Renal Mitochondrial ATP Transporter Ablation Ameliorates Obesity-Induced CKD.

SIGNIFICANCE STATEMENT: This study sheds light on the central role of adenine nucleotide translocase 2 (ANT2) in the pathogenesis of obesity-induced CKD. Our data demonstrate that ANT2 depletion in renal proximal tubule cells (RPTCs) leads to a shift in their primary metabolic program from fatty acid oxidation to aerobic glycolysis, resulting in mitochondrial protection, cellular survival, and preservation of renal function. These findings provide new insights into the underlying mechanisms of obesity-induced CKD and have the potential to be translated toward the development of targeted therapeutic strategies for this debilitating condition. BACKGROUND: The impairment in ATP production and transport in RPTCs has been linked to the pathogenesis of obesity-induced CKD. This condition is characterized by kidney dysfunction, inflammation, lipotoxicity, and fibrosis. In this study, we investigated the role of ANT2, which serves as the primary regulator of cellular ATP content in RPTCs, in the development of obesity-induced CKD. METHODS: We generated RPTC-specific ANT2 knockout ( RPTC-ANT2-/- ) mice, which were then subjected to a 24-week high-fat diet-feeding regimen. We conducted comprehensive assessment of renal morphology, function, and metabolic alterations of these mice. In addition, we used large-scale transcriptomics, proteomics, and metabolomics analyses to gain insights into the role of ANT2 in regulating mitochondrial function, RPTC physiology, and overall renal health. RESULTS: Our findings revealed that obese RPTC-ANT2-/- mice displayed preserved renal morphology and function, along with a notable absence of kidney lipotoxicity and fibrosis. The depletion of Ant2 in RPTCs led to a fundamental rewiring of their primary metabolic program. Specifically, these cells shifted from oxidizing fatty acids as their primary energy source to favoring aerobic glycolysis, a phenomenon mediated by the testis-selective Ant4. CONCLUSIONS: We propose a significant role for RPTC-Ant2 in the development of obesity-induced CKD. The nullification of RPTC-Ant2 triggers a cascade of cellular mechanisms, including mitochondrial protection, enhanced RPTC survival, and ultimately the preservation of kidney function. These findings shed new light on the complex metabolic pathways contributing to CKD development and suggest potential therapeutic targets for this condition.

In vitro and in vivo pharmacokinetic characterization, chiral conversion and PBPK scaling towards human PK simulation of S-MRI-1867, a drug candidate for Hermansky-Pudlak syndrome pulmonary fibrosis.

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.

Functional Pdgfra fibroblast heterogeneity in normal and fibrotic mouse lung.

Aberrant fibroblast function plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, a devastating disease of unrelenting extracellular matrix deposition in response to lung injury. Platelet-derived growth factor α-positive (Pdgfra+) lipofibroblasts (LipoFBs) are essential for lung injury response and maintenance of a functional alveolar stem cell niche. Little is known about the effects of lung injury on LipoFB function. Here, we used single-cell RNA-Seq (scRNA-Seq) technology and PdgfraGFP lineage tracing to generate a transcriptomic profile of Pdgfra+ fibroblasts in normal and injured mouse lungs 14 days after bleomycin exposure, generating 11 unique transcriptomic clusters that segregated according to treatment. While normal and injured LipoFBs shared a common gene signature, injured LipoFBs acquired fibrogenic pathway activity with an attenuation of lipogenic pathways. In a 3D organoid model, injured Pdgfra+ fibroblast-supported organoids were morphologically distinct from those cultured with normal fibroblasts, and scRNA-Seq analysis suggested distinct transcriptomic changes in alveolar epithelia supported by injured Pdgfra+ fibroblasts. In summary, while LipoFBs in injured lung have not migrated from their niche and retain their lipogenic identity, they acquire a potentially reversible fibrogenic profile, which may alter the kinetics of epithelial regeneration and potentially contribute to dysregulated repair, leading to fibrosis.

Cannabinoformins: Designing Biguanide-Embedded, Orally Available, Peripherally Selective Cannabinoid-1 Receptor Antagonists for Metabolic Syndrome Disorders.

