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From Peripheralized to Cell- and Organelle-Targeted Medicine: The 3rd Generation of Cannabinoid-1 Receptor Antagonists for the Treatment of Chronic Kidney Disease

Periodic Reporting for period 4 - CaNANObinoids (From Peripheralized to Cell- and Organelle-Targeted Medicine: The 3rd Generation of Cannabinoid-1 Receptor Antagonists for the Treatment of Chronic Kidney Disease)

Reporting period: 2020-10-01 to 2021-09-30

Chronic kidney disease (CKD), a distinct manifestation of diabetic renal disease that is seen in patients with both type 1 and type 2 diabetes, is also a significant epidemiologic problem and is thought to affect around 8% of the European population. A decade ago, most attention was focused on glomerular changes related to CKD. Since then, there is growing evidence that proximal tubular and mitochondrial injuries are key features in the pathogenesis of this disease. This shift in paradigms will lead to new approaches for a cure.

This ERC-funded study introduces the novel hypothesis that activation of the cannabinoid-1 receptor (CB1R)/endocannabinoid (eCB) system explicitly in renal proximal tubule cells (RPTCs) and their mitochondria may lead to kidney dysfunction and to the development of CKD. Therefore, specific blockade of CB1Rs on these cells or organelles could serve as novel therapeutic strategies to treat CKD and its comorbidities.
The specific objectives are:
1. Characterize the contribution of RPTC CB1R to the pathogenesis of CKD
2. Elucidate the mechanism by which CB1R modulates mitochondrial function in RPTCs
3. Develop efficient and selective CB1R antagonists that target RPTCs and their mitochondria
To prove the hypothesis that proximal tubular CB1Rs are critically involved in the pathogenesis of obesity- or diabetes-induced CKD, we have utilized our novel mouse strain that lacks CB1R in the RPTCs (RPTC-CB1R-/-), and subjected the KO mice and their littermate controls to either obesity or diabetes. In both cases, the null mice develop the same degree of obesity or diabetes as their littermate controls, BUT their kidney was completely protected from the development of dysfunction, inflammation and fibrosis.

Next, we elucidated the downstream cellular pathways involved in CB1R-induced CKD during obesity or diabetes. Our findings show that during obese conditions CB1R governs intracellular lipid accumulation in the RPTCs by modulating the PKA/LKB1/AMPK/ACC signaling pathway. During diabetes conditions, CB1R regulates Ca2+-dependent PKC-β1 activation, which, in turn, modulates the expression and translocation of the facilitative glucose transporter 2 (GLUT2) in RPTCs. We also revealed the existence of a CB1R/mTORC1 signaling axis in RPTCs and its importance in regulating kidney function in health and disease.

We also reported a novel kidney-to-bone axis modulated via RPTC-CB1R. Specifically, we showed that nullification of CB1R from the RPTCs preserved bone mass under hyperglycemic conditions via affecting both osteoclastogenesis and bone formation mediated by erythropoietin (EPO).

In addition, we have found that stimulating CB1R results in mitochondrial fragmentation in RPTCs, and this effect is associated with the progression of obesity-induced CKD, and most likely related to the increased phosphorylation of dynamin-related protein 1 (DRP-1) that regulates mitochondrial fission. CB1R-induced mitochondrial fission was found to be associated with mitochondrial dysfunction, as documented by reduced oxygen consumption and ATP production, increased reactive oxygen species and cellular lactate levels, as well as a decline in mitochondrial biogenesis.

Moreover, we show that pharmacological activation/blockade and genetic overexpression/deletion of hepatic CB1R modulates soluble leptin receptor (sOb-R) levels and hepatic leptin resistance. Interestingly, peripheral CB1R blockade failed to reverse diet-induced reduction of sOb-R levels, increased fat mass and dyslipidemia, and hepatic steatosis in mice lacking C/EBP homologous protein (CHOP), whereas direct activation of CB1R in wild-type hepatocytes reduced sOb-R levels in a CHOP-dependent manner.

Taken together, we have established the first complete view of the involvement of proximal tubular CB1R in the development of CKD, and delineated the cascade of events underlying the activation of CB1R in RPTCs that lead to diabetic renal dysfunction. In addition, we have uncovered a new role for CB1R as a direct modulator of mitochondrial function in RPTCs by regulating mitochondrial shape, biogenesis integrity and membrane physiology as well as identified its role in regulating bone remodeling during diabetes. Additionally, we highlight a novel molecular aspect by which the hepatic eCB/CB1R system is involved in the development of hepatic leptin resistance and in the regulation of sOb-R levels via CHOP.
Our achievements so far have paved the way for us to try to use novel methodologies to identify new molecules and/or drug delivery systems which would allow us to target the CB1R for the treatment of CKD and metabolic diseases (obesity, diabetes, fatty liver). Therefore, we are currently trying to design novel, efficient and selective CB1R antagonists that specifically target the diseases organ or cell by utilizing two methodologies: (i) Nanotechnology (e.g. polymeric nanoparticles, liposomes, micelles), as nanoscaled drug delivery systems (DDSs) , and (ii) Virtual Screening, the need for methods enabling faster discovery of effective drug candidates is obvious.Towards that goal, we use a computerized platform for screening and finding novel and diverse bioactive molecules that interact with the CB1R in peripheral organs.

In the 1st methodology, we developed a novel DDS for repurposing the abandoned first-in-class centrally acting and water-insoluble CB1R blocker rimonabant for the treatment of NAFLD and T2D. We hypothesized that encapsulating rimonabant in polymeric nanoparticles for its peripheralization distribution would allow us to create the "next generation" of drugs that target the CB1R receptor only in peripheral organs, such as the liver, without the side effects associated with blocking the same receptor in the brain. Indeed, by using a nano-DDS, we were able to efficiently deliver rimonabant at a high dose to the liver, reduce its toxic centrally mediated side effects, and demonstrate its therapeutic potential in reducing obesity-induced hepatic steatosis and liver injury, improving insulin sensitivity, and reversing hypertriglyceridemia. Our study has important translational/therapeutic implications, since it further supports the clinical evaluation of peripheral CB1R antagonists to treat obese individuals and combat the obesity epidemic and its sequelae. It also offers a unique way to repurpose old drugs by reducing their CNS-targeted toxicity and to support their immediate translation into clinical testing.

In the 2nd methodology employed here, we applied our in-house algorithm, iterative stochastic elimination, to produce a ligand-based model that distinguishes between CB1R antagonists and random molecules by physicochemical properties only. We screened ∼2 million commercially available molecules and found that about 500 of them are potential candidates to antagonize the CB1R. We applied a few criteria for peripheral activity and narrowed that set down to 30 molecules, out of which 15 could be purchased. Ten out of those 15 showed good affinity to the CB1R and two of them with nanomolar affinities (Ki of ∼400 nM). The eight molecules with top affinities were tested for activity: two compounds were pure antagonists, and five others were inverse agonists. These molecules are currently being examined in vivo for their peripheral versus central distribution and subsequently will be tested for their effects on obesity in small animals.
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