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Sanyal Biotechnology and Innovate Biopharmaceuticals Present Data at AASLD Validating a Proprietary “Leaky Gut” Assay Demonstrating Reduced Intestinal Permeability with Larazotide Acetate Treatment in a NASH Preclinical Study

A Novel Non-Lethal Technique for Serial Measurement of “Leaky Gut” And Relationship to  NASH Liver Pathology in the DIAMOND™ Mouse Model

AASLD 2018.

Jonathon Marioneaux, Debbie Mayo, Mike Mostert, and Rebecca Caffrey

Background and Aims: Decline in gut integrity (“leaky gut”) occurs in the progression of NAFLD and NASH, and is associated with and partially driven by the leakage of bacterial products into the bloodstream from the gut. In mice this is usually measured by oral gavage with FITC-dextran, exsanguination of the mouse, and measurement of fluorescence in a large volume of serum. We developed a technique for measurement of FITC-dextran in 20 µL of serum from tail-vein nick that enables serial measurements for monitoring changes in gut integrity in individual mice.

Method:  77 DIAMOND™ mice were placed on either normal chow (NCNW) or Western Diet (WDSW) and aged up to 8, 20, 28, 36 and 40 weeks, then dosed with 4 kDa FITC-dextran @ 600 mg/kg body weight by gavage; at least 20 µL of serum was taken by tail vein nick 4 hours later when FITC-dextran was absorbed by the gut into the bloodstream. An optimized competition ELISA technique which measures small dextran polymers of 4 kDa in mouse serum was developed based on an ELISA originally used to measure contamination by large polymers in aqueous sugar solutions.

Results: Current literature shows FITC-dextran in mouse serum from 0-10 microliters/ml in healthy animals, to about 70 microliters/ml is animals treated to cause leaky gut (see Brandl 2009).  The range of this assay is 1 to 1000 microliters/ml when FITC-dextran was spiked into mouse serum, and the assay was linear over that range.  FITC-dextran levels in serum from NCNW and WDSW groups for all timepoints fell within this dynamic range. DIAMOND™ mice on NCNW like other mouse models with no gut/liver pathology, had serum dextran concentrations from 0 to ≤10 µg/ml; interestingly, DIAMOND™ mice on WDSW had significantly higher levels of serum dextran than other induced mouse models at all timepoints, indicating that this strain develops significant gut leakage that likely plays a role in driving the NASH pathology.  WDSW mice had average serum dextran concentrations of 143 (8 weeks, NASH F0), 254 (20 wks, NASH F1), 282 (28 wks, NASH F1/2), 300 (36 wks, NASH F3), and 355 (40 week, NASH F3 w tumorigenesis). At every timepoint the serum dextran was significantly higher in the WDSW group vs. the NCNW group (P ≤ 0.02). Repeat studies with unconjugated 4kD dextran polymer show comparable results.

Conclusion: DIAMOND™ mice are an excellent model for the role of gut integrity in driving NASH pathogenesis due to high gut permeability on WDSW. This new technique will enable serial measurements of gut permeability in the same mouse, allowing investigators to assess effectiveness of their compounds in treating this driver of NASH.