Optimization of Pharmacokinetic and In Vitro Safety Profile of a Series of Pyridine Diamide Indirect AMPK Activators

A set of focused analogues have been generated around a lead indirect adenosine monophosphate-activated kinase (AMPK) activator to improve the rat clearance of the molecule. Analogues were focused on inhibiting amide hydrolysis by the strategic placement of substituents that increased the steric environment about the secondary amide bond between 4-aminopiperidine and pyridine-5-carboxylic acid. It was found that placing substituents at position 3 of the piperidine ring and position 4 of the pyridine could all improve clearance without significantly impacting on-target potency. Notably, trans-3-fluoropiperidine 32 reduced rat clearance from above liver blood flow to 19 mL/min/kg and improved the hERG profile by attenuating the basicity of the piperidine moiety. Oral dosing of 32 activated AMPK in mouse liver and after 2 weeks of dosing improved glucose handling in a db/db mouse model of Type II diabetes as well as lowering fasted glucose and insulin levels

Optimization of Pharmacokinetic and In Vitro Safety Profile of a Series of Pyridine Diamide Indirect AMPK Activators

A set of focused analogues have been generated around a lead indirect adenosine monophosphate-activated kinase (AMPK) activator to improve the rat clearance of the molecule. Analogues were focused on inhibiting amide hydrolysis by the strategic placement of substituents that increased the steric environment about the secondary amide bond between 4-aminopiperidine and pyridine-5-carboxylic acid. It was found that placing substituents at position 3 of the piperidine ring and position 4 of the pyridine could all improve clearance without significantly impacting on-target potency. Notably, trans-3-fluoropiperidine 32 reduced rat clearance from above liver blood flow to 19 mL/min/kg and improved the hERG profile by attenuating the basicity of the piperidine moiety. Oral dosing of 32 activated AMPK in mouse liver and after 2 weeks of dosing improved glucose handling in a db/db mouse model of Type II diabetes as well as lowering fasted glucose and insulin levels

AMPK Activation through Mitochondrial Regulation Results in Increased Substrate Oxidation and Improved Metabolic Parameters in Models of Diabetes

<div><p>Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK <i>in vitro</i> via complex I inhibition at much lower concentrations than metformin (IC₅₀ 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production <i>in vitro</i> was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the <i>db/db</i> mouse diabetes model. <i>In vivo</i> metabolite profiling of R419-treated <i>db/db</i> mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both ¹³C-palmitate and ¹³C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO₂ in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function <i>in vivo</i>. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function <i>in vitro</i> and in diabetic animals <i>in vivo</i>. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models. </p> </div

Effects on fatty acid, BCAA, and amino acid pathways in <i>db/db</i> mice treated with R419.

<p>Male <i>db/db</i> mice (8 weeks old) were PO QD dosed with vehicle or 10 mg/kg R419. Thirty minutes after oral dosing, liver, muscle, and adipose tissues and plasma were collected at 3 days (n = 6) of compound administration and analyzed at Metabolon for metabolite profiles. <i>A</i>: Heat map of fatty acid oxidation metabolites in liver, muscle, adipose, and plasma from R419-treated mice shown relative to vehicle. <i>B</i>: Heat map of intermediates of branched chain amino acid catabolism in liver, muscle, adipose, and plasma from R419-treated mice shown relative to vehicle. <i>C</i>: Heat map of amino acids in liver, muscle, adipose, and plasma from R419-treated mice shown relative to vehicle. Biochemical data were analyzed using Welch’s two-sample t-tests.</p

Scheme illustrating possible metabolic route of ¹³C-glucose to ¹³CO₂ occurring in skeletal muscle from R419-treated <i>db/db</i> mice.

<p>Input glucose is broken down via glycolysis to pyruvate, which is decarboxylated to CO₂ and acetyl CoA. ¹³C-acetyl CoA can be condensed with oxaloacetate to form citrate, used to elongate short-chain fatty acids or routed for BHBA production. Skeletal muscle mitochondrial short chain fatty acid elongation utilizes acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, enoyl-CoA reductase, and NADH as a reducing agent. Although in general, R419-treatment resulted in reduction of palmitate and myristate levels in the skeletal muscle, these fatty acid pools were enriched in ¹³C-labeled palmitate and myristate. ¹³C-palmitate and ¹³C-myristate can be broken down by β-oxidation, releasing ¹³C-acetyl CoA, which can enter the TCA cycle and be released as ¹³CO₂ by either isocitrate dehydrogenase or α-ketoglutarate dehydrogenase after multiple cycles. Asterisks indicate a molecule is ¹³C-labeled. Dashed arrows indicate multistep conversions.</p

Effects on glucose metabolism in <i>db/db</i> mice treated with R419.

<p>Male <i>db/db</i> mice (8 weeks old) were PO QD dosed with vehicle, 5 mg/kg R419, or 10 mg/kg R419 for 8 days. On day 8, mice were given an intraperitoneal injection of 0.5 mg/kg [U-¹³C]-D-glucose 30 minutes after compound dosing. Liver, skeletal muscle, adipose, and plasma samples were collected at 60 and 90 minutes following glucose injection (n=4). <i>A</i>: ¹³CO₂ enrichment (¹³CO₂/¹²CO₂ ratio) in skeletal muscle, liver, and adipose. <i>B</i>: ¹³C-labeled palmitate or myristate enrichment (% of palmitate or myristate) and normalized palmitate or myristate measured following saponification of skeletal muscle acylglycerols and acylcarnitines. % of total fatty acid was obtained using the following formula, (integrated peak area of ¹³C-labeled fatty acid)/ (integrated peak area of ¹³C-labeled plus unlabeled fatty acid). Total fatty acid (labeled plus unlabeled) was normalized by tissue weight. The data are presented as mean (bar) ± SEM (line). Statistical analyses between an R419-treated group and the corresponding vehicle control were performed using the unpaired 2-tailed Student <i>t</i> test. Asterisks *, ***, #, and ## represent p < 0.05, p <0.001, p < 0.0001, and p < 0.00001 respectively for R419 treatment compared to vehicle.</p