Peripheral Hybrid CB1R and iNOS Antagonist MRI-1867 Displays Anti-Fibrotic Efficacy in Bleomycin-Induced Skin Fibrosis.

Charles N Zawatsky,George Kunos,Jasmina Abdalla,Malliga R Iyer,Resat Cinar,Joshua K Park

doi:10.3389/fendo.2021.744857

Abstract

Scleroderma, or systemic sclerosis, is a multi-organ connective tissue disease resulting in fibrosis of the skin, heart, and lungs with no effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) and increased activity of inducible NO synthase (iNOS) promote tissue fibrosis including skin fibrosis, and joint targeting of these pathways may improve therapeutic efficacy. Recently, we showed that in mouse models of liver, lung and kidney fibrosis, treatment with a peripherally restricted hybrid CB1R/iNOS inhibitor (MRI-1867) yields greater anti-fibrotic efficacy than inhibiting either target alone. Here, we evaluated the therapeutic efficacy of MRI-1867 in bleomycin-induced skin fibrosis. Skin fibrosis was induced in C57BL/6J (B6) and Mdr1a/b-Bcrp triple knock-out (KO) mice by daily subcutaneous injections of bleomycin (2 IU/100 µL) for 28 days. Starting on day 15, mice were treated for 2 weeks with daily oral gavage of vehicle or MRI-1867. Skin levels of MRI-1867 and endocannabinoids were measured by mass spectrometry to assess target exposure and engagement by MRI-1867. Fibrosis was characterized histologically by dermal thickening and biochemically by hydroxyproline content. We also evaluated the potential increase of drug-efflux associated ABC transporters by bleomycin in skin fibrosis, which could affect target exposure to test compounds, as reported in bleomycin-induced lung fibrosis. Bleomycin-induced skin fibrosis was comparable in B6 and Mdr1a/b-Bcrp KO mice. However, the skin level of MRI-1867, an MDR1 substrate, was dramatically lower in B6 mice (0.023 µM) than in Mdr1a/b-Bcrp KO mice (8.8 µM) due to a bleomycin-induced increase in efflux activity of MDR1 in fibrotic skin. Furthermore, the endocannabinoids anandamide and 2-arachidonylglycerol were elevated 2-4-fold in the fibrotic vs. control skin in both mouse strains. MRI-1867 treatment attenuated bleomycin-induced established skin fibrosis and the associated increase in endocannabinoids in Mdr1a/b-Bcrp KO mice but not in B6 mice. We conclude that combined inhibition of CB1R and iNOS is an effective anti-fibrotic strategy for scleroderma. As bleomycin induces an artifact in testing antifibrotic drug candidates that are substrates of drug-efflux transporters, using Mdr1a/b-Bcrp KO mice for preclinical testing of such compounds avoids this pitfall.

Highlights

Scleroderma, or systemic sclerosis (SSc), is a connective tissue disease with multiple clinical manifestations, including autoimmunity, vascular dysfunction, and tissue fibrosis [1], and a prevalence in the United States of around 240 cases per 1 million adults [2]
To determine whether a similar mechanism is triggered by bleomycin in the skin, we first assessed the skin levels of the peripherally restricted hybrid CB1R/Inducible nitric oxide synthase (iNOS) inhibitor MRI-1867, a substrate of P-gp [16], in a bleomycin-induced murine model of skin fibrosis
Skin levels of anandamide (AEA) and 2-arachidonoyl glycerol (2AG) were higher in fibrotic compared to normal skin (Figure 1E), suggesting an upregulated endocannabinoid system in the fibrotic tissue

Summary

Introduction

Scleroderma, or systemic sclerosis (SSc), is a connective tissue disease with multiple clinical manifestations, including autoimmunity, vascular dysfunction, and tissue fibrosis [1], and a prevalence in the United States of around 240 cases per 1 million adults [2]. In SSc, the expression of iNOS in the endothelium, smooth muscle cells, fibroblasts, macrophages and many other cell types is robustly induced by inflammatory mediators and cytokines and its activity is increased at inflammatory sites [8]. The iNOS-mediated formation of NO is increased in inflammatory cells such as macrophages or activated fibroblasts [10]. In SSc patients, increased production of NO is suggested by the increased expression of iNOS in endothelial cells, fibroblasts and mononuclear cells infiltrating the fibrotic skin [12] as well as in alveolar macrophages [13]. The role of NO synthases and especially iNOS is elegantly dissected by the work of Cotton et al, which proposes an active role of iNOS-induced NO production in endothelial cell damage and advances the concept of iNOS inhibition as a viable therapeutic strategy for SSc [14]

Methods

Results

Conclusion