Hepatic targeting of the centrally active cannabinoid 1 receptor (CB1R) blocker rimonabant via PLGA nanoparticles for treating fatty liver disease and diabetes

Abstract

Over-activation of the endocannabinoid/CB₁R system is a hallmark feature of obesity and its related comorbidities, most notably type 2 diabetes (T2D), and non-alcoholic fatty liver disease (NAFLD). Although the use of drugs that widely block the CB₁R was found to be highly effective in treating all metabolic abnormalities associated with obesity, they are no longer considered a valid therapeutic option due to their adverse neuropsychiatric side effects. Here, we describe a novel nanotechnology-based drug delivery system for repurposing the abandoned first-in-class global CB₁R antagonist, rimonabant, by encapsulating it in polymeric nanoparticles (NPs) for effective hepatic targeting of CB₁Rs, enabling effective treatment of NAFLD and T2D. Rimonabant-encapsulated NPs (Rimo-NPs) were mainly distributed in the liver, spleen, and kidney, and only negligible marginal levels of rimonabant were found in the brain of mice treated by iv/ip administration. In contrast to freely administered rimonabant treatment, no CNS-mediated behavioral activities were detected in animals treated with Rimo-NPs. Chronic treatment of diet-induced obese mice with Rimo-NPs resulted in reduced hepatic steatosis and liver injury as well as enhanced insulin sensitivity, which were associated with enhanced cellular uptake of the formulation into hepatocytes. Collectively, we successfully developed a method of encapsulating the centrally acting CB₁R blocker in NPs with desired physicochemical properties. This novel drug delivery system allows hepatic targeting of rimonabant to restore the metabolic advantages of blocking CB₁R in peripheral tissues, especially in the liver, without the negative CB₁R-mediated neuropsychiatric side effects.

Keywords: CB1 receptor blocker; Drug delivery system; Hepatic steatosis; Insulin resistance; Nanomedicine; Obesity; Polymeric nanoparticles.

Graphical abstract

Fig. 1

The physicochemical properties of Rimo-NPs. (a) Formulation. (b) Transmission electron microscopy (TEM) micrographs of Rimo-NPs immediately after lyophilization (Uranyl acetate negative staining, magnification 25 K, scale bar = 500 nm). (c) Long-term stability (size and content) of lyophilized Rimo-NPs stored at −20 °C for 2 years. (d) Stability of Rimo-NPs in terms of size following incubation in 10% FBS solution in comparison to PBS solution. Data represent the mean ± SEM from 3 independent formulations per condition.

Fig. 2

Rimo-NP biodistribution in mice. (a) Rimo-NPs (Cy5-PLGA labeled) were administered ip to C57Bl/6JHsd mice, and examined 1, 4, 8, and 24 h after injection. Representative organ micrographs harvested 1 h after treatment (left column) and scanning micrographs by means of a Typhoon scanner (right columns). (b) The quantified fluorescence intensities in each organ are normalized to unlabeled NP-treated animals. (c) The accumulation of rimonabant in organs was also evaluated by analyzing the rimonabant levels in the liver, kidney, spleen, fat, lung, and brain 1 h post-injection (1 mg/kg, ip or iv). Data represent the mean ± SEM of 3 mice per group. *p < 0.05 relative to free rimonabant levels in the same tissue and the route of administration.

