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    • PF is orally bioavailable and has appropriate pharmacokineti

    2023-05-29

    PF-06409577 is orally bioavailable and has appropriate pharmacokinetic properties for use in rodents, allowing us to evaluate its impact on lipid and cholesterol biosynthesis in vivo (Cameron et al., 2016). PF-06409577 lowered the incorporation of 14C-acetate into hepatic lipids in vivo in a dose dependent manner (Fig. 2E). Plasma mevalonic acid, a direct product of HMGCR, was also shown to be reduced acutely following a single dose of PF-06409577, similar to the HMGCR inhibitor Rosuvastatin (Fig. 2F). We evaluated PF-06409577 in a rodent model of diet induced metabolic syndrome that lacked AMPK specifically in the hepatocytes, allowing us to understand which drug actions were the result of hepatocyte AMPK activation. AMPK α1/α2 mice were fed a high fat diet for an extended period of 24weeks prior to infection with adeno-associated virus expressing either GFP or Cre specifically in the hepatocytes. Livers from mice infected with Cre virus (AMPK-HepKO) showed a near complete removal of total and phospho-threonine 172 AMPK alpha protein, as well as a decrease in the levels of the AMPK phosphorylation site on ACC, serine 79 (Fig. 3A), indicating efficient deletion of the AMPK α1 and α2 Methyllycaconitine citrate australia following viral expression of Cre recombinase. Unbound plasma concentrations of PF-06409577 were measured in these animals and averaged 20nM 4h after being dosed. We did not observe a change following AMPK knockout or activation with PF-06409577 in the reported AMPK-mediated phosphorylation site on SREBP1, serine 372 (Fig. 3A). AMPK HepKO and control mice were both treated with PF-06409577 for either one dose of 100mg/kg or 42days of consecutive once daily oral dosing of 100mg/kg, and sacrificed 4h after treatment for evaluation of the impact of drug action. PF-06409577 caused AMPK activation after acute (Fig. 3B-C) and chronic dosing (Fig. 3D-E), as determined by measurement of AMPK phosphorylation at Thr172 and ACC phosphorylation at Ser79 using antibody based MesoScale Diagnostics (MSD) assays which function similar to ELISAs. Despite the daily activation of AMPK for 42days there was no impact of PF-06409577 on body weight, body composition, or glucose and insulin levels before and after a glucose tolerance challenge, results consistent with studies using a similar AMPK β1 biased activator (Supplemental Fig. 2) (Cokorinos et al., 2017). PF-06409577 had a large impact on hepatic lipids, in contrast to the modest impact on weight and glycemic traits. PF-06409577 treatment resulted in lower hepatic triacylglycerol (TAG) and hepatic cholesterol in the control AAV-GFP infected animals (Fig. 3F-G). In AMPK HepKO animals there was no impact of PF-06409577 on liver TAG or cholesterol (Fig. 3F-G). Staining of liver samples with Oil Red O and histological analysis of stain area confirmed these results (Supplemental Fig. 2I-J). Plasma levels of the alanine liver transaminase (ALT), a marker for general liver health that is elevated in NAFLD and NASH, was also lowered in control AAV-GFP infected animals treated with PF-06409577, but not AMPK HepKO animals, consistent with the general reduction in hepatic lipid content (Fig. 3H). These effects suggested the benefit of PF-06409577 treatment resulted from AMPK activation in hepatocytes and not from other locations such as the adipose tissue or macrophages. Evaluation of systemic circulating lipids revealed only modest changes following chronic PF-06409577 treatment, in contrast to the large impact on hepatic lipid levels. Nonetheless, a significant reduction in circulating triglycerides was observed following PF-06409577 treatment in control but not AMPK HepKO mice (Fig. 3I). In contrast to small changes in circulating triglycerides, PF-06409577 resulted in a large increase in plasma β-hydroxybutyrate, after 42days of dosing as well as after a single dose of compound (Fig. 3K-L). PF-06409577 failed to cause a change in circulating β-hydroxybutyrate in HepKO mice, suggesting that the effects were driven by β-hydroxybutyrate production in the hepatocyte (Fig. 3K-L).