Introduction

Serum 7AlphaC4 (7-alpha-hydroxy-4-cholesten-3-one) is a diagnostic test for bile acid (BA) malabsorption (BAM) or bile acid diarrhea (BAD) and the only available blood-based test for BAM/BAD. Concentrations >48 ng/mL have a high positive predictive value (PPV) of 82% for BAD and high likelihood of responding to BA sequestrants.¹ In BAM, less BAs re-enter enterohepatic circulation and more BAs spill into the colon increasing motility and secretion. Less reabsorbed BA means less negative feedback and an ensuing increase in hepatic BA synthesis. As BA synthesis increases, blood levels of 7AlphaC4, a BA precursor, also increase. This 7AlphaC4 test performed on a random blood collection as a surrogate measure of stool BA aims to simplify the investigation and thereby, improve recognition of BAM/BAD which may often be overlooked in IBD and other patients with more than one possible etiology of diarrhea.

Aims & Methods

We assessed the distribution of numeric serum 7AlphaC4 results (in ng/mL) in 712 patient samples using an in-house developed method utilizing protein precipitation followed by liquid chromatography with tandem mass spectrometry. Reference range of this validated assay is 1.8 – 57 as determined using sera of 120 non-disease individuals.

Results

Analysis of 712 patient samples yielded a mean (± standard deviation) 7AlphaC4 of 36.2 ± 50.1 ng/mL (range, undetectable (<0.5) to 549); 37.6% were low at <15 (associated with a NPV of 85%); 21.3% were high at >48. Most 7AlphaC4 was ordered in the general or urgent medical setting (42.8%), by gastroenterologists ( 27.8%) or infectious disease specialists (9.3%). Other testing in order of frequency included: tTG IgA or other celiac tests, fecal calprotectin, fecal pancreatic elastase, stool PCR for 22 GI pathogens, fecal fat, C. difficile toxin, Giardia, H.pylori, C. and stool lactoferrin. Most common International Classification of Diseases (ICD) codes were: diarrhea unspecified, noninfectious gastroenteritis and colitis, abdominal distension, unspecified abdominal pain, irritable bowel syndrome with diarrhea (IBS-D), change in bowel habit, other fatigue, gastroesophageal reflux, functional diarrhea.
ICD codes are not known for all samples. However, of 50 patients with the highest 7AlphaC4 levels (>100), at least 20% had CD (ileal resection status unknown). In 86 patients with known diagnoses, those with CD with small intestine involvement had the highest mean 7AlphaC4 of 83.9 ± 77.1 (n=27) compared to 46.1 ± 83.7 in functional diarrhea (n=20), 37.3 ± 58.9 in unspecified diarrhea (n=191), 31.4 ± 22.3 in IBS-D (n=31), and 22.1 ± 10.8 in UC (n=8).

Table 1. Distribution of All Serum 7AlphaC4 Results

Conclusion

More than half (58.9%) of all 7AlphaC4 results were low (< 15) or high (>48) and as such, potentially diagnostic, carrying a high NPV or high PPV for bile acids contributing to diarrhea. BAM/BAD may account for 30% of all chronic diarrhea cases, thereby, affecting 1-2% of the general population. Disruptions in BA homeostasis and reabsorption occur more frequently in IBD (up to 40%, with or without ileal resection). We observed that many of the highest 7AlphaC4 levels occurred in Crohn’s patients. In summary, 7AlphaC4 is a new blood-based marker that provides quantitative evidence of the contribution of colonic BA toward diarrhea (and hence, a higher likelihood of responding to BA sequestrants), especially in patients with persistent diarrhea of more than one possible etiology.

References

1. Walters JRF. Making the Diagnosis of Bile Acid Diarrhea. Am J Gastroenterol. 2020 Dec;115(12):1974-1975.

Introduction

In perianal fistulizing Crohn’s disease (PFCD), higher infliximab (IFX) serum trough concentrations are associated with improved outcomes. Most PFCD patients require dose intensification to achieve therapeutic target concentrations. IFX pharmacokinetics (PK) in PFCD have not been previously characterized. We hypothesized that IFX clearance (CL) is increased in PFCD compared to Crohn’s disease with luminal disease only (CD).

