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

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 Mdr1_a/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 Mdr1_a/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 Mdr1_a/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 Mdr1_a/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 Mdr1_a/b-Bcrp KO mice for preclinical testing of such compounds avoids this pitfall.

Keywords: ATP-binding cassette transporters; P-gp (P-glycoprotein); bleomycin; cannabinoid (CB) receptor 1; endocannabinoids; peripheral CB1R antagonist; polypharmacology; skin fibrosis.

Figure 1

Bleomycin-induced skin fibrosis minimizes skin exposure and target engagement by MRI-1867 in C57BL6/J mice. (A) Schematic presentation of bleomycin-induced skin fibrosis development and therapeutic treatment regime. (B) Dermal thickness quantification for histological images. (C) Representative histological images from skin biopsies stained with H&E Ep, Epidermis; D, Dermis; Hy, Hypodermis and adipose tissue; Pc, Panninculus carnosus. Double arrow indicates dermal length that used for quantification. (D) Levels of MRI-1867 in control and bleomycin-treated skin biopsy specimens following acute or 14-day chronic administration of MRI-1867 at 10 mg/kg, respectively. (E) Levels of endocannabinoids AEA and 2AG in skin biopsies. Data represent means ± SEM from 3-10 mice per group. Data were analyzed by 1-way ANOVA followed by Dunnett’s multiple comparisons test. Significant difference from corresponding values in controls (saline and vehicle treated group) (*P < 0.05).

Figure 2

Bleomycin induces drug efflux transporter P-gp in skin of C57BL6/J mice. (A) Representative histological images for P-gp immunohistochemistry staining in skin biopsies of control and bleomycin-induced fibrotic skin. A, adipocytes; BV, blood vessels; D, Dermis; PC, Panninculus carnosus. (B) Quantification of P-gp protein expression in skin biopsies. Data represent mean ± SEM from five subjects in each group. Data were analyzed by t-test for comparison of histological scoring. * (p <0.05) indicates significant difference from the control group.

Figure 3

Dose-dependent systemic and skin exposure of MRI-1867 in $\text{Mdr}{1}_{\text{a}/\text{b}}^{(-/-)}$ -Bcrp^(-/-) mice. (A) Serum and (B) Skin levels of MRI-1867 at 1 hour after the last dose of 14 days oral administration at 1, 3, 10, 30, and 60 mg/kg doses in bleomycin-treated $\text{Mdr}{1}_{\text{a}/\text{b}}^{(-/-)}$ -Bcrp^(-/-) mice. Data represent means ± SEM from 4-10 mice per group.

Figure 4

MRI-1867 (30 mg/kg) attenuates bleomycin-induced fibrosis and dermal thickness in $\text{Mdr}{1}_{\text{a}/\text{b}}^{(-/-)}$ -Bcrp^(-/-) mice. (A) Hydroxyproline levels in biopsied skin. (B) Dermal thickness analyses from skin biopsies. (C) Representative histological images from skin biopsies stained with H&E. (D) AEA and 2AG levels in biopsied skin. Six mm skin biopsies are used for the analysis. Dermal thickness assessed from left scapular injection site biopsies, Hydroxyproline and endocannabinoid measurements performed using right scapular injection site biopsies from each mouse. Data represent means ± SEM from 3-13 mice per group. Data were analyzed by 1-way ANOVA followed by Dunnett’s multiple comparisons test. Significant difference from corresponding values in control (saline and vehicle treated group) (*P < 0.05) or from values in bleomycin and vehicle-treated group (^#P < 0.05).

Figure 5

Comparison of the anti-fibrotic effects of MRI-1867 and rimonabant in $\text{Mdr}{1}_{\text{a}/\text{b}}^{(-/-)}$ -Bcrp^(-/-) mice. (A) Dermal thickness analysis, (B) Hydroxyproline levels, (C) AEA and 2AG levels from skin biopsies in control and bleomycin-injected Mdr1a/b(-/-) -Bcrp^(-/-) mice. Rimonabant (10 mg/kg) or MRI-1867 (1, 3, 10 mg/kg) were orally administered daily for the last 14 days of bleomycin injections. Data represent means ± SEM from 4-10 mice per group. Data were analyzed by 1-way ANOVA followed by Dunnett’s multiple comparisons test. Significant difference from corresponding values in control (saline and vehicle treated group) (*P < 0.05), or from values in bleomycin and vehicle-treated group (^#P < 0.05), or from values in bleomycin and rimonabant-treated group (⁺P < 0.05).