Novel Small Molecule Agonist of TGR5 Possesses Anti-Diabetic Effects but Causes Gallbladder Filling in Mice.

Activation of TGR5 via bile acids or bile acid analogs leads to the release of glucagon-like peptide-1 (GLP-1) from intestine, increases energy expenditure in brown adipose tissue, and increases gallbladder filling with bile. Here, we present compound 18, a non-bile acid agonist of TGR5 that demonstrates robust GLP-1 secretion in a mouse enteroendocrine cell line yet weak GLP-1 secretion in a human enteroendocrine cell line. Acute administration of compound 18 to mice increased GLP-1 and peptide YY (PYY) secretion, leading to a lowering of the glucose excursion in an oral glucose tolerance test (OGTT), while chronic administration led to weight loss. In addition, compound 18 showed a dose-dependent increase in gallbladder filling. Lastly, compound 18 failed to show similar pharmacological effects on GLP-1, PYY, and gallbladder filling in Tgr5 knockout mice. Together, these results demonstrate that compound 18 is a mouse-selective TGR5 agonist that induces GLP-1 and PYY secretion, and lowers the glucose excursion in an OGTT, but only at doses that simultaneously induce gallbladder filling. Overall, these data highlight the benefits and potential risks of using TGR5 agonists to treat diabetes and metabolic diseases

Compound 18 demonstrates potent cAMP production and GLP-1 secretion in mouse cells, but weaker cAMP production and GLP-1 secretion in human cells.

<p>The compound structure for both INT-777 and compound 18 are shown (A). HEK-293 cells (white circles) or HEK-293 cells stably transfected with the mouse TGR5 gene (black circles) were treated with compound 18 and assayed for cAMP production (B). Human (NCI-H716, black circles) or mouse (STC-1, white circles) enteroendocrine cells were treated with compound 18 and assayed for cAMP production (C) and GLP-1 secretion (D).</p

Compound 18 improves glucose tolerance in an OGTT in C57Bl/6 mice.

<p>Overnight-fasted C57BL/6 mice were orally dosed with vehicle (white circles), sitagliptin (sita) at 10 mg/kg (black squares), or INT-777 at 100 mg/kg (red squares) (A), or with vehicle (white circles), compound 18 at 1 mg/kg (yellow triangles), 3 mg/kg (green triangles), 10 mg/kg (blue triangles), 30 mg/kg (orange triangles), or 100 mg/kg (purple triangles) (B), followed 15 minutes later with glucose (3 g/kg). Glucometers were used to measure glucose at 0, 20, 40, 60, and 120 minutes post glucose (A and B), and the resulting raw data used to generate area under the curve analyses (AUCs) (C). <i>n</i> = 5; mean +/- SEM. * <i>P < 0</i>.<i>01</i>.</p

Chronic administration of compound 18 to mice does not cause desensitization and also leads to weight loss.

<p>Diet-induced obese (DIO) C57BL/6 mice were either dosed acutely or chronically for two weeks with vehicle (white circles, white bars) or compound 18 at 60 mg/kg (black triangles, black bars). An OGTT on over-night fasted DIO mice was performed after either a single dose (A, acute) or after two weeks of dosing (B, chronic), using 2 g/kg glucose dosed 30 minutes after compound dosing. Changes in body weights (C) and cumulative food intake (D) were recorded for the two week period. <i>n</i> = 8; mean +/- SEM. * <i>P < 0</i>.<i>01</i>.</p

Expression of TGR5 in human and mouse tissues and enteroendocrine cell lines NCI-H715 and STC-1.

<p>TaqMan gene expression assay using total RNA isolated from human (A) and mouse (B) tissues as well as human NCI-H716 (A) and mouse STC-1 cells (B). β-actin was used as an internal control. Data represent the fold over ileum for each species independently, since different probe/primer sets were used. Values represent the mean +/- SEM.</p

Compound 18 increases GLP-1 and PYY and lowers the glucose and insulin excursions as compared to sitagliptin in an OGTT in C57BL/6 mice.

<p>Overnight-fasted C57BL/6 mice were orally dosed with vehicle (white circles), compound 18 at 30 mg/kg (white triangles), or sitagliptin at 10 mg/kg (black diamonds), followed 15 minutes later by an oral dose of glucose (3 g/kg). Plasma was analyzed for active GLP-1 (A), total PYY (B), and insulin (D), while blood glucose was measured by glucometers (C). Resulting raw data was used to generate area under the curve (AUC) analyses. <i>n</i> = 5 per time-point; mean +/- SEM. * <i>P < 0</i>.<i>05</i> versus vehicle. <i>$ P < 0</i>.<i>05</i> versus sitagliptin.</p

Compound 18 causes gallbladder filling at doses that do not induce GLP-1 secretion.

<p>C57BL/6 mice were orally dosed with vehicle, compound 18 at 1, 3, 10, 30, or 100 mg/kg, or INT-777 at 100 mg/kg, for 3 days twice a day (BID), with over-night fasting performed on the last evening. On the third day, compound was dosed in the morning followed 30 minutes later by measurement of bile filling (A) by use of a needle and syringe and GLP-1 (x-36) amide (B) by cardiac puncture. Representative photos of gallbladder filling in animals treated 3 days with vehicle, compound 18 at 30 mg/kg, and INT-777 at 100 mg/kg; gallbladders highlighted with dotted circles (C). <i>n</i> = 5; mean +/- SEM. * <i>P < 0</i>.<i>05</i>. ** <i>P < 0</i>.<i>01</i></p

Compound 18 stimulates GLP-1 and PYY secretion in C57Bl/6 mice.

<p>Overnight-fasted C57BL/6 mice were orally dosed with vehicle (white circles), compound 18 at 3 mg/kg (white squares), 10 mg/kg (black squares), 30 mg/kg (white triangles), or 100 mg/kg (black triangles), or INT-777 at 100 mg/kg (stars), followed 0.25, 0.50, 1.50, or 3.00 hours post dose by cardiac puncture. Plasma was analyzed for GLP-1 (X-36) amide (A), total PYY (B), or compound levels of INT-777 or compound 18 (C). Resulting raw data was used to generate area under the curve (AUCs) analyses. <i>n</i> = 5 per time-point; mean +/- SEM. * <i>P < 0</i>.<i>05</i>. ** <i>P < 0</i>.<i>01</i>.</p