Efficacy in diet-induced obese mice of the hepatotropic, peripheral cannabinoid 1 receptor inverse agonist TM38837.

BACKGROUND AND PURPOSE: The cannabinoid CB1 receptor has a well-established role in appetite regulation. Drugs antagonizing central CB1 receptors, most notably rimonabant, induced weight loss and improved the metabolic profile in obese individuals but were discontinued due to psychiatric side effects. However, metabolic benefits were only partially attributable to weight loss, implying a role for peripheral receptors, and peripherally restricted CB1 receptor antagonists have since been of interest. Herein, we describe the evaluation of the peripherally restricted potent CB1 receptor inverse agonists TM38837 and TM39875, with acidic functionality, which were administered daily to diet-induced obese (DIO) mice for 5 weeks at doses for which CNS-mediated effects were minimal. EXPERIMENTAL APPROACH: Compounds were tested in dose-response in acute studies to compare efficacy (gastric transport) and extent of CNS exposure (hypothermia and satiety sequence) to demonstrate peripheral restriction and select doses for the subsequent chronic DIO study. KEY RESULTS: TM38837 but not TM39875 produced considerable (26%) weight loss, linked to a sustained reduction in food intake, together with improvements in plasma markers of inflammation and glucose homeostasis. Pharmacokinetic analysis indicated high plasma and low brain levels for both compounds with high liver levels for TM38837 (but not TM39875) due to hepatic uptake. CONCLUSION AND IMPLICATIONS: Weight loss and metabolic benefits of TM38837 are likely not CNS-mediated but could be linked to enhanced liver exposure, which implicates intracellular CB1 receptors in hepatocytes as a possible driver of obesity and co-morbidities.

A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation.

The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

Immunohistochemical analysis of cannabinoid receptor 1 expression in steatotic rat livers.

The primary aim of the present study was to determine the expression levels of cannabinoid receptor type 1 (CB1) in steatotic rat livers. The secondary aim was to clarify whether steatosis and inflammation are more marked in areas with increased CB1 overexpression. For ethical and economic reasons, the present study investigated tissue from archived liver blocks, which were obtained from 38 rats that had been euthanized during the course of previous research at the Karolinska Institute of the Karolinska University Hospital (Stockholm, Sweden) and Lund University (Malmö, Sweden). Liver tissue fixed in formalin and embedded in paraffin was used that had been sourced from 36 male Sprague Dawley rats (age, 7 weeks) and 2 rats (age, 180 days) lacking normal leptin receptors. The rat liver tissue was stained with antibodies against CB1 and counterstained with hematoxylin. The expression of CB1 and the number of cells overexpressing CB1 were determined. Steatosis was scored according to the Dixon scoring system. CB1 overexpression and steatosis were detected in hepatocytes from all 38 livers sampled. The expression of CB1 was more marked in hepatocytes localized next to portal triads. Near the central veins, the expression was significantly weaker. Steatosis was more marked in areas of increased CB1 overexpression. Lymphocyte infiltration was more commonly observed in areas of increased CB1 overexpression. Therefore, the present results indicate that CB1 receptors are overexpressed in areas with steatosis, and indicate that CB1 in hepatocytes contributes to the formation of steatosis in rats, even prior to its progression to steatohepatitis. These results are consistent with publications reporting that CB1 in hepatocytes increases lipogenesis and contributes to inflammation.

Structure activity relationships of fused bicyclic and urea derivatives of spirocyclic compounds as potent CCR1 antagonists.

A series of fused bicyclic and urea derivatives of spirocyclic compounds were designed, synthesised and evaluated in vitro as potent CCR1 antagonists. In particular, 4 (7nM), 44 (1.3nM), 48 (0.89nM) and 50 (0.63nM) were the most potent hCCR1 antagonists in this series of compounds. Moreover, some of these substances demonstrated good rodent cross-over, especially 46 which exhibited very high rat CCR1 binding affinity with an IC50 value of 16nM.

The hepatic cannabinoid 1 receptor as a modulator of hepatic energy state and food intake.

The cannabinoid 1 receptor (CB1R) has a well-established role in appetite regulation. Central CB1R antagonists, notably rimonabant, induced weight loss and improved the metabolic profile in obese individuals, but were discontinued due to psychiatric side-effects. The CB1R is also expressed peripherally, where its effects include promotion of liver fat accumulation, which consumes ATP. Type 2 diabetes in obese subjects is linked to excess liver fat, whilst there is a negative correlation between hepatic ATP content and insulin resistance. A decreased hepatic ATP/AMP ratio increases food intake by signals via the vagus nerve to the brain. The hepatic cannabinoid system is highly upregulated in obesity, and the effects of hepatic CB1R activation include increased activity of lipogenic and gluconeogenic transcription factors. Thus, blockade of hepatic CB1Rs could contribute significantly to the weight-reducing and insulin-sensitizing effects of CB1R antagonists. Additionally, upregulation of the hepatic CB1R may contribute to chronic liver inflammation, fibrosis and cirrhosis from causes including obesity, alcoholism and viral hepatitis. Peripheral CB1R antagonists induce weight loss and metabolic improvements in obese rodents; however, as there is evidence that hepatic CB1Rs are predominately intracellular, due to high intrinsic clearance, many drugs may not effectively block these receptors and therefore have limited efficacy. Hepatoselective CB1R antagonists may be effective at reducing hepatic steatosis, insulin resistance and bodyweight in obese, diabetic patients, with far fewer side-effects than first-generation CB1R antagonists. Additionally, such compounds may be effective in treating inflammatory liver disease, such as non-alcoholic steatohepatitis, reducing the likelihood of disease progression to cirrhosis or cancer.

Design, synthesis and structure activity relationships of spirocyclic compounds as potent CCR1 antagonists.

A series of CCR1 antagonists based upon spirocyclic compounds 1b and 2b were synthesised in which substituted aniline moiety was replaced with substituted benzamides. In vitro data revealed that CCR1 potency could be retained in such compounds.

Optimization of monocarboxylate transporter 1 blockers through analysis and modulation of atropisomer interconversion properties.

We have previously described a novel series of potent blockers of the monocarboxylate transporter, MCT1, which show potent immunomodulatory activity in an assay measuring inhibition of PMA/ionomycin-induced human PBMC proliferation. However, the preferred compounds had the undesirable property of existing as a mixture of slowly interconverting rotational isomers. Here we show that variable temperature NMR is an effective method of monitoring how alteration to the nature of the amide substituent can modulate the rate of isomer exchange. This led to the design of compounds with increased rates of rotamer interconversion. Moreover, some of these compounds also showed improved potency and provided a route to further optimization.