Fig. 3

Rimo-NP uptake by liver cells. (a) FACS gating strategy demonstrating the increased accumulation of Rimo-NPs in the primary hepatocytes containing the parenchymal fraction (CD45-negative cells) as well as in NPCs (CD45-positive cells). (b) Representative images of Rimo-NP (Cy5-PLGA labeled) uptake by liver hepatocytes (arrows) in mice 1 h post-treatment (ip; 140 mg/kg NPs, 200 μL). (c) Representative image of Rimo-NP (Cy5-PLGA labeled) uptake by Kupffer cells (arrows) or non-Kupffer cells (arrowheads) in mice 1 h post-treatment (ip; 140 mg/kg NPs, 200 μL). For b and c, representative confocal microscopy images are shown [red, Rimo-NPs; green, hepatocytes (albumin-positive cells); dark yellow, Kupffer cells (F4/80-positive cells)], the co-localization of the NPs in hepatocytes or Kupffer cells is shown in the merged images. Magnification x60, scale bar = 20 μm. (d) Qualitative and (e) quantitative assessments of Rimo-NP (Cy5-PLGA labeled) uptake by primary mouse hepatocytes in culture [green, Cy5; blue, nuclei (DAPI)]. The fluorescence intensities are normalized to cells treated with unlabeled NPs; magnification x60, scale bar = 25 μm. Data represent the mean ± SEM of five independent experiments, *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Rimo-NPs do not induce centrally mediated side effects. (a) Free rimonabant, but not Rimo-NPs increased the ambulatory activity both after ip and iv administration of 1 mg/kg. Data represent the mean ± SEM of 4 mice per group. *p < 0.05 relative to vehicle (Veh; free PLGA-NPs), ^#p < 0.05 relative to free rimonabant. (b) Free rimonabant at 1 and 10 mg/kg ip, but not Rimo-NPs at 1 mg/kg ip, inhibited WIN-55,212 (3 mg/kg, ip)-induced catalepsy as measured by the bar assay. Data represent the mean ± SEM from 7 to 26 mice per group. *p < 0.05 relative to the corresponding vehicle (4% DMSO, 1% Tween80, 95% saline) w/o WIN-55,212, ^#p < 0.05 relative to the corresponding vehicle with WIN-55,212. (c, d) Free rimonabant at 10 mg/kg ip, but not Rimo-NPs or free rimonabant, both at 1 mg/kg ip, induced an anxiogenic effect in the EPM. Data represent the mean ± SEM from 10 to 24 mice per group. *p < 0.05 relative to the corresponding vehicle (4% DMSO, 1% Tween80, 95% saline), ^#p < 0.05 relative to free rimonabant at 10 mg/kg. (e, f) Free rimonabant at 10 mg/kg ip, but not Rimo-NPs or free rimonabant at 1 mg/kg ip, inhibited acute food and water intakes. Data represent the mean ± SEM from 4 mice per group. *p < 0.05 relative to the vehicle (Veh; free PLGA-NPs), ^#p < 0.05 relative to free rimonabant at 10 mg/kg.

Fig. 5

Metabolic effects of chronic treatment with free rimonabant or Rimo-NPs in DIO mice. Mice on STD or HFD for 14 weeks were treated with vehicle (Veh; free PLGA-NPs) or 1 mg/kg/d, ip, of free rimonabant or Rimo-NPs for 28 days. Free rimonabant significantly reduced body weight (a) and fat mass (b), increased lean mass (c), without affecting the food intake (d). Serum leptin levels were significantly reduced by free rimonabant and to a lesser extent by Rimo-NPs (e). Indirect calorimetry assessment over a 12 h period in the dark period revealed that free rimonabant but not Rimo-NPs resulted in an upregulation of oxygen consumption (VO₂; f), total energy expenditure (TEE; g), and fat oxidation (FO; h) without affecting the ambulatory activity (i), voluntary activity (j), and the total distance the mice traveled in the cage (k). Data represent the mean ± SEM from 7 mice per group, *p < 0.05 relative to STD-Veh, ^#p < 0.05 relative to HFD-Veh, ^$p < 0.05 relative to HFD-free rimonabant.

Fig. 6

Weight-independent effects of Rimo-NPs in ameliorating obesity-induced dyslipidemia and hepatic steatosis. Mice on STD or HFD for 14 weeks were treated with vehicle (Veh; free PLGA-NPs) or 1 mg/kg/d, ip, of free rimonabant or Rimo-NPs for 28 days. (a) Serum triglycerides. (b) Serum total cholesterol. (c) The HDL-to-LDL ratio. Both free rimonabant and Rimo-NPs significantly reversed the HFD-induced hepatic steatosis, as measured by reductions in liver weight (d), liver-to-body weight ratio (e), hepatic triglyceride content (f), and circulating ALT levels (g). Fat deposition assessed by H&E (i) and Oil Red O (j) staining and quantification (h). Scale bar = 20 or 100 μm for H&E staining and 20 or 50 μm for Oil Red-O staining, as indicated on the images. Data represent the mean ± SEM from 7 mice per group, *p < 0.05 relative to STD-Veh, ^#p < 0.05 relative to HFD-Veh, ^$p < 0.05 relative to HFD-free rimonabant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Weight-independent effects of Rimo-NPs in ameliorating obesity-induced insulin resistance. Mice on STD or HFD for 14 weeks were treated with vehicle (Veh; free PLGA-NPs) or 1 mg/kg/d, ip, of free rimonabant or Rimo-NPs for 28 days. Free rimonabant, but not Rimo-NPs, reversed obesity-induced glucose intolerance (a-c). Both Rimo-NPs and free rimonabant improved insulin sensitivity (d-e), reduced hyperinsulinemia (f), HOMA-IR (g), and ISI (h). Data represent the mean ± SEM from 7 mice per group, *p < 0.05 relative to STD-Veh, ^#p < 0.05 relative to HFD-Veh, ^$p < 0.05 relative to HFD-free rimonabant.