Identification of a promising drug candidate for the treatment of type 2 diabetes based on a P2Y(1) receptor agonist

Abstract

The activation by extracellular nucleotides of pancreatic P2Y receptors, particularly, the P2Y(1)R subtype, increases insulin secretion. Therefore, we developed analogues of the P2Y(1)R receptor agonist 2-MeS-ADP, as potential antidiabetic drugs. Analogue 3A was found to be a potent P2Y(1)R agonist (EC(50) = 0.038 μM vs 0.0025 μM for 2-MeS-ADP) showing no activity at P2Y(2/4/6)Rs. Analogue 3A was stable at pH 1.4 (t(1/2) = 7.3 h) and resistant to hydrolysis vs 2-MeS-ADP by alkaline phosphatase (t(1/2) = 6 vs 4.5 h), human e-NPP1 (4% vs 16% hydrolysis after 20 min), and human blood serum (30% vs 50% hydrolysis after 24 h). Intravenous administration of 3A in naive rats decreased blood glucose from 155 mg/dL to normal values, ca. 87 mg/dL, unlike glibenclamide, leading to subnormal values (i.e., 63 mg/dL). Similar observations were made for streptozotocin (STZ)-treated and db(+)/db(-) mouse models. Furthermore, 3A inhibits platelet aggregation in vitro and elongates bleeding time in mice (iv administration of 30 mg of 3A/kg), increasing bleeding time to 16 vs 9 min for Prasugrel. Oral administration of 30 mg/kg 3A to rats increased tail bleeding volume, similar to aspirin. These findings suggest that 3A may be an effective treatment for type 2 diabetes by reducing both blood glucose levels and platelet aggregation.

Figure 1

Nucleotide-based insulin secretagogue

Figure 2

ADP(α-B) analogues tested as P2Y₁R agonists.

Figure 3

Conformation of P-diastereomers of analogue 3.

Figure 4

Concentration-response curves for analogues 2–4 determined by measuring analogue-induced increases in [Ca²⁺]i in human 1321N1 astrocytoma cells stably transfected with the turkey P2Y₁R.

Figure 5

Hydrolysis of ADP, 2-MeS-ADP, and analogue 3A in human blood serum.

Figure 6

Hydrolysis of analogue 3A at pH 1.4, 37 °C.

Figure 7

Glucose tolerance tests in naïve mice (n = 5) treated with saline, analogue 3A, or glibenclamide. Fasting rats (A) and mice (B, C) were treated iv with the indicated compounds at 2.5 mg/kg (A), 10 mg/kg (B) or 30 mg/kg body weight (B, C) 10 min after glucose challenge. Glibenclamide was given orally as a positive control in (A) at 2.5 mg/kg body weight or (B) at 3 mg/kg body weight, 30 min before glucose challenge. Error bars are ± standard deviation.

Figure 8

Glucose tolerance tests in the STZ diabetic mouse model. Diabetic (STZ) and normal (blue) mice (n = 5) were administered an OGTT, as described in Figure 7. Three doses of analogue 3A, 30 (purple), 15 (light blue), and 7.5 (orange) mg/kg body weight, were administered intravenously (iv) and glibenclamide (12 mg/kg body weight, green) was given PO. Saline administration to diabetic mice (red) was used as a control. Error bars are ± standard deviation.

Figure 9

Glucose tolerance tests in the db⁺/db⁻ diabetic mouse model. The db⁺/db⁻ mice were selected for elevated glucose levels. Analogue 3A (iv, 30 mg/kg body weight, green) and glibenclamide (PO, 20 mg/kg body weight, red) were administered with an OGTT (n = 5), as described in Figure 7. Error bars are ± standard deviation.

Figure 10

Analogue 3A inhibits ADP-induced platelet aggregation in human plasma. Analogue 3A was tested for inhibition of ADP (10 μM) induced platelet aggregation in PRP obtained from one subject in duplicate samples.

Figure 11

Anti-platelet aggregation activity of analogue 3A in vivo. An increase in mouse tail-bleed time was used as a model for the in vivo anti-platelet aggregation activity of analogue 3A in mg/kg body weight. Mice were treated iv with different doses of analogue 3A or R-361015, a compound with known anti-platelet aggregation activity, and then the tail was incised and the time to cessation of bleeding was measured. Animal numbers are above bars. Error bars are standard deviation.

Figure 12

Oral anti-platelet aggregation activity of analogue 3A in vivo. Analogue 3A (30 mg/kg) or aspirin (10 mg/kg body weight) was administered PO to Lewis rats (n = 5) prior to tail incision, as in Figure 10. Time to cessation of bleeding (A) and total volume of exsanguinated blood (B) was measured. Error bars are ± standard deviation.

Figure 13

Concentration-response curves for analogue 3A and ADP were determined by measuring analogue-induced increases in [Ca²⁺]i in U-205 cells stably transfected with hP2Y₁₂R.

Scheme 1

One-Pot Synthesis of 2-MeS-Adenosine 5′-O-(α-Borano)diphosphate, Analogue 3A^a

Scheme 2

Synthesis of 2-MeS-AP₃(′-B)-A-2-MeS, Analogue 4^{a a}Reaction conditions: (a) CDI (5 equiv), DMF, RT, 3 h; (b) dry MeOH (5 equiv), RT, 8 min; (c) BPi (1 equiv), DMF, MgCl₂ (8 equiv), RT, overnight.