Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo

Abstract

An inhibitor of human liver glycogen phosphorylase a (HLGPa) has been identified and characterized in vitro and in vivo. This substance, [R-(R*, S*)]-5-chloro-N-[3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)pr opyl]-1H-indole-2-carboxamide (CP-91149), inhibited HLGPa with an IC50 of 0.13 microM in the presence of 7.5 mM glucose. CP-91149 resembles caffeine, a known allosteric phosphorylase inhibitor, in that it is 5- to 10-fold less potent in the absence of glucose. Further analysis, however, suggests that CP-91149 and caffeine are kinetically distinct. Functionally, CP-91149 inhibited glucagon-stimulated glycogenolysis in isolated rat hepatocytes (P < 0.05 at 10-100 microM) and in primary human hepatocytes (2.1 microM IC50). In vivo, oral administration of CP-91149 to diabetic ob/ob mice at 25-50 mg/kg resulted in rapid (3 h) glucose lowering by 100-120 mg/dl (P < 0.001) without producing hypoglycemia. Further, CP-91149 treatment did not lower glucose levels in normoglycemic, nondiabetic mice. In ob/ob mice pretreated with 14C-glucose to label liver glycogen, CP-91149 administration reduced 14C-glycogen breakdown, confirming that glucose lowering resulted from inhibition of glycogenolysis in vivo. These findings support the use of CP-91149 in investigating glycogenolytic versus gluconeogenic flux in hepatic glucose production, and they demonstrate that glycogenolysis inhibitors may be useful in the treatment of type 2 diabetes.

Figure 1

Synthetic route to CP-91149. a, HCl-MeOH, 60 h, 23°C, then NaHCO₃/H₂O, 77%; b, 1.0 equiv. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl, 1.5 equiv. 1-hydroxybenzotriazole, dichloromethane, 23°C, 18 h, 100% crude; c, 2.0 equiv. NaOH, MeOH, H₂O, 25°C, 2 h, 80%; d, as for b, but 1.1 equiv. dimethylamine HCl and 1.1 equiv. triethylamine in dimethylformamide, 18 h, 25°C, 62%.

Figure 2

Inhibition of HLGPa by CP-91149 and caffeine. HLGPa activity was measured at varied concentrations of CP-91149 (▪) or caffeine (•). The data are plotted as percent inhibition vs. concentration of compound. The data (±SEM) are from a representative experiment performed in triplicate.

Figure 3

The effect of glucose on the potency of CP-91149 (▪) and caffeine (•). IC₅₀ values for HLGPa inhibition were determined at varied glucose concentrations, and then normalized by dividing the values by the IC₅₀ value obtained in the absence of glucose. The normalized results are plotted as function of glucose concentration.

Figure 4

Synergistic inhibition of HLGPa by CP-91149, caffeine and theophylline. (A) HLGPa activity was measured in the presence of varied concentrations of CP-91149 under control conditions (▪), or with 240 μM caffeine (•) or 480 μM theophylline (▴) in the assay buffer. (B) HLGPa activity was measured in the presence of varied caffeine concentration under control conditions (▪), or with 1.0 μM CP-91149 (•) or 490 μM theophylline (▴) in the assay buffer.

Figure 5

Inhibition of glycogenolysis by CP-91149 in isolated rat hepatocytes. Isolated rat hepatocytes were preincubated with (A) 100 μM CP-91149 or DMSO or (B) 1–100 μM CP-91149 or DMSO for 5 min, then in the absence (filled bars) or presence (open bars) of 0.3 nM GGN for 60 min in buffer containing 7.5 mM glucose, followed by glycogen determination. Results (μmol/g) are the mean ± SD of triplicate determinations. ∗∗, P < 0.01; ∗∗∗, P < 0.001 between treatment (GGN, CP-91149) vs. control (no GGN); †, P < 0.05; ‡, P < 0.01; §, P < 0.001 between CP-91149 + GGN vs. control + GGN.

Figure 6

Inhibition of glycogenolysis by CP-91149 in primary human hepatocytes. Primary human hepatocytes were incubated with [¹⁴C-U]glucose plus 1 μM insulin, then with 10 mM glucose and (A) 50 μM forskolin (F) or 25 nM GGN (G), plus 30 μM CP-91149 as indicated, or (B) in the absence (filled bars) or presence (open bars) of 25 nM GGN, plus CP-91149 as indicated for 2 h. (A) Results (dpm ¹⁴C-glycogen) are the mean ± SEM of triplicate determinations from a representative experiment. ∗, P < 0.001 between GGN, forskolin, or CP-91149 vs. untreated control; ‡, P < 0.01 between control + GGN or forskolin vs. CP-91149 + GGN or forskolin. (B) Results are expressed relative (%) to untreated control in the absence of GGN (100%) and are the mean ± SEM from 3 to 10 independent experiments, each performed in triplicate. ∗, P < 0.05 between CP-91149 vs. untreated control; ‡, P < 0.001 between CP-91149 + GGN vs. GGN-treated control.

Figure 7

Dose-response of glucose lowering activity by CP-91149 in (A) obese, diabetic ob/ob mice, and (B) lean, nondiabetic control mice. Mice were treated with vehicle or CP-91149 as indicated, and plasma glucose concentration was determined 3 h later. Values (mg/dl) represent the mean ± SEM for 10–60 mice per group. ∗, Significant (P < 0.01) decrease by CP-91149 compared with the vehicle-treated group.