We have designed orally bioavailable, non-brain-penetrant antagonists of the cannabinoid-1 receptor (CB1R) with a built-in biguanide sensor to mimic 5'-adenosine monophosphate kinase (AMPK) activation for treating obesity-associated co-morbidities. A series of 3,4-diarylpyrazolines bearing rational pharmacophoric pendants designed to limit brain penetration were synthesized and evaluated in CB1R ligand binding assays and recombinant AMPK assays. The compounds displayed high CB1R binding affinity and potent CB1R antagonist activities and acted as AMPK activators. Select compounds showed good oral exposure, with compounds 36, 38-S, and 39-S showing <5% brain penetrance, attesting to peripheral restriction. In vivo studies of 38-S revealed decreased food intake and body weight reduction in diet-induced obese mice as well as oral in vivo efficacy of 38-S in ameliorating glucose tolerance and insulin resistance. The designed "cannabinoformin" four-arm CB1R antagonists could serve as potential leads for treatment of metabolic syndrome disorders with negligible neuropsychiatric side effects.

Cannabinoid receptor type 1 (CB1R) inhibits hypothalamic leptin signaling via ß-arrestin1 in complex with TC-PTP and STAT3.

Molecular interactions between anorexigenic leptin and orexigenic endocannabinoids, although of great metabolic significance, are not well understood. We report here that hypothalamic STAT3 signaling in mice, initiated by physiological elevations of leptin, is diminished by agonists of the cannabinoid receptor 1 (CB1R). Measurement of STAT3 activation by semi-automated confocal microscopy in cultured neurons revealed that this CB1R-mediated inhibition requires both T cell protein tyrosine phosphatase (TC-PTP) and ß-arrestin1 but is independent of changes in cAMP. Moreover, ß-arrestin1 translocates to the nucleus upon CB1R activation and binds both STAT3 and TC-PTP. Consistently, CB1R activation failed to suppress leptin signaling in ß-arrestin1 knockout mice in vivo, and in neural cells deficient in CB1R, ß-arrestin1 or TC-PTP. Altogether, CB1R activation engages ß-arrestin1 to coordinate the TC-PTP-mediated inhibition of the leptin-evoked neuronal STAT3 response. This mechanism may restrict the anorexigenic effects of leptin when hypothalamic endocannabinoid levels rise, as during fasting or in diet-induced obesity.

A cortico-amygdala neural substrate for endocannabinoid modulation of fear extinction.

Preclinical and clinical studies implicate endocannabinoids (eCBs) in fear extinction, but the underlying neural circuit basis of these actions is unclear. Here, we employed in vivo optogenetics, eCB biosensor imaging, ex vivo electrophysiology, and CRISPR-Cas9 gene editing in mice to examine whether basolateral amygdala (BLA)-projecting medial prefrontal cortex (mPFC) neurons represent a neural substrate for the effects of eCBs on extinction. We found that photoexcitation of mPFC axons in BLA during extinction mobilizes BLA eCBs. eCB biosensor imaging showed that eCBs exhibit a dynamic stimulus-specific pattern of activity at mPFC→BLA neurons that tracks extinction learning. Furthermore, using CRISPR-Cas9-mediated gene editing, we demonstrated that extinction memory formation involves eCB activity at cannabinoid CB1 receptors expressed at vmPFC→BLA synapses. Our findings reveal the temporal characteristics and a neural circuit basis of eCBs' effects on fear extinction and inform efforts to target the eCB system as a therapeutic approach in extinction-deficient neuropsychiatric disorders.

An Integrative Multiomics Framework for Identification of Therapeutic Targets in Pulmonary Fibrosis.

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 (CB1 R) 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.

The endocannabinoid system promotes hepatocyte progenitor cell proliferation and maturation by modulating cellular energetics.

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 ß-catenin, with subsequent induction of its downstream targets. Systemic analyses of cells after CRISPR-mediated knockout of the ß-catenin-regulated transcriptome revealed that AEA modulates ß-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 ß-catenin pathways while modulating the metabolic activity of their precursor cells.

The effects of acute alcohol administration on circulating endocannabinoid levels in humans.

