Introduction

Limiting exogenous fat intake is proposed as a preventive measure against obesity^[1]. Despite the elucidation of the molecular process of fatty acid uptake in enterocytes, there is no specific inhibitor that has been proven to effectively prevent obesity by inhibiting fatty acid absorption. Sterol O-acyltransferase 2 (SOAT2) is an enzyme responsible for esterifying free cholesterol and fatty acids into cholesteryl esters^{[2, 3]}. Evidence suggests the importance of intestinal SOAT2 in controlling dietary fatty acid absorption, hinting that targeting intestinal SOAT2 could be a promising approach to modulating the incidence of diet-induced obesity. Nanoparticles are vehicles that augment the efficacy of drug delivery and therapy ^{[4 -7]}. A novel nanoparticle system specifically designed to deliver SOAT2 siRNAs into the small intestine would be a promising therapeutic option to modulate intestinal fatty acid uptake and prevent obesity.

Aims & Methods

The role of intestinal SOAT2 in obesity was evaluated using intestine-specific SOAT2 knockout (Soat2^I-KO) mice through biochemical analyses, isotopic tracer methods, fluorescent palmitic acid assays, and western blot. Subsequently, a novel nanoparticle system composed of poly (D, L-lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-b-PEG) polymer as the core and chitosan (CS) shell, which serves as a protective layer against stomach acid, was developed to facilitate intestinal delivery and Soat2 inhibition. Mice were orally administered SOAT2 siRNA/CS-PLGA nanoparticles to evaluate their therapeutic efficacy against obesity. To elucidate the underlying mechanisms of how intestinal SOAT2 regulates fatty acid uptake, a SOAT2 knockdown Caco-2 (shSOAT2) cell model was established. Fatty acid uptake, immunofluorescence, mRNA expression profiling, siRNA library screening, subcellular distributions, and ubiquitination of CD36 protein were employed in shSOAT2 cells to assess the ubiquitination and degradation of CD36. Co-immunoprecipitation, immunofluorescence, and western blot were carried out to explore the underlying mechanism by which SOAT2 knockdown regulates CD36 ubiquitination.

Results

Soat2^I-KO mice were capable of preventing the development of dietary-induced obesity due to reduced intestinal lipid absorption. Then, the Soat2 siRNA/CS-PLGA nanoparticle system can specifically deliver SOAT2 siRNAs into the small intestines of mice and effectively inhibit intestinal lipid uptake, thus resolving obesity. In revealing the underlying mechanism by which intestinal SOAT2 regulates fatty acid uptake, we found enhanced CD36 ubiquitination and degradation in enterocytes upon SOAT2 inhibition. Insufficient free cholesterol esterification promotes endoplasmic reticulum stress and the recruitment of E3 ligase RNF5, which activates CD36 ubiquitination in SOAT2 knockdown enterocytes.

Conclusion

The SOAT siRNA/CS-PLGA nanoparticle system offers advantages such as being non-invasive, having low systemic toxicity, and ensuring higher compliance. Our work shows the promising translational relevance of intestinal-specific SOAT2 siRNA-based therapy to prevent obesity. Although SOAT2 is known as a regulator of cholesterol homeostasis, our data provide additional evidence for the role and mechanism of SOAT2 in regulating intestinal fatty acid absorption, suggesting that inhibition of this enzyme might serve as 'a stone to hit two birds'.

References

[1] S. B. Heymsfield, T. A. Wadden, The New England journal of medicine. 2017, 376, 254-266
[2] Y. Azuma, T. Kawasaki, K. Ikemoto, K. Obata, K. Ohno, N. Sajiki, T. Yamada, M. Yamasaki, Y. Nobuhara, Japanese journal of pharmacology. 1998, 78, 355-64.
[3] C. A. Marzetta, Y. E. Savoy, A. M. Freeman, C. A. Long, J. L. Pettini, R. E. Hagar, P. B. Inskeep, K. Davis, A. F. Stucchi, R. J. Nicolosi, et al., Journal of lipid research. 1994, 35, 1829-38.
[4] X. Zhang, V. Goel, G. J. Robbie, Journal of clinical pharmacology. 2020, 60, 573-585.
[5] K. K. Ray, R. S. Wright, D. Kallend, W. Koenig, L. A. Leiter, F. J. Raal, J. A. Bisch, T. Richardson, M. Jaros, P. L. J. Wijngaard, J. J. P. Kastelein, The New England journal of medicine. 2020, 382, 1507-1519.
[6] A. R. Sousa, A. V. Oliveira, M. J. Oliveira, B. Sarmento, International journal of pharmaceutics. 2019, 568, 118530.
[7] M. Song, S. Zhang, Z. Tao, J. Li, Y. Shi, Y. Xiong, W. Zhang, C. Liu, S. Chen, Biomaterials. 2021, 278, 121183.

