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 (CB
1
R) 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 CB
1
R/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 CB
1
R 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.
Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder that affects lysosome-related organelles, often leading to fatal pulmonary fibrosis (PF). The search for a treatment for HPS ...pulmonary fibrosis (HPSPF) is ongoing. S-MRI-1867, a dual cannabinoid receptor 1 (CB1R)/inducible nitric oxide synthase (iNOS) inhibitor, has shown great promise for the treatment of several fibrotic diseases, including HPSPF. In this study, we investigated the in vitro ADME characteristics of S-MRI-1867, as well as its pharmacokinetic (PK) properties in mice, rats, dogs, and monkeys. S-MRI-1867 showed low aqueous solubility (< 1 µg/mL), high plasma protein binding (>99%), and moderate to high metabolic stability. In its preclinical PK studies, S-MRI-1867 exhibited moderate to low plasma clearance (CLp) and high steady-state volume of distribution (Vdss) across all species. Despite the low solubility and P-gp efflux, S-MRI-1867 showed great permeability and metabolic stability leading to a moderate bioavailability (21-60%) across mouse, rat, dog, and monkey. Since the R form of MRI-1867 is CB1R-inactive, we investigated the potential conversion of S-MRI-1867 to R-MRI-1867 in mice and found that the chiral conversion was negligible. Furthermore, we developed and validated a PBPK model that adequately fits the PK profiles of S-MRI-1867 in mice, rats, dogs, and monkeys using various dosing regimens. We employed this PBPK model to simulate the human PK profiles of S-MRI-1867, enabling us to inform human dose selection and support the advancement of this promising drug candidate in the treatment of HPSPF.
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•S-MRI-1867: CB1R/iNOS inhibitor shows low solubility and high protein binding, impacting formulation and bioavailability.•There is minimal chiral conversion of S-MRI-1867 to the R- inactive form, ensuring sustained efficacy of the stereoisomer.•S-MRI-1867 exhibits high permeability, moderate clearance, and large volume of distribution in preclinical studies.•The PBPK model accurately predicts PK profiles across species, supporting interspecies scalability.•The validated PBPK model enables simulation of human PK profiles, achieving effective concentrations at manageable doses.
Experimental evidence points out that the activation of the endocannabinoid system induces neuroprotective effects and reduces mood disorders. In the hippocampus of patients with mesial temporal lobe ...epilepsy (MTLE), studies indicated augmented cannabinoid 1 receptor (CB
R) binding, in spite of its low mRNA and protein expressions. Although this situation suggests an enhanced CB
R-induced neurotransmission in patients with MTLE, especially those with pharmacoresistant seizures, which present important neuronal damage and high comorbid mood disorders. The present study focused to investigate the status of CB
R and the endocannabinoid system by obtaining CB
R-induced G-protein signaling efficacy and measuring the tissue levels of endocannabinoids in the hippocampus and the temporal neocortex of patients with pharmacoresistant MTLE. Furthermore, the obtained results were correlated with comorbid anxiety and depression. The experiments revealed that patients with MTLE present increased CB
R-induced G-protein signaling efficacy (Emax) as well as an augmented tissue content of anandamide and oleoylethanolamine and low 2-arachidonoylglycerol. Some of these changes were more evident in patients with MTLE without mood disorders. The current findings indicate that pharmacoresistant MTLE is associated with increased CB
R-induced transductional mechanisms as well as augmented tissue content of specific endocannabinoids in the hippocampus and the temporal neocortex. The enhanced endocannabinoid neurotransmission may be involved in the absence of comorbid mood disorders in some patients with MTLE.
HPS-1 is a genetic type of Hermansky-Pudlak syndrome (HPS) with highly penetrant pulmonary fibrosis (HPSPF), a restrictive lung disease that is similar to idiopathic pulmonary fibrosis (IPF). Hps1
...(pale ear) is a naturally occurring HPS-1 mouse model that exhibits high sensitivity to bleomycin-induced pulmonary fibrosis (PF). Traditional methods of administering bleomycin as an intratracheal (IT) route to induce PF in this model often lead to severe acute lung injury and high mortality rates, complicating studies focusing on pathobiological mechanisms or exploration of therapeutic options for HPSPF.
To develop a murine model of HPSPF that closely mimics the progression of human pulmonary fibrosis, we investigated the pulmonary effects of systemic delivery of bleomycin in Hps1
mice using a subcutaneous minipump and compared results to oropharyngeal delivery of bleomycin.