Aims & Methods

Patients with CD and PFCD starting standard-dose intravenous IFX (5 mg/kg at week 0, 2, 6, thereafter every 8 weeks) were enrolled. Pre-infusion samples were collected from week 2 to 26 to measure IFX trough levels (TL), and anti-drug antibodies if IFX TL was below 1.0 μg/mL. Target TL were 20 μg/mL (week 2), 15 μg/mL (week 6), and 10 μg/mL (week 14) for PFCD, and 5.0 μg/mL (week 14) for CD. A population PK model was constructed using nonlinear mixed-effects modeling and adjusted for clinically relevant covariates.

Results

A total of 148 patients were included (63 PFCD, 85 CD), contributing 761 serum samples. Baseline demographics were comparable, except for higher luminal disease activity and concomitant immunomodulator use in CD. During induction, target IFX TL were achieved in significantly fewer PFCD patients (43% vs. 57%, P = .038). At week 14, median IFX TLs were lower in PFCD (2.8 μg/mL [IQR 1.0-5.4] than in CD (4.8 μg/mL [IQR 2.0–6.6], P = .014), with target TL attainment in only 9% of PFCD versus 46% of CD patients (P < .001). In multivariate regression analysis, PFCD was independently associated with a sevenfold increased risk of underdosing at week 14 (adjusted risk ratio [aRR] = 7.14, P < .001). Anti-drug antibodies (ADA) against infliximab were detected in 10/19 (53%) of PFCD patients and 9/25 (36%) CD patients with IFX TL below 1 μg/mL (P = .36) after a median of 20 weeks, with 35% of cases being transient during follow-up. While more PFCD patients had very low infliximab levels at week 14 (<1 μg/mL; 22% vs. 10%, P = .07), ADA presence and median ADA levels at that time did not differ significantly between these groups (P = .99 and P = .25, respectively).
IFX PK was described by a two-compartment model with linear elimination. Population PK modeling of the first 14 weeks revealed an 18% increase in IFX CL among patients with active perianal fistulas adjusted for dose, weight, serum albumin and presence of anti-drug antibodies (P < 0.01).

Conclusion

Active perianal fistulas are independently associated with increased infliximab clearance in patients with Crohn’s disease, resulting in significantly lower and frequently subtherapeutic serum trough levels despite standard induction dosing. At week 14, patients with PFCD had a sevenfold higher risk of underexposure compared to those with luminal CD only. These findings highlight a critical need for intensified or personalized infliximab dosing strategies during induction in PFCD to optimize therapeutic exposure and improve clinical outcomes.

Introduction

Ustekinumab is a recombinant human monoclonal IgG1 antibody targeting the interleukins IL-12 and IL-23. Ustekinumab is approved for treatment of moderately to severely active inflammatory bowel disorders, i.e. Crohn’s disease and ulcerative colitis, besides its approved use in moderate-to-severe plaque psoriasis and active psoriatic arthritis. The presented study provides the basis for the marketing authorisation of the proposed ustekinumab biosimilar FYB202, together with an extensive analytical characterisation and the results of an efficacy and safety trial in patients with moderate-to-severe plaque psoriasis.

Aims & Methods

The primary objective of the three-arm trial RUSTIC was A) to prove pharmacokinetic (PK) similarity in terms of AUC_0-inf and C_max of the proposed ustekinumab biosimilar FYB202 to both EU-approved (EU-Ref) and US-licensed ustekinumab (US-Ref) and B) to prove PK similarity of both reference drugs. PK similarity was concluded if the 90% confidence interval (CI) of the geometric mean ratios was contained within the acceptance interval of 80.00–125.00% (analysis of covariance [ANCOVA] with body weight as covariate). Treatments included a single 45-mg subcutaneous injection. The multi-centric clinical trial was conducted at four German trial sites. Safety analyses comprised immunogenicity (anti-drug antibodies [ADAs] and neutralising antibodies [NAbs]), adverse events (AEs), vital signs, body temperature, 12-lead electrocardiograms, and local tolerability (subject- and investigator-assessed). Healthy male and female subjects (18–55 years, body weight 60–90 kg, body-mass index 20.5–30.0 kg/m²) were included in the trial and observed for up to 112 days after dosing.

