In Vitro Evaluation of a Novel Synthetic Bilirubin Analog as an Antioxidant and Cytoprotective Agent for Pancreatic Islet Transplantation

Abstract

Bilirubin is a natural cytoprotective agent and physiologic doses have proven to be beneficial in various models of organ and cellular transplantation. Recently, we showed that bilirubin has protective effects in models of pancreatic islet transplantation, preventing cell death associated with islet stress and suppressing the release of damage-associated molecular patterns. Despite these promising therapeutic attributes, the natural bilirubin used in these research studies is animal-derived (porcine), making it unsuitable for clinical application. In the current study, we synthesized two bilirubin analogs that can be produced without the use of animal-derived products. Antioxidant activity for the analogs was measured using the ferric-reducing-ability-of-plasma (FRAP) and 2,2V-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) assays. Dose-dependent cytotoxicity and cytoprotective effects were then demonstrated in isolated rat islets. Compound 1 showed similar antioxidant activity to natural bilirubin. Dose-dependent cytotoxicity was seen following treatment with Compound 1 and natural bilirubin at doses >40 μM, resulting in significantly increased cell death when compared to control islets ( P < 0.05) or islets treated with doses ≤20 μM ( P < 0.05). Following hypoxic challenge, islet cell death was reduced in islets treated with Compound 1 at 10 μM (17.27% ± 0.26%) compared to natural bilirubin at 10 μM (51.36% ± 0.71%; P < 0.0001) or 20 μM (59.02% ± 0.83%; P < 0.0001) and control islets (36.51% ± 0.44%; P < 0.0001). Compound 1 was found to have promising antioxidant and cytoprotective effects, limiting islet cell death in a model of islet transplantation hypoxic stress. Compound 1 may serve as a synthetic drug lead for clinical islet transplantation and further evaluation of this molecule and its analogs is warranted.

Pro- and Antioxidant Effect of Food Items and Matrices during Simulated In Vitro Digestion

“…Interestingly, the digestion of the blank samples without food constituents already showed a certain antioxidant as well as pro-oxidant activity in our study. While there is a lack of research on the pro-oxidant effects of bile and digestive enzymes, several studies have found bile components, such as bilirubin, to have an antioxidant effect due to their ability to bind free radicals, e.g., reactive nitrogen species (RNS) [ 43 , 44 ]. For example, a recent study investigated the in vitro antioxidant effects of two different types of synthetic bilirubin using FRAP and ABTS assays [ 44 ].…”

“…While there is a lack of research on the pro-oxidant effects of bile and digestive enzymes, several studies have found bile components, such as bilirubin, to have an antioxidant effect due to their ability to bind free radicals, e.g., reactive nitrogen species (RNS) [ 43 , 44 ]. For example, a recent study investigated the in vitro antioxidant effects of two different types of synthetic bilirubin using FRAP and ABTS assays [ 44 ]. The authors reported that both compounds exhibited similar potent antioxidant effects [ 44 ].…”

“…For example, a recent study investigated the in vitro antioxidant effects of two different types of synthetic bilirubin using FRAP and ABTS assays [ 44 ]. The authors reported that both compounds exhibited similar potent antioxidant effects [ 44 ]. Bilirubin can be oxidized to biliverdin, which is immediately reduced by biliverdin reductase to bilirubin in biological systems [ 45 ].…”

Case Report: Hyperbilirubinemia in Gilbert Syndrome Attenuates Covid-19-Induced Metabolic Disturbances

“…The GS patient with Covid-19 showed a rapid clinical improvement and short hospital stay as compared with a Covid-19 patient. Indeed, it has been proven that bilirubin exerts potent antioxidant effects which might alleviates Covid-19 induced-oxidative stress ( 9 ). Also, it has been reported that bilirubin has cardioprotective effects, improves endothelial function and provokes the nitric oxide (NO) release ( 10 ), thus, preventing from endothelial dysfunction and cardiovascular complications in COVID-19 ( 11 ), as evident of hypertension in Covid-19 case compared to GS patient with Covid-19.…”