, and vedolizumab). Disease duration will be obtained from the participant's clinical records and measured in years from initial IBD diagnosis to study enrollment. Correlation analysis will assess the relationship between gene expression response (measurement tool: RNA-seq with DESeq2; unit of measure: log2FC) and disease duration (measurement source: clinical records; unit of measure: years). |
| Correlation between ex vivo gene expression response to ATTO-1091 and Total Mayo Score in participants with Ulcerative Colitis | Through study completion, up to 12 months | Gene expression response to ATTO-1091 will be measured by RNA sequencing (RNA-seq) in intestinal mucosal biopsies following 16 hours of ex vivo exposure and analyzed using DESeq2. For each analyzed gene, gene expression response will be expressed as log2 fold change (log2FC) following ATTO-1091 exposure compared with the corresponding control condition and with each individual pathway inhibitor (tulisokibart, risankizumab, and vedolizumab). Ulcerative Colitis disease activity will be assessed using the Total Mayo Score, expressed in points. The Total Mayo Score ranges from 0 to 12 points, with higher scores indicating greater disease activity. Participants enrolled in this study are required to have a Total Mayo Score of 6 to 12 points. Correlation analysis will assess the relationship between gene expression response (measurement tool: RNA-seq with DESeq2; unit of measure: log2FC) and Ulcerative Colitis disease activity (measurement tool: Total Mayo Score; unit of measure: points). |
| Correlation between ex vivo gene expression response to ATTO-1091 and Crohn's Disease Activity Index in participants with Crohn's Disease | Through study completion, up to 12 months | Gene expression response to ATTO-1091 will be measured by RNA sequencing (RNA-seq) in intestinal mucosal biopsies following 16 hours of ex vivo exposure and analyzed using DESeq2. For each analyzed gene, gene expression response will be expressed as log2 fold change (log2FC) following ATTO-1091 exposure compared with the corresponding control condition and with each individual pathway inhibitor (tulisokibart, risankizumab, and vedolizumab). CD activity will be assessed using the Crohn's Disease Activity Index (CDAI) and expressed in points. The CDAI ranges from 0 to approximately 600 points, with higher scores indicating greater disease activity. Participants enrolled in this study are required to have a CDAI score between 220 and 600 points. Correlation analysis will assess the relationship between gene expression response (measurement tool: RNA-seq with DESeq2; unit of measure: log2FC) and Crohn's Disease activity (measurement tool: CDAI; unit of measure: points). |
| Correlation between ex vivo gene expression response to ATTO-1091 and participant age | Through study completion, up to 12 months | Gene expression response to ATTO-1091 will be measured by RNA sequencing (RNA-seq) in intestinal mucosal biopsies following 16 hours of ex vivo exposure and analyzed using DESeq2. For each analyzed gene, gene expression response will be expressed as log2 fold change (log2FC) following ATTO-1091 exposure compared with the corresponding control condition and with each individual pathway inhibitor (tulisokibart, risankizumab, and vedolizumab). Participant age at study enrollment will be obtained from the participant's clinical records and measured in years. Correlation analysis will assess the relationship between gene expression response (measurement tool: RNA-seq with DESeq2; unit of measure: log2FC) and participant age (measurement source: clinical records; unit of measure: years). |
Countries
Italy
Contacts
CONTACTSilvia D'Alessio, PhD
PRINCIPAL_INVESTIGATORSilvio Danese, MD, PhD
IRCCS San Raffaele
Outcome results
None listed