Structure activity relationships leading to the identification of the indirect activator of AMPK, R419.

Using an in-cell AMPK activation assay, we have developed structure-activity relationships around a hit pyridine dicarboxamide 5 that resulted in 40 (R419). A particular focus was to retain the on-target potency while also improving microsomal stability and reducing off-target activities, including hERG inhibition. We were able to show that removing a tertiary amino group from the piperazine unit of hit compound 5 improved microsomal stability while hERG inhibition was improved by modifying the substitution of the central core pyridine ring. The SAR resulted in 40, which continues to maintain on-target potency. Compound 40 was able to activate AMPK in vivo after oral administration and showed efficacy in animal models investigating activation of AMPK as a therapy for glucose control (both db/db and DIO mouse models).

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.

Metabolism of fostamatinib, the oral methylene phosphate prodrug of the spleen tyrosine kinase inhibitor R406 in humans: contribution of hepatic and gut bacterial processes to the overall biotransformation.

The metabolism of the spleen tyrosine kinase inhibitor N4-(2,2-dimethyl-3-oxo-4-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine (R406) and its oral prodrug N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine disodium hexahydrate (R788, fostamatinib) was determined in vitro and in humans. R788 was rapidly converted to R406 by human intestinal microsomes, and only low levels of R788 were observed in plasma of human subjects after oral administration of (14)C-R788. R406 was the major drug-related compound in plasma from human subjects, and only low levels of metabolites were observed in plasma. The plasma metabolites of R406 were identified as a sulfate conjugate and glucuronide conjugate of the para-O-demethylated metabolite of R406 (R529) and a direct N-glucuronide conjugate of R406. Elimination of drug-related material into the urine accounted for 19% of the administered dose, and the major metabolite in urine from all the human subjects was the lactam N-glucuronide of R406. On average, 80% of the total drug was recovered in feces. Two drug-related peaks were observed; one peak was identified as R406, and the other peak was identified as a unique 3,5-benzene diol metabolite of R406. The 3,5-benzene diol metabolite appeared to result from the subsequent O-demethylations and dehydroxylation of R529 by anaerobic gut bacteria because only R529 was converted to this metabolite after the in vitro incubation with human fecal samples. These data indicate that the major fecal metabolite of R406 observed in humans is a product of a hepatic cytochrome P450-mediated O-demethylation and subsequent O-demethylations and dehydroxylation by gut bacteria.

AMPK activation through mitochondrial regulation results in increased substrate oxidation and improved metabolic parameters in models of diabetes.

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 in vitro 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 in vitro 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 db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both (13)C-palmitate and (13)C-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 in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. 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.

Single- and multiple-dose disposition kinetics of sunitinib malate, a multitargeted receptor tyrosine kinase inhibitor: comparative plasma kinetics in non-clinical species.

PURPOSE: The purpose of these extensive non-clinical studies was to assess pharmacokinetics and dispositional properties of sunitinib and its primary active metabolite (SU12662). METHODS: Sunitinib was administered in single and repeat oral doses in mice, rats, and monkeys. Assessments were made using liquid-chromatography-tandem mass spectrometric methods, radioactive assays, and quantitative whole body autoradiography. RESULTS: Sunitinib was readily absorbed with good oral bioavailability and linear kinetics at clinically-relevant doses. SU12662 plasma levels were less than those of sunitinib in mice and monkeys, but greater in rats. Sunitinib was extensively distributed with moderate-to-high systemic clearance and eliminated primarily into feces. Single- and repeat-dosing kinetics were similar. A prolonged half-life allowed once-daily dosing, enabling adequate systemic exposure with limited-to-moderate accumulation. In multiple-dose studies with cyclic dosing, drug plasma concentrations cleared from one cycle to the next. CONCLUSIONS: Sunitinib exhibited advantageous pharmacokinetic and dispositional properties in non-clinical species, translating into favorable properties in humans.