Results

Overall, 491 subjects were randomised (FYB202: n=164, EU-Ref: n=163, US-Ref: n=164); 486 subjects completed the trial, and 478 subjects were included in the PK analysis set. PK similarity was demonstrated for all three comparisons: all 90% CIs of the respective geometric mean ratios for AUC_0-inf and C_max were contained within the acceptance interval of 80.00–125.00% (Table 1). After administration of FYB202, fewer subjects had at least one positive ADA result at trial end compared to the reference groups (FYB202: 20%, EU-Ref: 42%, US-Ref: 51%). ADA titre levels were similar between all three groups. The percentage of subjects with at least one positive NAb-reactive result at trial end was slightly lower in the FYB202 group compared to the reference groups (FYB202: 11%, EU-Ref: 15%, US-Ref: 18%). More than half of the subjects had at least one treatment-related AE (FYB202: 53%, EU-Ref: 55%, US-Ref: 57%); no treatment-related serious AEs occurred. Local tolerability at the injection site was comparable in all treatment groups.

Table 1: Point estimates and 90% confidence intervals [%] for the primary pharmacokinetic parameters of ustekinumab (PK analysis set; N=478)

Conclusion

The RUSTIC trial demonstrated bioequivalent PK characteristics between FYB202 and both EU-approved and US-licensed ustekinumab and for the two reference drugs. No clinically meaningful differences with respect to the overall safety, immunogenicity and local tolerability were observed.

Disclosure

The trial is registered in the EU Clinical Trials Register (EudraCT no. 2022-000832-43).

The clinical trial was financed by Formycon AG, Martinsried, Germany and conducted by SocraTec R&D GmbH, Oberursel/Erfurt, Germany (data management: SocraMetrics GmbH, Erfurt, Germany). During conduct of the trial and preparation of the abstract, S.B. and K.N. were employees of Formycon AG. J.K. and M.A. were employees of SocraTec R&D GmbH. R-S.W. was an employee of SocraMetrics GmbH. B.S. was cofounder and managing director of SocraTec R&D GmbH and SocraMetrics GmbH.

Introduction

Aloe barbadensis Mill. (Aloe) with its complex polysaccharide composition has been suggested to have prebiotic properties, however no human intervention trials have previously been undertaken to study this effect.

Aims & Methods

With the aim of designing prebiotic strategies for human gut health promotion, this randomized, placebo-controlled study evaluated the effects of increasing doses of chemically characterized Aloe extract on gastrointestinal function and overall gastrointestinal microenvironment, including fecal microbiota and metabolite composition in healthy adults.
Fecal samples were collected from healthy subjects (56 females and 21 males, age 19 - 69) at baseline, and week 3 and week 6 after intervention with placebo or 0.5 g, 1.0 g or 3.0g of Aloe extract/day. Gastrointestinal symptoms were assessed using validated questionnaires and stool consistency was evaluated using the Bristol Stool Form Scale. A food questionnaire was used to evaluate dietary quality, adapted using an index created by the Swedish National Food Agency. Fecal microbiota was evaluated by GA-map^TM Dysbiosis Test, while H-1 nuclear magnetic resonance (NMR) spectroscopy was used for metabolomic profiling. Multivariate Orthogonal Projections to Latent Structures Discriminatory Analysis (OPLS-DA) was applied to fecal microbiota and metabolite data sets. Statistical analyses were carried out using non-parametric tests.

Results

All participants completed the study and demographic parameters such as age, sex, diet quality and BMI did not differ between the study groups at baseline. Intervention with increasing doses of Aloe was safe and well tolerated and did not cause gastrointestinal symptoms, as assessed using the gastrointestinal symptoms rating scale. Stool consistency changed after supplementation with Aloe, (n= 57; 3.9 (3.2 – 4.4) vs 3.6 (2.8 – 4.2), P = 0.005), but not for placebo (n= 20; 3.9 (3.2 – 4.5) vs. 3.7 (3.2 – 4.3), P = 0.82), comparing baseline vs. end of intervention, without difference between the groups (P = 0.33).
No alterations were seen in the fecal microbiota profiles before vs. after intervention, in either of the groups, indicative of a stable overall gut microbiota. Furthermore, the fecal metabolite profiles remained unaltered after intervention with Aloe. However, Aloe modulated the fecal metabolite profile in healthy subjects with BMI ≥25 (n = 24; R2 = 0.87, Q2 = 0.51), but not healthy subjects with BMI ≤24 (n = 32; R2 = 0.44, Q2 = 0.12), when comparing baseline vs. end of intervention by OPLS-DA. Moreover, Aloe intervention showed potential to reduce the difference seen in the fecal metabolite profiles between subjects with BMI ≤24 and those with BMI ≥25 from R2 = 0.57 and Q2 = 0.30 at baseline to R2 = 0.38 and Q2 = -0.11 at end of intervention. No such tendencies were seen in the smaller placebo group (BMI ≤24, n = 12 vs. BMI ≥25, n =6).