Supplementary Infographic ↗

Introduction

Redx Pharma is developing RXC008, a gastrointestinal (GI)-restricted highly potent and selective ‘pan-ROCK’ inhibitor (inhibits rho-associated coiled-coil kinase ROCK1 and ROCK2) to treat fibrostenosis due to Crohn’s disease. ROCK1/2 are key signalling kinases in fibrosis and suppression of ROCK activity is highly efficacious in multiple rodent models of fibrotic disease. Systemic inhibition of both ROCK1/2 leads to hypotension limiting its therapeutic use; RXC008 is therefore designed to be restricted to the GI tract with minimal systemic exposure.

Aims & Methods

RXC008 potency was determined against ROCK1 and ROCK2 isolated enzymes in a biochemistry assay, by surface plasmon resonance (SPR) against ROCK2 and in a T853-pMYPT1 (Thr853 phospho-Myosin Phosphatase Target Subunit 1) cell assay. Anti-fibrotic efficacy was determined in vivo using the 9-week chronic dextran sulphate sodium (DSS) and the 45-day adoptive T-cell transfer (AT) murine models of colitis, by staining with Masson’s Trichrome and by Magnetic Resonance Imaging (MRI). Gene expression changes were assessed by RNAseq. T853-pMYPT1 was measured in tissue and cells by ELISA. RXC008 concentrations were measured by Liquid Chromatography with Tandem Mass Spectrometry in colon tissue and plasma.

Results

RXC008 inhibited ROCK1 and ROCK2 with 1.19/1.3nM potencies respectively in biochemical assays, displayed picomolar affinity for ROCK2 binding in SPR, and suppressed T853-pMYPT1 levels in HAP1 cells expressing ROCK1 and ROCK2 with a potency of 4.6nM. Blood pressure provides a sensitive biomarker for peripheral pan-ROCK inhibition. Minimal plasma exposure of RXC008 and no clinical signs associated with hypotension were detected in rats administered oral doses up to 1000 mg/kg/day for 28-days. In a dog telemetry study, minimal plasma exposure of RXC008 and no effect on blood pressure were detected at oral doses up to 1000mg/kg, while an intra-venous dose of 1mg/kg gave plasma compound concentrations ten-fold above the HAP1 cell IC50 together with a significant reduction in blood pressure.
In the DSS model, oral dosing (po) of 10mg/kg RXC008 once daily (QD) attenuated the disease activity index (diarrhea, body weight, hematochezia) and significantly (p<0.0001) suppressed colon fibrosis measured by Trichrome staining and MRI. In this model, RXC008 modulated the expression of >2000 genes (p≤0.01, log2 fold change) in the colon, including fibrotic genes Col1a1 and TGFβ, the extra-cellular matrix gene Wisp2 and tissue repair genes AhR and IL-22Rα. Monotherapy RXC008 (10, 30 or 100 mg/kg po QD) reduced the fibrosis score in the AT model (P<0.05), in contrast the anti-inflammatory agents anti-p40 or anti-TNFα alone had no effect on fibrosis. RXC008 (3, 10 and 30 mg/kg po QD) also significantly (p<0.05) reduced fibrosis in combination with anti-TNFα. Minimal to no plasma exposure of RXC008 was detected in the AT model despite efficacious GI tissue exposure. RXC008 significantly reduced T853-pMYPT1 levels in colon tissue in healthy and AT mice. Ex-vivo, RXC008 suppressed T853-pMYPT1 in fibroblasts (pIC50s: 7.83, 9.55, 9.02) isolated from fibrostenotic resection tissue from three Crohn’s Disease patients.

