A novel insulin secretagogue based on a dinucleoside polyphosphate scaffold

Abstract

Dinucleoside polyphosphates exert their physiological effects via P2 receptors (P2Rs). They are attractive drug candidates, as they offer better stability and specificity compared to nucleotides, the most common P2 receptor ligands. The activation of pancreatic P2Y receptors by nucleotides increases insulin secretion. Therefore, in the current study, dinucleoside polyphosphate analogues (di-(2-MeS)-adenosine-5',5''-P(1),P(4),alpha,beta-methylene-tetraphosphate), 8, (di-(2-MeS)-adenosine-5',5''-P(1),P(4),beta,gamma-methylene-tetraphosphate), 9, and di-(2-MeS)-adenosine-5',5''-P(1),P(3),alpha,beta-methylene triphosphate, 10, were developed as potential insulin secretagogues. Analogues 8 and 9 were found to be agonists of the P2Y(1)R with EC(50) values of 0.42 and 0.46 microM, respectively, whereas analogue 10 had no activity. Analogues 8-10 were found to be completely resistant to hydrolysis by alkaline phosphatase over 3 h at 37 degrees C. Analogue 8 also was found to be 2.5-fold more stable in human blood serum than ATP, with a half-life of 12.1 h. Analogue 8 administration in rats caused a decrease in a blood glucose load from 155 mg/dL to ca. 100 mg/dL and increased blood insulin levels 4-fold as compared to basal levels. In addition, analogue 8 reduced a blood glucose load to normal values (80-110 mg/dL), unlike the commonly prescribed glibenclamide, which reduced glucose levels below normal values (60 mg/dL). These findings suggest that analogue 8 may prove to be an effective and safe treatment for type 2 diabetes.

Figure 1

P2Y₁ receptor agonists.

Figure 2

A series of novel dinucleoside polyphosphate analogues.

Figure 3

Agonist potency profile of compounds 8, 9, and 2-MeS-ADP for the increase in [Ca²⁺]_i in 1321N1 astrocytoma cells expressing recombinant P2Y₁ receptors. Cells loaded with Fura-2 as described in Experimental Section were exposed to 8, 9, and 2-MeS-ADP at the indicated concentrations. Each value shown is a mean ± SE of three experiments.

Figure 4

Overnight fasting Wistar Han rats (5 per group) were administered (iv) 2.5 mg/kg bodyweight of analogue 4 or analogue 8, 10 min postglucose challenge. Glibenclamide was administered orally at −30 min. (A) The effect of analogues 4 or 8 (2.5 mg/kg body weight) on blood glucose levels over time were compared to glibenclamide (1 mg/kg body weight) or saline administration. (B) The effect of analogue 8 (2.5 mg/kg body weight) or saline on blood insulin concentrations over time.

Figure 5

Hydrolysis of analogues 8−10 and ATP by alkaline phosphatase monitored by HPLC. Hydrolysis of 4 mM nucleotide by alkaline phosphatase (12.5 units) at 37 °C was monitored for 3 h.

Figure 6

Enzymatic hydrolysis of analogue 8 and ATP in human blood serum monitored by HPLC. Hydrolysis of 0.25 mM analogue 8 or ATP in human blood serum (180 μL) and RPMI-1640 (540 μL) at 37 °C was monitored for 24 h. (A) HPLC chromatograms of analogue 8 hydrolysates at 3, 7, and 24 h. The location of the peaks was determined in comparison to nucleotide standards. (B) Percentage of analogue 8 and ATP in human blood serum over time (hydrolysis t_1/2 = 12.1 and 4.9 h, respectively).

Scheme 1^a

^aReaction conditions: (a) CH₂Cl₂, DMAP, TsCl, RT, 12 h. (b) Analogue 13: tetra-(n-butylammonium)methylenediphosphonate in dry DMF, RT, 48 h. (c) Analogue 14: tetra-(n-butylammonium)pyrophosphate in dry DMF, RT, 48 h. (d) 18% HCl, pH 2.3, RT for 3 h followed by 24% NH₄OH, pH9, RT for 45 min. (e) CDI, DMF, RT, 3 h; f) MgCl₂, RT, 12 h.