Several lines of evidence suggest that endocannabinoid signalling may influence alcohol consumption. Preclinical studies have found that pharmacological blockade of cannabinoid receptor 1 leads to reductions in alcohol intake. Furthermore, variations in endocannabinoid metabolism between individuals may be associated with the presence and severity of alcohol use disorder. However, little is known about the acute effects of alcohol on the endocannabinoid system in humans. In this study, we evaluated the effect of acute alcohol administration on circulating endocannabinoid levels by analysing data from two highly-controlled alcohol administration experiments. In the first within-subjects experiment, 47 healthy participants were randomized to receive alcohol and placebo in a counterbalanced order. Alcohol was administered using an intravenous clamping procedure such that each participant attained a nearly identical breath alcohol concentration of 0.05%, maintained over 3 h. In the second experiment, 23 healthy participants self-administered alcohol intravenously; participants had control over their exposure throughout the paradigm. In both experiments, circulating concentrations of two endocannabinoids, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), were measured at baseline and following alcohol exposure. During the intravenous clamping procedure, acute alcohol administration reduced circulating AEA but not 2-AG levels when compared to placebo. This finding was confirmed in the self-administration paradigm, where alcohol reduced AEA levels in an exposure-dependent manner. Future studies should seek to determine whether alcohol administration has similar effects on brain endocannabinoid signalling. An improved understanding of the bidirectional relationship between endocannabinoid signalling and alcohol intake may deepen our understanding of the aetiology and repercussions of alcohol use disorder.

Chronic and Binge Alcohol Ingestion Increases Truncated Oxidized Phosphatidylcholines in Mice Lungs Due to Increased Oxidative Stress.

Heavy alcohol drinking has negative health effects in multiple organs. It predisposes lungs to inflammatory conditions associated with acute lung injury and increased incidence of pneumonia and sepsis, which may lead to death due to acute respiratory distress syndrome in some individuals with alcohol use disorder (AUD). In general, rodent models of alcohol exposure either do not recapitulate multiple organ injuries as seen in humans or require longer duration to establish tissue injury and inflammation. The recently introduced NIAAA model of alcohol-induced liver injury, characterized by a marked increase in steatosis and liver damage with 10 days of a liquid diet containing 5% ethanol followed by a single ethanol binge (5 g/kg). Therefore, we employed this model to explore the status of surfactant phospholipids, oxidative stress, tissue injury markers and inflammatory cytokines in lungs. In lungs of C57BL/6J mice, the alcohol feeding significantly increased levels of the surfactant phospholipid dipalmitoyl phosphatidylcholine (DPPC) as well as the truncated oxidized phosphatidylcholines palmitoyl oxovaleryl phosphatidyl-choline (POVPC), palmitoyl glutaryl phosphatidyl-choline (PGPC), palmitoyl oxo-nonanoyl phosphatidyl-choline (ALDO-PC), and palmitoyl azelaoyl phosphatidyl-choline (PAzePC) at 9 h post-binge. Additionally, gene expression of the enzymes catalyzing lipid oxidation, such as arachidonate 15-lipoxygenase (Alox15), prostaglandin synthase 2 (Ptgs2), Cytochrome P450 2E1 (Cyp2E1) and NADPH oxidase 1 (Nox1) were significantly increased. Furthermore, ethanol increased levels of the inflammatory cytokine Interleukin-17 in bronchoalveolar lavage fluid. In conclusion, the NIAAA alcohol feeding model might be suitable to study alcohol-induced lung injury and inflammation.

Deficiency in endocannabinoid synthase DAGLB contributes to early onset Parkinsonism and murine nigral dopaminergic neuron dysfunction.

Endocannabinoid (eCB), 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain, regulates diverse neural functions. Here we linked multiple homozygous loss-of-function mutations in 2-AG synthase diacylglycerol lipase ß (DAGLB) to an early onset autosomal recessive Parkinsonism. DAGLB is the main 2-AG synthase in human and mouse substantia nigra (SN) dopaminergic neurons (DANs). In mice, the SN 2-AG levels were markedly correlated with motor performance during locomotor skill acquisition. Genetic knockdown of Daglb in nigral DANs substantially reduced SN 2-AG levels and impaired locomotor skill learning, particularly the across-session learning. Conversely, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, DAN activity and dopamine release and rescued the locomotor skill learning deficits. Together, we demonstrate that DAGLB-deficiency contributes to the pathogenesis of Parkinsonism, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neuronal activity and dopamine release, and suggest potential benefits of 2-AG augmentation in alleviating Parkinsonism.

Progressive pulmonary fibrosis in a murine model of Hermansky-Pudlak syndrome.

BACKGROUND: 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). Hps1ep/ep (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. METHODS: 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 Hps1ep/ep mice using a subcutaneous minipump and compared results to oropharyngeal delivery of bleomycin. RESULTS: 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. CONCLUSION: 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.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of 3,4-Diarylpyrazoline Series of Compounds as Potent, Nonbrain Penetrant Antagonists of Cannabinoid-1 (CB1R) Receptor with Reduced Lipophilicity.

