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by Bhasker Radaram, Tinamarie Skedzielewski, Stephen Lenhard, Tolulope Aweda, Shih-Hsun Cheng, Glenn Moran, Andrew Gehman, Simon Campbell, Hasan Alsaid on behalf of the TRISTAN Consortium

WMIC 2023

Abstract

Introduction: Bile acids are amphiphilic steroid derivatives produced by hepatocytes that play a key role in digestion of lipids and uptake of fat-soluble vitamins. Bile Salt Export Pump (BSEP) is an efflux transporter that plays a critical role in the secretion of bile salts into the bile. In many species such as humans and rodents, cholic acid is the largest key constituent of the bile acid spectrum. Inhibition of BSEP function by drugs such as Bosentan has resulted in the buildup of bile salts in the liver and lead to drug-induced liver injury (DILI). Formulation of drugs in DMSO (Dimethyl Sulfoxide) poses a safety risk in rodents . To better mitigate the formulation challenges for animal safety, herein, we report the formulation effects of Bosentan in PEG400 and assessed by 18F-3β-Fluorocholic acid (18F-3β-FCA) to examine hepatobiliary transport in vivo in the presence and absence of BSEP inhibitor Bosentan.

Methods: All animal procedures complied with the guidelines of the Institutional Animal Care and Use Committee at GSK following the guidance of Animal Use. 18F-3β-FluoroCholic Acid (18F-FCA) was produced as per literature procedure  using an automated ELIXYS Box (Sofie Biosciences, Inc). Wistar (Han) IGS white (albino) outbred male rats (strain 273), 6-11 weeks old with the body weights of 250-410 g were randomized in control (no treatment) and Bosentan treated groups. All animals were fasted for at least 4 hrs before PET imaging to reduce tracer metabolic variability. Rats in the Bosentan treated group (n=4) received 50 mg/Kg Bosentan in PEG400 (iv slow bolus infusion (0.2 mL/min) 1 hr prior to PET imaging. Dynamic PET imaging was acquired using the Mediso LFER150 PET scanner for 1 hr starting with iv injection of 18F-3β-FCA (400 µCi per rat in 200 µL 8% EtOH/PBS). At the end of the PET data acquisition, blood samples were collected for total bile salt analysis. Statistical analysis was performed using two sample t-test with equal variances.

Results: 18F-FCA radiosynthesis was achieved with radiochemical yields (RCY) of 6.64±0.64% (d.c) (n=9) with high radiochemical purity (>99%). Formulation of Bosentan in PEG400 was tolerated in all animals with no sign of pain or distress. However, the Bosentan treated group did not show any inhibition (mean 18F-FCA uptake) compared to the control group (figure 1a) and effluxed to the gallbladder and intestines. The total bile salt analysis  was two folds greater in the Bosentan treated group compared to the control group although this difference was not statistically significant.

Conclusions: In summary, Bosentan in the PEG400 formulation improved the tolerability and animal safety compared to the 100% DMSO formulation. However, it did not show a BSEP inhibition compared to the previously published Bosentan formulation in 100% DMSO.