Introduction

Gut microbiota in Crohn's disease (CD) exhibits considerable variability, with conflicting associations with disease severity and response to therapeutic intervention. We aimed to longitudinally investigate the dynamics of microbial composition in a cohort of patients with newly diagnosed, treatment-naïve Crohn's disease (ndCD) over a one-year period.

Aims & Methods

A prospective real-world study was conducted, enrolling patients with ndCD who were treatment-naïve. Treatment decisions were made by physicians with expertise in CD management. Key one-year clinical outcomes included corticosteroid-free clinical remission (CSFR) and biochemical remission (BR), defined as C-reactive protein levels <1mg/dL and fecal calprotectin levels <150ug/g. Paired stool samples were subjected to 16S rRNA gene sequencing (V4 region) at diagnosis and after one year, followed by analysis for microbial diversity and dysbiosis indices. A linear mixed-effect model was constructed to identify factors associated with microbial alterations and specific taxa driving these alterations, while controlling for confounding variables.

Results

Seventy-five patients completed the one-year follow-up (49.3% female, median age 27 years [IQR 22-38.5]); 45 (60%) received biologics (95.5% anti-tumor necrosis factor [TNF]). Rates of CSFR, BR, and combined CSFR-BR at one-year post-diagnosis were 64%, 56%, and 45.3%, respectively. Analysis of microbial composition revealed significant improvements in microbial indices among patients achieving all desired clinical outcomes, characterized by increased microbial diversity (Shannon index, p=0.004, p=0.002, p=0.004, respectively) and decreased dysbiosis (microbial dysbiosis index [MDI], p=0.003, p=0.04, p=0.01, respectively). Such improvements were not observed in patients failing to achieve CSFR, BR or combined CSFR-BR. Notably, patients treated with biologics, irrespective of outcome achievement, exhibited prominent shifts (improvement) in microbial composition (both indices p<0.001), whereas non-treated patients did not demonstrate significant changes (Shannon index, p=0.53; MDI, p=0.81). Furthermore, microbial composition at baseline was more disrupted among patients treated with biologics compared to non-treated patients (Shannon index, p=0.04; MDI, p=0.03). The linear mixed model analysis confirmed a positive association between biologics and favorable changes in microbial indices (Shannon index, p=0.02; MDI, p=0.04), identifying an increase in 20 gut commensal taxa (considered beneficial) and a decrease in Escherichia coli among the biologics-treated subgroup. Conversely, no taxa exhibited significant alterations in the non-biologics treated subgroup.

Conclusion

In a real-world cohort of patients with treatment-naïve ndCD, clinical improvement over one year was associated with improvement in microbial composition. Treatment with anti-TNF agents was associated with improvement in microbial composition irrespective of clinical outcomes. These findings suggest that microbial composition reflects inflammatory activity and may be positively and independently modified by anti-TNF treatment.

Introduction

The human gut microbiome consists of a diverse range of microorganisms, including not only bacteria but also viruses, archaea and fungi. Among these, bacteria have been shown to have a significant impact on the health or disease status of the host. However, despite increasing evidence for the role of fungi in liver disease, inflammatory bowel disease and cancer, little is known about the interactions between bacteria and fungi.

Aims & Methods

In this study, we aimed to investigate in depth the interkingdom relationship between fungal and bacterial communities in the gut of healthy monozygotic and dizygotic twins using a novel merging analysis method. Faecal samples from 106 twin pairs were analysed using 16S rRNA gene sequencing for bacteria and ITS sequencing for fungi. To enable a comprehensive analysis of the interactions between these two microbial communities, we adapted a novel merging normalisation method and linked it to systematic dietary and environmental characteristics.

Results

The implementation of the merging normalisation method allowed for an in-depth comparison of the fungal and bacterial communities independent of the low read counts. In direct comparison, bacteria were significantly more abundant and diverse than fungi, with Bacteroides and Prevotella being the most dominant bacterial genera. Within the fungal community, Candida was the most abundant genus in the cohort, followed by Geotrichum, while Saccharomyces was less abundant. Other fungal genera were detected sporadically between individuals. Most bacterial taxa showed a negative correlation with both Candida and Geotrichum at some level of abundance. For example, Bacteroides showed no antagonism towards Candida when its relative abundance was below 5%, whereas genera such as Prevotella (rho=-0.16, p=0.02), Alistipes (rho=-0.16, p=0.02) and Faecalibacterium (rho=-0.17, p=0.01) showed antagonistic interactions with Candida at relative abundances as low as 0.1%. These correlations were calculated across different abundances of the interacting fungi and bacteria, showing that negative associations increased with higher abundance (up to rho=-0.6 for Alistipes). An interkingdom network analysis further revealed a complex dynamic involving the three most abundant genera - Bacteroides, Prevotella and Candida - which showed mutually antagonistic relationships.