Conclusion

RXC008 suppresses fibrosis in animal models of colitis and is being developed as a novel first-in-class therapy to inhibit fibrotic stricture formation in patients with Crohn’s Disease. A first-in-human clinical trial (ISRCTN82038982) commenced in February 2024 and will include assessment of T853-pMYPT1 in tissue biopsy samples for target engagement.

Disclosure

Kirsty Houslay, Amy Marshall, Manuela La Montagna, Katie Anderson, Kay Eckersley, Clifford D. Jones, Andrew Belfield, Helen Mckeever, Helen Timmis, Richard Armer, Caroline Phillips, Elaine Kilgour are employees of Redx Pharma. Debby Laukens has received research ​funding from ​Redx Pharma.

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Introduction

Locally advanced esophageal cancer (EC) is treated with neoadjuvant chemo(radio)therapy (nCRT) followed by surgery, but only a modest percentage of patients (16-43%) achieves a pathological complete response post-nCRT. The addition of immune checkpoint inhibition (ICI) targeting programmed death-1 or its ligand (PD-1/PD-L1) is explored to increase response rates, yet adequate patient selection methods are lacking. PD-L1 expression’s predictive value in biopsies is inconsistent due to intra- and intertumoral heterogeneity, and the influence of nCRT on ICI response is unclear. Novel imaging methods are crucial to understand PD-L1 heterogeneity in order to ultimately select patients for ICI.

Aims & Methods

This ongoing study aims to assess the safety and feasibility of the innovative technique ultrasound guided quantitative fluorescence molecular endoscopy (US-qFME) using the fluorescently labelled ICI drug durvalumab-680LT before and after nCRT in EC patients to visualize, for the first time, PD-L1 drug distribution from the macroscopic to the cellular level. A durvalumab-680LT dose-optimization was performed in which up to now fifteen EC patients scheduled for nCRT were included in either the control (not receiving durvalumab-680LT), 4.5 mg, or 15 mg cohort. The next five patients will receive 25 mg durvalumab-680LT, after which the most optimal dose group will be expanded with 16 patients. US-qFME procedures were performed both before and after nCRT, consisting of in vivo fluorescence signal visualization, quantification by mucosal and ultrasound-guided spectroscopy measurements of healthy esophageal tissue, tumor and/or lymph nodes, and collection of biopsies. Subsequently, ex vivo analyses were performed for quantification of fluorescence signals, correlation to histology and visualization of tissue drug distribution and drug target cells.

Results

Higher fluorescence signals were visualized in vivo in tumor compared to healthy tissue. Ex vivo quantification of the fluorescence signals revealed higher median fluorescence signal intensities in tumor biopsies compared to healthy esophageal tissue pre-nCRT in both the 4.5 mg and 15 mg dose cohorts with a broad range in signals in the tumor (94.8 a.u. [41.0-131.7] vs. 31.0 a.u. [24.6-38.1] (p=0.125), and 103.9 a.u. [40.7-197.6] vs. 27.1 a.u. [25.8-40.8] (p=0.0625), respectively). Similar results were found during both mucosal and ultrasound-guided spectroscopy with higher signals in tumor tissue both in vivo and ex vivo compared to healthy tissue. The signals in healthy tissues of patients receiving the tracer were comparable to both tumor and healthy tissue in the control cohort (26.2 a.u. [21.5-37.6] and 11.7 a.u. [11.3-19.0]). An SDS-PAGE experiment confirmed the tracer’s integrity and stability in the mucosa. During fluorescence microscopy, only a dose of 15 mg durvalumab-680LT was sufficient to visualize membrane-bound durvalumab. Analyses post-nCRT are still ongoing. No (serious) adverse events occurred.

Conclusion

Durvalumab-680LT administration was safe and specifically targeted the tumor tissue as higher signals were found in tumor than in healthy tissue. In addition, the broad range in signals in the tumor tissue indicates, as expected, an inter- and intra-patient difference in PD-L1 expression. These preliminary findings show the potential of the novel combination of durvalumab-680LT and US-qFME to guide future patient selection for ICI therapy. Further analyses are ongoing, including correlation to PD-L1 staining and fluorescence microscopy to visualize drug distribution and potential drug-target interactions.

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