Our study revealed that systemic delivery of bleomycin induced limited, acute inflammation that resolved. The distinct inflammatory phase preceded a slow, gradually progressive fibrogenesis that was shown to be both time-dependent and dose-dependent. The fibrosis phase exhibited characteristics that better resembles human disease with focal regions of fibrosis that were predominantly found in peribronchovascular areas and in subpleural regions; central lung areas contained relatively less fibrosis.
This model provides a preclinical tool that will allow researchers to study the mechanism of pulmonary fibrosis in HPS and provide a platform for the development of therapeutics to treat HPSPF. This method can be applied on studies of IPF or other monogenic disorders that lead to pulmonary fibrosis.
The mammalian liver regenerates upon tissue loss, which induces quiescent hepatocytes to enter the cell cycle and undergo limited replication under the control of multiple hormones, growth factors, ...and cytokines. Endocannabinoids acting via cannabinoid type 1 receptors (CBâR) promote neural progenitor cell proliferation, and in the liver they promote lipogenesis. These findings suggest the involvement of CBâR in the control of liver regeneration. Here we report that mice lacking CBâR globally or in hepatocytes only and wild-type mice treated with a CBâR antagonist have a delayed proliferative response to two-thirds partial hepatectomy (PHX). In wild-type mice, PHX leads to increased hepatic expression of CBâR and hyperactivation of the biosynthesis of the endocannabinoid anandamide in the liver via an in vivo pathway involving conjugation of arachidonic acid and ethanolamine by fatty-acid amide hydrolase. In wild-type but not CBâRâ»/â» mice, PHX induces robust up-regulation of key cell-cycle proteins involved in mitotic progression, including cyclin-dependent kinase 1 (Cdk1), cyclin B2, and their transcriptional regulator forkhead box protein M1 (FoxM1), as revealed by ultrahigh-throughput RNA sequencing and pathway analysis and confirmed by real-time PCR and Western blot analyses. Treatment of wild-type mice with anandamide induces similar changes mediated via activation of the PI3K/Akt pathway. We conclude that activation of hepatic CBâR by newly synthesized anandamide promotes liver regeneration by controlling the expression of cell-cycle regulators that drive M phase progression.
Pulmonary fibrosis (PF) is a heterogeneous disease with a poor prognosis. Therefore, identifying additional therapeutic modalities is required to improve outcome. However, the lack of biomarkers of ...disease progression hampers the preclinical to clinical translational process. Here, this work assesses and identifies progressive alterations in pulmonary function, transcriptomics, and metabolomics in the mouse lung at 7, 14, 21, and 28 days after a single dose of oropharyngeal bleomycin. By integrating multi‐omics data, this work identifies two central gene subnetworks associated with multiple critical pathological changes in transcriptomics and metabolomics as well as pulmonary function. This work presents a multi‐omics‐based framework to establish a translational link between the bleomycin‐induced PF model in mice and human idiopathic pulmonary fibrosis to identify druggable targets and test therapeutic candidates. This work also indicates peripheral cannabinoid receptor 1 (CB1R) antagonism as a rational therapeutic target for clinical translation in PF. Mouse Lung Fibrosis Atlas can be accessed freely at https://niaaa.nih.gov/mouselungfibrosisatlas.
Multi‐omics approach helps to establish a translational link in idiopathic pulmonary fibrosis (IPF) between human and its experimental model in mice. Multi‐omics‐based framework assists the identification of druggable targets in IPF. Systems pharmacology endorses cannabinoid CB1R antagonism as a rational therapeutic strategy in IPF.
The proliferation and differentiation of hepatic progenitor cells (HPCs) drive the homeostatic renewal of the liver under diverse conditions. Liver regeneration is associated with an increase in ...Axin2
Cnr1
HPCs, along with a marked increase in the levels of the endocannabinoid anandamide (AEA). But the molecular mechanism linking AEA signaling to HPC proliferation and/or differentiation has not been explored. Here, we show that in vitro exposure of HPCs to AEA triggers both cell cycling and differentiation along with increased expression of Cnr1, Krt19, and Axin2. Mechanistically, we found that AEA promotes the nuclear localization of the transcription factor β-catenin, with subsequent induction of its downstream targets. Systemic analyses of cells after CRISPR-mediated knockout of the β-catenin-regulated transcriptome revealed that AEA modulates β-catenin-dependent cell cycling and differentiation, as well as interleukin pathways. Further, we found that AEA promotes OXPHOS in HPCs when amino acids and glucose are readily available as substrates, but AEA inhibits it when the cells rely primarily on fatty acid oxidation. Thus, the endocannabinoid system promotes hepatocyte renewal and maturation by stimulating the proliferation of Axin2
Cnr1
HPCs via the β-catenin pathways while modulating the metabolic activity of their precursor cells.
