Abstract
BackgroundRecombinant human brain natriuretic peptide (rhBNP) is an important peptide-based therapeutic drug indicated for the treatment of acute heart failure. Accurate determination of the potency of therapeutic rhBNP is crucial for the safety and efficacy of the drug. The current bioassay involves use of rabbit aortic strips, with experiments being complicated and time-consuming and markedly variable in results. Animal-less methods with better precision and accuracy should be explored. We have therefore developed an alternative cell-based assay, which relies on the ability of BNP to induce cGMP production in HEK293 cells expressing BNP receptor guanylyl cyclase-A.Methodology/Principal FindingsAn alternative assay based on the measurement of BNP-induced cGMP production was developed. Specifically, the bioassay employs cells engineered to express BNP receptor guanylyl cyclase-A (GCA). Upon rhBNP stimulation, the levels of the second messager cGMP in these cells drastically increased and subsequently secreted into culture supernatants. The quantity of cGMP, which corresponds to the rhBNP activity, was determined using a competitive ELISA developed by us. Compared with the traditional assay, the novel cell-based assay demonstrated better reproducibility and precision.Conclusion/SignificanceThe optimized cell-based assay is much simpler, more rapid and precise compared with the traditional assay using animal tissues. To our knowledge, this is the first report on a novel and viable alternative assay for rhBNP potency analysis.
Highlights
The natriuretic peptides (NPs) are a family of ring shaped vasoactive hormones with considerable sequence homology among themselves
The results indicated that the pCMV6-ENTRY-guanylyl cyclase-A (GCA) transfected HEK293 cells produced increased levels of cyclic guanosine monophosphate (cGMP) in response to the ascending concentrations of Recombinant human brain natriuretic peptide (rhBNP), while, clearly, the mocktransfected HEK293 failed to respond to rhBNP stimulation (Fig. 1B)
Accurate determination of the potency of therapeutic rhBNP is crucial for the safety and efficacy of the drug
Summary
The natriuretic peptides (NPs) are a family of ring shaped vasoactive hormones with considerable sequence homology among themselves. The B-type natriuretic peptide (BNP), called brain natriuretic peptide, a 32 amino acid polypeptide, is the most clinically potent type, which secrets from the ventricles in response to volume expansion and pressure overload [5]. It counterbalances actions of the reninangiotensin-aldosterone and neurohormonal systems, and plays a central role in cardiovascular regulation [6]. The use of it as an effective therapeutic intervention was well established in patients with acute decompensated heart failure [10,11,12]. Recombinant human brain natriuretic peptide (rhBNP) is an important peptide-based therapeutic drug indicated for the treatment of acute heart failure. We have developed an alternative cell-based assay, which relies on the ability of BNP to induce cGMP production in HEK293 cells expressing BNP receptor guanylyl cyclase-A
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