In the present report, we describe the synthesis and structure-activity relationships of novel "four-arm" dihydropyrazoline compounds designed as peripherally restricted antagonists of cannabinoid-1 receptor (CB1R). A series of racemic 3,4-diarylpyrazolines were synthesized and evaluated initially in CB1 receptor binding assays. The novel compounds, designed to limit brain penetrance and decreased lipophilicity, showed high affinity for CB1R and potent in vitro CB1R antagonist activities. Promising compounds with potent CB1R activity were evaluated in tissue distribution studies. Compounds 6a, 6f, and 7c showed limited brain penetrance attesting to its peripheral restriction. The 4S-enantiomer of these compounds further showed a stereoselective affinity for the CB1 receptor and behaved as inverse agonists. In vivo studies on food intake and body weight reduction in diet-induced obese (DIO) mice showed that these compounds could serve as potential leads for the development of selective CB1R antagonists with improved potency and peripheral restriction.

Dual inhibition of CB1 receptors and iNOS, as a potential novel approach to the pharmacological management of acute and long COVID-19.

COVID-19 (SARS-CoV-2) causes multiple inflammatory complications, resulting not only in severe lung inflammation but also harm to other organs. Although the current focus is on the management of acute COVID-19, there is growing concern about long-term effects of COVID-19 (Long Covid), such as fibroproliferative changes in the lung, heart and kidney. Therefore, the identification of therapeutic targets not only for the management of acute COVID-19 but also for preventing Long Covid are needed, and would mitigate against long-lasting health burden and economic costs, in addition to saving lives. COVID-19 induces pathological changes via multiple pathways, which could be targeted simultaneously for optimal effect. We discuss the potential pathologic function of increased activity of the endocannabinoid/CB1 receptor system and inducible NO synthase (iNOS). We advocate a polypharmacology approach, wherein a single chemical entity simultaneously interacts with CB1 receptors and iNOS causing inhibition, as a potential therapeutic strategy for COVID-19-related health complications. LINKED ARTICLES: This article is part of a themed issue on The second wave: are we any closer to efficacious pharmacotherapy for COVID 19? (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.10/issuetoc.

Targeting Endocannabinoid System in Epilepsy: For Good or for Bad.

Epilepsy is a neurological disorder with a high prevalence worldwide. Several studies carried out during the last decades indicate that the administration of cannabinoids as well as the activation of the endocannabinoid system (ECS) represent a therapeutic strategy to control epilepsy. However, there are controversial studies indicating that activation of ECS results in cell damage, inflammation and neurotoxicity, conditions that facilitate the seizure activity. The present review is focused to present findings supporting this issue. According to the current discrepancies, it is relevant to elucidate the different effects induced by the activation of ECS and determine the conditions under which it facilitates the seizure activity.

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

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.

Effects of a Peripherally Restricted Hybrid Inhibitor of CB1 Receptors and iNOS on Alcohol Drinking Behavior and Alcohol-Induced Endotoxemia.

Alcohol consumption is associated with gut dysbiosis, increased intestinal permeability, endotoxemia, and a cascade that leads to persistent systemic inflammation, alcoholic liver disease, and other ailments. Craving for alcohol and its consequences depends, among other things, on the endocannabinoid system. We have analyzed the relative role of central vs. peripheral cannabinoid CB1 receptors (CB1R) using a "two-bottle" as well as a "drinking in the dark" paradigm in mice. The globally acting CB1R antagonist rimonabant and the non-brain penetrant CB1R antagonist JD5037 inhibited voluntary alcohol intake upon systemic but not upon intracerebroventricular administration in doses that elicited anxiogenic-like behavior and blocked CB1R-induced hypothermia and catalepsy. The peripherally restricted hybrid CB1R antagonist/iNOS inhibitor S-MRI-1867 was also effective in reducing alcohol consumption after oral gavage, while its R enantiomer (CB1R inactive/iNOS inhibitor) was not. The two MRI-1867 enantiomers were equally effective in inhibiting an alcohol-induced increase in portal blood endotoxin concentration that was caused by increased gut permeability. We conclude that (i) activation of peripheral CB1R plays a dominant role in promoting alcohol intake and (ii) the iNOS inhibitory function of MRI-1867 helps in mitigating the alcohol-induced increase in endotoxemia.

