A Highly Potent, Orally Bioavailable Pyrazole-Derived Cannabinoid CB2 Receptor- Selective Full Agonist for In Vivo Studies.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein-mediated efflux from the brain. 3H and 14C labeled RNB-61 showed apparent K d values of <4 nM toward human CB2R in both cell and tissue experiments. The 6,800-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

Targeting Oxidative Stress in Neurodegenerative Disorders: A Novel Role for PCSK9 Inhibition?

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protein that regulates cholesterol levels by lysosomal low-density lipoprotein receptor (LDLR) degradation and has recently been associated with the production of neuronal oxidative stress and age-associated cardiovascular dysfunction. Since increased oxidative stress and vascular dysfunction are implicated in the pathology of aging and various neurodegenerative disorders, targeting PCSK9 may offer a promising therapeutic avenue for addressing these conditions. While the precise mechanisms through which PCSK9 contributes to vascular and neuronal oxidative stress in the brain remain elusive, preclinical studies have highlighted a neuroprotective effect linked to PCSK9 inhibition. This inhibition has shown promise in reducing oxidative stress, mitigating neuroinflammation, and alleviating neuropathological changes, thus underscoring the therapeutic potential of this approach in addressing neurodegenerative conditions.

Coordinated action of a gut-liver pathway drives alcohol detoxification and consumption.

Alcohol use disorder (AUD) affects millions of people worldwide, causing extensive morbidity and mortality with limited pharmacological treatments. The liver is considered as the principal site for the detoxification of ethanol metabolite, acetaldehyde (AcH), by aldehyde dehydrogenase 2 (ALDH2) and as a target for AUD treatment, however, our recent data indicate that the liver only plays a partial role in clearing systemic AcH. Here we show that a liver-gut axis, rather than liver alone, synergistically drives systemic AcH clearance and voluntary alcohol drinking. Mechanistically, we find that after ethanol intake, a substantial proportion of AcH generated in the liver is excreted via the bile into the gastrointestinal tract where AcH is further metabolized by gut ALDH2. Modulating bile flow significantly affects serum AcH level and drinking behaviour. Thus, combined targeting of liver and gut ALDH2, and manipulation of bile flow and secretion are potential therapeutic strategies to treat AUD.

A highly potent, orally bioavailable pyrazole-derived cannabinoid CB2 receptor-selective full agonist for in vivo studies.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent K d values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

Patent review of cannabinoid receptor type 2 (CB2R) modulators (2016-present).

INTRODUCTION: Cannabinoid receptor type 2 (CB2R), predominantly expressed in immune tissues, is believed to play a crucial role within the body's protective mechanisms. Its modulation holds immense therapeutic promise for addressing a wide spectrum of dysbiotic conditions, including cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, and autoimmune diseases, as well as lung disorders, cancer, and pain management. AREAS COVERED: This review is an account of patents from 2016 up to 2023 which describes novel CB2R ligands, therapeutic applications, synthesis, as well as formulations of CB2R modulators. EXPERT OPINION: The patents cover a vast, structurally diverse chemical space. The focus of CB2R ligand development has shifted from unselective dual-cannabinoid receptor type 1 (CB1R) and 2 agonists toward agonists with high selectivity over CB1R, particularly for indications associated with inflammation and tissue injury. Currently, there are at least eight CB2R agonists and one antagonist in active clinical development. A better understanding of the endocannabinoid system (ECS) and in particular of CB2R pharmacology is required to unlock the receptor's full therapeutic potential.

Mitochondrial fatty acid beta-oxidation: a possible therapeutic target for skeletal muscle lipotoxicity in peripheral artery disease myopathy.