Conclusion

The implementation of the novel, easy-to-use merging analysis provided an in-depth view of the interactions between bacterial and fungal communities in the human gut, which are predominantly antagonistic in nature. The bacterial community appears to use several mechanisms to protect the gut environment from fungal colonisation.

Disclosure

Nothing to disclose

Introduction

Current treatment options for IBD are limited by low clinical remission rates and loss of response. Most therapies target single pathogenic pathways and while combinations of biologics targeting different pathogenic cytokines can increase clinical remission rates, dosing schedules are inconvenient. JAK inhibitors, target multiple cytokine pathways, but are limited by safety concerns.
Salt-inducible kinases (SIKs) are a subfamily of serine/threonine kinases in the AMP-activated protein kinase (AMPK) family and act as key regulators of immune cell signaling. SIK inhibitors have been shown to target multiple cytokine pathways.
Three SIK isoforms exist: SIK1, SIK2 and SIK3. SIK inhibitors targeting multiple isoforms have been investigated in clinical trials. Although there have been signals of activity in patients with ulcerative colitis and psoriasis, limited efficacy and safety concerns related to SIK1 and 2 inhibition have hampered their development.

Aims & Methods

We performed structure based drug design to identify highly potent and selective SIK3 inhibitors that could offerbest in class efficacy and safety for the treatment of various auto-immune diseases. Our effort led to to the discovery of 100s of compounds spanning SIK3 binding sites. Compounds were tested in protein and cell-based assays to evaluate potency and selectivity against SIK3 and in a phenotypic human whole blood TNFα assay. Lead optimization involving rigorous testing in safety and pharmacology panels, resulted in the selection of lead candidates that were tested in a variety of translational models to identify the most efficacious compounds. Human PK predictions were performed using non-clinical in vivo PK assessments and allometric scaling leading to the selection of the drug candidate, which entered GLP toxicology studies.

Results

O3R-5671 is a highly potent and specific SIK3 inhibitor with sub-nanomolar potency for SIK3 and low activity against SIK1 and 2 and over all human kinases. It demonstrated potent activity in a panel of human primary phenotypic cell assays inhibiting TNFα, IL-12, and IL-23 release in LPS-stimulated myeloid cells, and inhibiting TNFa release in LPS-stimulated human whole blood. O3R-5671 also inhibited activation of TCR-stimulated human primary T cells and induced IL-10 secretion from myeloid cells.
In vivo, O3R-5671 demonstrated dose-dependent activity in a mouse LPS challenge model, dose-dependent efficacy in two distinct mouse models of IBD, and dose-dependent efficacy in mouse models of IL-23-induced psoriasis and psoriatic arthritis. Cross-species PK and allometric scaling were used to predict O3R-5671’s human PK profile- which is expected to be conducive to potent SIK3 inhibition and clinical efficacy in patients with IBD.
O3R-5671 demonstrated a highly attractive profile in ADME and in vitro safety panels. Six-week GLP toxicity studies in rats and dogs established no observed adverse effect levels (NOAELs) which support dosing in humans with doses that are predicted to be pharmacologically active with a large safety margin.

Conclusion

O3R-5671 is a highly specific and potent SIK3 inhibitor that is predicted to have a flat PK profile in humans. These properties should allow O3R-5671 to apply strong drug pressure on SIK3 while sparing SIK1 and 2. This is expected to result in the inhibition of multiple pathogenic cytokines including TNFa and IL-23, both of which have been validated as IBD targets. A clinical trial is ongoing to establish the safety and PK profile of O3R-5671 and understand its effect on pharmacodynamic biomarkers.

Disclosure

All authors were employed by Galapagos who discovered O3R-5671 or Novalix, a CRO that was employed by Galapagos. Some authors are employed by Onco3R therapeutics, who are currently developing O3R-5671