National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: IV. The 2020 Highly morbid forms report.

Chronic graft-versus-host disease (GVHD) can be associated with significant morbidity, in part because of nonreversible fibrosis, which impacts physical functioning (eye, skin, lung manifestations) and mortality (lung, gastrointestinal manifestations). Progress in preventing severe morbidity and mortality associated with chronic GVHD is limited by a complex and incompletely understood disease biology and a lack of prognostic biomarkers. Likewise, treatment advances for highly morbid manifestations remain hindered by the absence of effective organ-specific approaches targeting "irreversible" fibrotic sequelae and difficulties in conducting clinical trials in a heterogeneous disease with small patient numbers. The purpose of this document is to identify current gaps, to outline a roadmap of research goals for highly morbid forms of chronic GVHD including advanced skin sclerosis, fasciitis, lung, ocular and gastrointestinal involvement, and to propose strategies for effective trial design. The working group made the following recommendations: (1) Phenotype chronic GVHD clinically and biologically in future cohorts, to describe the incidence, prognostic factors, mechanisms of organ damage, and clinical evolution of highly morbid conditions including long-term effects in children; (2) Conduct longitudinal multicenter studies with common definitions and research sample collections; (3) Develop new approaches for early identification and treatment of highly morbid forms of chronic GVHD, especially biologically targeted treatments, with a special focus on fibrotic changes; and (4) Establish primary endpoints for clinical trials addressing each highly morbid manifestation in relationship to the time point of intervention (early versus late). Alternative endpoints, such as lack of progression and improvement in physical functioning or quality of life, may be suitable for clinical trials in patients with highly morbid manifestations. Finally, new approaches for objective response assessment and exploration of novel trial designs for small populations are required.

CB1 R and iNOS are distinct players promoting pulmonary fibrosis in Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is a rare genetic disorder which, in its most common and severe form, HPS-1, leads to fatal adult-onset pulmonary fibrosis (PF) with no effective treatment. We evaluated the role of the endocannabinoid/CB1 R system and inducible nitric oxide synthase (iNOS) for dual-target therapeutic strategy using human bronchoalveolar lavage fluid (BALF), lung samples from patients with HPS and controls, HPS-PF patient-derived lung fibroblasts, and bleomycin-induced PF in pale ear mice (HPS1ep/ep ). We found overexpression of CB1 R and iNOS in fibrotic lungs of HPSPF patients and bleomycin-infused pale ear mice. The endocannabinoid anandamide was elevated in BALF and negatively correlated with pulmonary function parameters in HPSPF patients and pale ear mice with bleomycin-induced PF. Simultaneous targeting of CB1 R and iNOS by MRI-1867 yielded greater antifibrotic efficacy than inhibiting either target alone by attenuating critical pathologic pathways. Moreover, MRI-1867 treatment abrogated bleomycin-induced increases in lung levels of the profibrotic interleukin-11 via iNOS inhibition and reversed mitochondrial dysfunction via CB1 R inhibition. Dual inhibition of CB1 R and iNOS is an effective antifibrotic strategy for HPSPF.

Functional Selectivity of a Biased Cannabinoid-1 Receptor (CB1R) Antagonist.

Seven-transmembrane receptors signal via G-protein- and ß-arrestin-dependent pathways. We describe a peripheral CB1R antagonist (MRI-1891) highly biased toward inhibiting CB1R-induced ß-arrestin-2 (ßArr2) recruitment over G-protein activation. In obese wild-type and ßArr2-knockout (KO) mice, MRI-1891 treatment reduces food intake and body weight without eliciting anxiety even at a high dose causing partial brain CB1R occupancy. By contrast, the unbiased global CB1R antagonist rimonabant elicits anxiety in both strains, indicating no ßArr2 involvement. Interestingly, obesity-induced muscle insulin resistance is improved by MRI-1891 in wild-type but not in ßArr2-KO mice. In C2C12 myoblasts, CB1R activation suppresses insulin-induced akt-2 phosphorylation, preventable by MRI-1891, ßArr2 knockdown or overexpression of CB1R-interacting protein. MRI-1891, but not rimonabant, interacts with nonpolar residues on the N-terminal loop, including F108, and on transmembrane helix-1, including S123, a combination that facilitates ßArr2 bias. Thus, CB1R promotes muscle insulin resistance via ßArr2 signaling, selectively mitigated by a biased CB1R antagonist at reduced risk of central nervous system (CNS) side effects.