We report an unexpected link between aging, thermogenesis and weight gain via the orphan G protein-coupled receptor GPR3. Mice lacking GPR3 and maintained on normal chow had similar body weights ...during their first 5 months of life, but gained considerably more weight thereafter and displayed reduced total energy expenditure and lower core body temperature. By the age of 5 months GPR3 KO mice already had lower thermogenic gene expression and uncoupling protein 1 protein level and showed impaired glucose uptake into interscapular brown adipose tissue (iBAT) relative to WT littermates. These molecular deviations in iBAT of GPR3 KO mice preceded measurable differences in body weight and core body temperature at ambient conditions, but were coupled to a failure to maintain thermal homeostasis during acute cold challenge. At the same time, the same cold challenge caused a 17-fold increase in Gpr3 expression in iBAT of WT mice. Thus, GPR3 appears to have a key role in the thermogenic response of iBAT and may represent a new therapeutic target in age-related obesity.
Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease without effective treatment, highlighting the need for identifying new targets and treatment modalities. The pathogenesis of IPF is ...complex, and engaging multiple targets simultaneously might improve therapeutic efficacy. To assess the role of the endocannabinoid/cannabinoid receptor 1 (endocannabinoid/CB1R) system in IPF and its interaction with inducible nitric oxide synthase (iNOS) as dual therapeutic targets, we analyzed lung fibrosis and the status of the endocannabinoid/CB1R system and iNOS in mice with bleomycin-induced pulmonary fibrosis (PF) and in lung tissue and bronchoalveolar lavage fluid (BALF) from patients with IPF, as well as controls. In addition, we investigated the antifibrotic efficacy in the mouse PF model of an orally bioavailable and peripherally restricted CB1R/iNOS hybrid inhibitor. We report that increased activity of the endocannabinoid/CB1R system parallels disease progression in the lungs of patients with idiopathic PF and in mice with bleomycin-induced PF and is associated with increased tissue levels of interferon regulatory factor-5. Furthermore, we demonstrate that simultaneous engagement of the secondary target iNOS by the hybrid CB1R/iNOS inhibitor has greater antifibrotic efficacy than inhibition of CB1R alone. This hybrid antagonist also arrests the progression of established fibrosis in mice, thus making it a viable candidate for future translational studies in IPF.
Abstract Objective Extreme obesity is a core phenotypic feature of Prader–Willi syndrome (PWS). Among numerous metabolic regulators, the endocannabinoid (eCB) system is critically involved in ...controlling feeding, body weight, and energy metabolism, and a globally acting cannabinoid-1 receptor (CB1 R) blockade reverses obesity both in animals and humans. The first-in-class CB1 R antagonist rimonabant proved effective in inducing weight loss in adults with PWS. However, it is no longer available for clinical use because of its centrally mediated, neuropsychiatric, adverse effects. Methods We studied eCB ‘tone’ in individuals with PWS and in the Magel2 -null mouse model that recapitulates the major metabolic phenotypes of PWS and determined the efficacy of a peripherally restricted CB1 R antagonist, JD5037 in treating obesity in these mice. Results Individuals with PWS had elevated circulating levels of 2-arachidonoylglycerol and its endogenous precursor and breakdown ligand, arachidonic acid. Increased hypothalamic eCB ‘tone’, manifested by increased eCBs and upregulated CB1 R, was associated with increased fat mass, reduced energy expenditure, and decreased voluntary activity in Magel2 -null mice. Daily chronic treatment of obese Magel2 -null mice and their littermate wild-type controls with JD5037 (3 mg/kg/d for 28 days) reduced body weight, reversed hyperphagia, and improved metabolic parameters related to their obese phenotype. Conclusions Dysregulation of the eCB/CB1 R system may contribute to hyperphagia and obesity in Magel2 -null mice and in individuals with PWS. Our results demonstrate that treatment with peripherally restricted CB1 R antagonists may be an effective strategy for the management of severe obesity in PWS.
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