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  • br Methods br Results br Discussion


    Discussion Peptide therapeutics represents a unique class of biopharmaceuticals that continues to evolve in drug discovery and development. We and others have been particularly interested in bioengineering innovative pGC-A and/or pGC-B peptide activators. This has been successfully accomplished by integrating key AAs from endogenous pGC ligands or utilizing knowledge based on genetic alterations and prohormone processing/degradation to create novel biotherapeutics whose biological actions extend beyond native ligands, as well as, to overcome certain limitations such as short circulating Imipenem [26,[34], [35], [36], [37], [38]]. In the current study, we took advantage of scientific knowledge related to proCNP processing [24,25] and the formation of a 53-AA intermediate peptide which is generally thought to be a storage form of CNP [2]. Our in vitro HEK293 cell studies clearly demonstrated that C53 can activate the pGC-B receptor as intracellular cGMP was significantly elevated in dose-dependent manner, with very minimal pGC-A activity at higher doses. These findings establish that C53 has biological activity that is selective for the pGC-B receptor, despite having an elongated amino terminus. The clinical development and application of peptide-based drugs has been limited, in part, due to short circulating half-life [39]. Indeed, studies have shown that CNP is rapidly degraded by NEP as well as catabolized by NPRC and therefore is short-lived in vivo [16,17,20]. Thus, we focused on the degradation and clearance of C53, as reduced NEP susceptibility and/or clearance by NPRC would enhance bioavailability. Elegant studies by Pankow and colleagues [18] have demonstrated that NEP-mediated degradation of NPs, the endogenous pGC-A or pGC-B ligands, is dependent in part, on the length of the amino and/or carboxyl termini. Our data demonstrated that C53, in the presence of NEP, mediated potent pGC-B activity and cGMP generation for 480 min. However CNP, in the presence of NEP, lost its ability to generate pGC-B mediated cGMP by 30 min, which is consistent with the known fact that CNP is highly susceptible to NEP degradation [18,19,40]. Interestingly in the presence of NEP, C53 generated >100% pGC-B mediated cGMP activity throughout the entire 480 min, thus suggesting that NEP cleaves, at least some of, C53 into unknown molecular forms that has ability to activate cGMP via pGC-B greater than that of C53 or CNP. In addition to NEP, NPRC is receptor-mediated mechanism for CNP metabolism and previous reports showed CNP had a high binding affinity to NPRC [41,42]. Our findings show that C53 interacted less with NPRC than CNP, thus providing evidence that C53 may be less likely to be metabolized by NPRC. Given this data, it is plausible to suggest that the extra 31 AAs on the amino terminus of C53 either lacks key NEP recognition sites and/or provides a protective structural conformation that prevents NEP and NPRC catabolism. Together, our in vitro data suggests C53 may have enhanced bioavailability, with superior biological properties, compared to CNP. However, future studies are warranted to elucidate the extent of the role of C53's amino terminus and its ability to confer resistance to breakdown. In our in vivo study using normal rats, we observed that acute IV infusion of C53 generated significantly higher CLL compared to equimolar infusion of CNP. This marked elevation in plasma CLL with C53 was paralleled with significantly elevated levels of plasma and urinary cGMP, the second messenger of pGC-B activation. Further, despite marked generation of cGMP with C53 infusion there was no significant effect on GFR, natriuresis, diuresis or the lowering of BP under these normal conditions. These results are consistent with previous studies in normal humans and canines demonstrating that cGMP generation with CNP infusion lacks significant renal actions or BP lowering effects [38,43]. However, recent and fascinating studies with mouse models of cell-specific deletion of CNP or the pGC-B receptor [[44], [45], [46]] have suggested this signaling pathway may indeed have a role in BP regulation. Thus, given complexity of the regulation of vascular tone and BP, further investigations are required to delineate whether C53 can alter BP chronically or under pathophysiology conditions such as hypertension [47]. Our current data supports the idea that the pGC-B/cGMP pathway may predominantly mediate cellular and tissue remodeling responses, unlike pGC-A/cGMP signaling which has immediate BP lowering and renal enhancing actions [1,35]. Nonetheless, our in vivo results provide evidence that C53 has a longer circulating half-life and enhanced cGMP-generating capabilities compared to CNP. However, additional pharmacokinetic studies are needed to calculate t1/2 and Cmax to further confirm its prolonged half-life.