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  • The physicochemical properties of the prodrugs provided impo

    2021-12-20

    The physicochemical properties of the prodrugs provided important clues for understanding a broad range of inhibitory activities in the cell assays (Table 2). Some physicochemical properties are useful in predicting drug absorption. Considering Lipinski’s rules and the polar surface area (PSA),l-alanine diamide prodrugs 11a–c and 15a–c appear to have different physicochemical parameters with respect to cLogP and PSA. In particular, compared to prodrugs 15a–c, which possess alkyl side chains and exhibited potent inhibitory effects in the cell assays, prodrugs 11a–c have high PSA values due to the presence of amide side chains. PSA is defined as the sum of the surfaces of the polar atoms in a molecule and correlate well with cell membrane permeability. A short preincubation of hepatocytes with prodrugs in the cell assays resulted in placing emphasis on the cell membrane permeability of the prodrugs. It was suggested that prodrugs 15a–c exhibited high inhibitory activities in the cell assays, presumably due to low PSA values and relatively high membrane permeability. Prodrug 15c exhibiting high inhibitory activity in the cell assays also demonstrated in vivo efficacy in an animal model (Fig. 4). In order to investigate the in vivo efficacy of the prodrugs, fasted cynomolgus monkeys were used as an animal model, considering that the fasting plasma SBHA level correlates with the hepatic glucose production originating in gluconeogenesis. Prodrug 15c was administered orally at 20mg/kg to fasted cynomolgus monkeys. The blood glucose levels of the monkeys were significantly decreased 4h after dosing compared to the vehicle-treated animals. Hypoglycemia, a safety concern of gluconeogenesis inhibition, was not observed. In addition, the plasma concentration of prodrug 15c and corresponding phosphonate 16 in this in vivo study showed that prodrug 15c disappeared rapidly and the corresponding parent phosphonate 16 remained for a long time at relatively high concentrations (Fig. 5). This result suggested that prodrug 15c was rapidly converted to corresponding phosphonate 16 in the body and l-alanine diamide prodrug moiety functioned as a prodrug. Reliable evidence of the in vivo efficacy of prodrug 15c has provided the possibility of developing new drugs for the treatment of T2DM.
    Conclusion In summary, we developed prodrugs of tricyclic-based FBPase inhibitors in order to investigate their effects in cells and in vivo. In contrast to prodrugs containing amide side chains, prodrugs containing alkyl side chains potently inhibited glucose production in monkey hepatocytes. The physicochemical properties of prodrugs suggested that low PSA values and relatively high membrane permeability was probably critical to the exhibition of high inhibitory activity in the cell assays. The in vivo study in fasted cynomolgus monkeys revealed that prodrug 15c lowered blood glucose levels and was rapidly converted to corresponding phosphonate 16. These results have provided the possibility of developing new drugs for the treatment of T2DM.
    Experimental
    Introduction A key reaction in the gluconeogenesis pathway is the cleavage of fructose-1,6-bisphosphate into fructose-6-phosphate and inorganic phosphate catalyzed by fructose-1,6-bisphosphatase (FBPase, E.C.3.1.3.11). In humans, two genes located on chromosome 9q22.32 encode this enzyme: FBP1 (611570) and FBP2 (603027). The FBP1 has eight exons, of which seven (exons 2–8) result in a transcript of approximately 1.5 kb that is translated into a protein with 338 amino acids that is expressed in the liver and kidney (El-Maghrabi et al., 1995). FBP2 is expressed in the muscles and presents a product of 1.3 kb in size that results in a protein of 339 amino acids (Tillmann et al., 2000). These isozymes catalyze the same reaction in different tissues, but their activities do not overlap; that is, when one is inactive, the other does not act as a substitute (Pagliara et al., 1972). To date, no study has shown a causal relationship between alterations in FBP2 gene and a specific disease. There is only one study of the association with gastric cancer and decreased FBP2 transcription (Li et al., 2013). However, only variants in FBP1 gene cause fructose-1,6-bisphosphatase deficiency (FBPase deficiency; FBP1D; OMIM#229700).