Conclusion

Supplementation with increasing doses of Aloe extract is safe and well tolerated in healthy adults. Overall, our findings support safety of Aloe extract, and show that supplementing diet with Aloe extract has the potential to modulate intestinal microbiota function, especially in healthy adults with a BMI ≥25, with an altered intestinal microenvironment.

Disclosure

Presenting Author - Bani Ahluwalia is employed by Calmino Group AB, while carrying out Post-doctoral research at the University of Gothenburg.

Introduction

Ulcerative colitis (UC) is a chronic, non-specific inflammatory disease of the bowel of unknown etiology. This disease is characterised by high difficulty in cure, high recurrence and cancer rates, which seriously endangers the lives and health of our population. Taraxasterol (TA), a major constituent of the traditional Chinese medicine dandelion, has been reported in several studies to have good anti-inflammatory and antioxidant activities as well as anti-tumour effects.

Aims & Methods

However, the molecular targets and mechanisms of action of TA against UC have not been fully elucidated.The Swiss Target Prediction, PharmMapper databases were used to obtain relevant targets for the drug TA. Targets for UC were obtained from GeneCards, OMIM, PharmGkb, DrugBank databases. Targets of TA anti-UC identified by intersection of drug target and disease target. The relevant targets of TA anti-UC were uploaded to the String database and the resulting data were imported into Cytoscape 3.9.1 software to construct a protein-protein interaction (PPI) network of drug-disease targets. At the same time, the top 10 hub targets were screened using CytoHubba software. Gene ontology (GO) and Kyoto Encyclopedia of Genomes (KEGG) pathway enrichment analyses were performed on TA anti-UC targets using R software, and molecular docking was performed by Autodock Vina 1.1.2. The GEO dataset was used to analyze the differential mRNA expression of the TA anti-UC top 10 hub gene in UC patients and the normal population. Lipopolysaccharide (LPS)-induced intestinal epithelial cells (NCM460) were used to construct an in vitro cellular inflammation model and a Dextran Sulfate Sodium Salt (DSS) induced acute mouse colitis model. The pivotal targets and key signaling pathways were also validated by RT-qPCR and Western blotting.

Results

A total of 129 relevant targets of TA against UC were obtained, of which the top 10 hub targets were GRB2, PTPN11, SRC, PIK3R1, LCK, EGFR, MAPK1, IGF1, ESR1 and JAK2. The PI3K-Akt pathway was significantly associated with TA treatment of UC as identified by KEGG enrichment analysis. GEO dataset analysis demonstrated that EGFR and GRB2 were upregulated in UC patients, while SRC, PIK3R1 and JAK2 were down-regulated. In vitro experiments revealed that TA treatment reversed the expression of EGFR, GRB2, PIK3R1 and JAK2 differential genes. Furthermore, TA was able to up-regulate the mRNA expression of pro-inflammatory factor IL-10 and decrease the mRNA levels of anti-inflammatory factors IL-6, IL-1β and TNF-α in LPS-induced NCM460 cells. In vivo assay confirmed that TA significantly alleviated DSS-induced colonic inflammation in mice, including reversal of weight loss and shortening of colon length, reduction of DAI score in mice, and improvement of intestinal barrier disruption. In vivo and in vitro experiments have confirmed that TA also inhibits the activation of the PI3K/Akt pathway, thus exerting an anti-inflammatory effect.

Conclusion

TA may play a therapeutic role in UC by inhibiting the PI3K-Akt pathway. EGFR, GRB2, PIK3R1 and JAK2 may be the most relevant potential therapeutic targets for TA in the treatment of UC.