Peripheral artery disease (PAD) is an atherosclerotic disease impacting over 200 million individuals and the prevalence increases with age. PAD occurs when plaque builds up within the peripheral arteries, leading to reduced blood flow and oxygen supply to the outer extremities. Individuals who experience PAD suffer from ischemia, which is typically accompanied by significant damage to skeletal muscles. Additionally, this tissue damage affects mitochondria, causing them to become dysregulated and dysfunctional, resulting in decreased metabolic rates. As there is no known cure for PAD, researchers are exploring potential therapeutic targets by examining coexisting cardiovascular conditions and metabolic risk factors, such as the aging process. Among these comorbidities, type-two diabetes mellitus and obesity are particularly common in PAD cases. These conditions, along with aging itself, are associated with an elevated accumulation of ectopic lipids within skeletal muscles, similar to what is observed in PAD. Researchers have attempted to reduce excess lipid accumulation by increasing the rate of fatty acid beta oxidation. Manipulating acetyl coenzyme A carboxylase 2, a key regulatory protein of fatty acid beta oxidation, has been the primary focus of such research. When acetyl coenzyme A carboxylase 2 is inhibited, it interrupts the conversion of acetyl-CoA into malonyl-CoA, resulting in an increase in the rate of fatty acid beta oxidation. By utilizing samples from PAD patients and applying the pharmacological strategies developed for acetyl coenzyme A carboxylase 2 in diabetes and obesity to PAD, a potential new therapeutic avenue may emerge, offering hope for improved quality of life for individuals suffering from PAD.

PCSK9 inhibition attenuates alcohol-associated neuronal oxidative stress and cellular injury.

Alcohol Use Disorder (AUD) is a persistent condition linked to neuroinflammation, neuronal oxidative stress, and neurodegenerative processes. While the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has demonstrated effectiveness in reducing liver inflammation associated with alcohol, its impact on the brain remains largely unexplored. This study aimed to assess the effects of alirocumab, a monoclonal antibody targeting PCSK9 to lower systemic low-density lipoprotein cholesterol (LDL-C), on central nervous system (CNS) pathology in a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for six weeks in 32 male rats subjected to a 35 % ethanol liquid diet or a control liquid diet (n = 8 per group). The study evaluated PCSK9 expression, LDL receptor (LDLR) expression, oxidative stress, and neuroinflammatory markers in brain tissues. Chronic ethanol exposure increased PCSK9 expression in the brain, while alirocumab treatment significantly upregulated neuronal LDLR and reduced oxidative stress in neurons and brain vasculature (3-NT, p22phox). Alirocumab also mitigated ethanol-induced microglia recruitment in the cortex and hippocampus (Iba1). Additionally, alirocumab decreased the expression of pro-inflammatory cytokines and chemokines (TNF, CCL2, CXCL3) in whole brain tissue and attenuated the upregulation of adhesion molecules in brain vasculature (ICAM1, VCAM1, eSelectin). This study presents novel evidence that alirocumab diminishes oxidative stress and modifies neuroimmune interactions in the brain elicited by chronic ethanol exposure. Further investigation is needed to elucidate the mechanisms by which PCSK9 signaling influences the brain in the context of chronic ethanol exposure.

NASH triggers cardiometabolic HFpEF in aging mice.

Both heart failure with preserved ejection fraction (HFpEF) and non-alcoholic fatty liver disease (NAFLD) develop due to metabolic dysregulation, has similar risk factors (e.g., insulin resistance, systemic inflammation) and are unresolved clinical challenges. Therefore, the potential link between the two disease is important to study. We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age dependent effects of NASH on cardiac function. C57Bl/6 J middle aged (10 months old) and aged mice (24 months old) were fed either control or choline deficient (CDAA) diet for 8 weeks. Before termination, echocardiography was performed. Upon termination, organ samples were isolated for histological and molecular analysis. CDAA diet led to the development of NASH in both age groups, without inducing weight gain, allowing to study the direct effect of NASH on cardiac function. Mice with NASH developed hepatomegaly, fibrosis, and inflammation. Aged animals had increased heart weight. Conventional echocardiography revealed normal systolic function in all cohorts, while increased left ventricular volumes in aged mice. Two-dimensional speckle tracking echocardiography showed subtle systolic and diastolic deterioration in aged mice with NASH. Histologic analyses of cardiac samples showed increased cross-sectional area, pronounced fibrosis and Col1a1 gene expression, and elevated intracardiac CD68+ macrophage count with increased Il1b expression. Conventional echocardiography failed to reveal subtle change in myocardial function; however, 2D speckle tracking echocardiography was able to identify diastolic deterioration. NASH had greater impact on aged animals resulting in cardiac hypertrophy, fibrosis, and inflammation.