Analysis of B-type natriuretic peptide impurities using label-free data-independent acquisition mass spectrometry technology.

Objectives: Synthetic B-type natriuretic peptide (BNP) is employed in most clinical testing platforms as a raw material of calibrator. Characterization of impurities with structures similar (BNPstrimp compounds) to that of BNP is a reasonable way to decrease clinical measurement errors and improve drug safety. Methods: A novel quantitative method targeted towards BNPstrimp compounds was developed. First, the peptide samples were separated and identified using ultra-performance liquid chromatography, coupled with high-resolution mass spectrometry (MS). To evaluate biological activity further, BNPstrimp immunoaffinity was investigated using western blot (WB) assays. Second, a quantitative label-free data-independent acquisition (DIA) MS approach was developed, and the internal standard peptide (ISP) was hydrolyzed. Absolute quantification was performed using an isotope dilution MS (ID-MS) approach. Third, method precision was investigated using the C-peptide reference material. Results: Seventeen BNPstrimp compounds were identified in synthetic BNP, and 10 of them were successfully sequenced. The immunoassay results indicated that deaminated, oxidized, and isomerized BNPstrimp compounds exhibited weaker immunoaffinity than intact BNP1-32. The mass fraction of the synthetic solid ISP1-16, quantified by ID-MS, was 853.5 (±17.8) mg/g. Validation results indicated that the developed method was effective and accurate for the quantitation of the well-separated BNP impurities. Conclusions: The developed approach was easy to perform, and it was suitable for the parallel quantification of low-abundance BNPstrimp compounds when they performed a good separation in liquid chromatography. The quantitative results were comparable and traceable. This approach is a promising tool for BNP product quality and safety assessment.

Comparative efficacy of pharmacological interventions for contrast-induced nephropathy prevention after coronary angiography: a network meta-analysis from randomized trials.

BACKGROUND: Contrast-induced nephropathy (CIN) is the major complication related to contrast media administration in patients after coronary angiography (CAG). However, inconsistent results have been published in the literature regarding the effects of pharmacological drugs on CIN prevention. We conducted a network meta-analysis to evaluate the relative efficacy of pharmacological interventions for the prevention of CIN. METHODS: We searched MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov from inception to July 2017. We included any randomized controlled trials of eleven pharmacological interventions that reported the prevention of CIN. RESULTS: We identified 3850 records through database searches, of which 107 studies comprising 21,450 participants were finally identified. Compared with intravenous saline, intravenous saline plus pharmacological drugs including statin [relative risk (RR) 0.57; 95% credibility interval (CrI) 0.39 to 0.83], N-acetylcysteine (NAC) (RR 0.84; 95% CrI, 0.71 to 0.98), vitamin and its analogues (RR 0.66; 95% CrI 0.45 to 0.97), brain natriuretic peptide (BNP) and its analogues (RR 0.46; 95% CrI 0.30 to 0.70), prostaglandin analogues (RR 0.37; 95% CrI 0.18 to 0.76), NAC plus sodium bicarbonate (SB) (RR 0.60; 95% CrI 0.39 to 0.90), and statin plus NAC (RR 0.39; 95% CrI 0.21 to 0.70), have helped to reduce the incidence of CIN in patients after CAG. The top four ranked treatments were statin plus NAC, BNP and its analogues, statin, and vitamin and its analogues, respectively. NAC plus intravenous saline was associated with lower incidence of short-term all-cause mortality than intravenous saline alone (RR 0.62; 95% CI, 0.40 to 0.96; P = 0.03). However, no evidence indicated that any of the pharmacological drugs were associated with a reduced requirement for dialysis and major adverse cardiac and cerebrovascular events (MACCE). CONCLUSIONS: Statin plus NAC plus intravenous saline seems to be the most effective treatment for the prevention of CIN in patients after CAG. NAC plus intravenous saline may have a protective role against short-term all-cause mortality. However, none of these drugs has effectively decreased the requirement for dialysis and MACCE.

Intramolecular azo-bridge as a cystine disulfide bond surrogate: Somatostatin-14 and brain natriuretic peptide (BNP) analogs.

Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst2 and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst2 in the nM range with best results obtained with peptide 2, that is, IC50 = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC50 = 60 nM.

Treatment with nesiritide, a recombinant B-type natriuretic Peptide, reduces vascular remodeling following balloon-induced endothelial injuries in rabbits.

Re-stenosis or remodeling of coronary and peripheral arteries remains a major complication following balloon-angioplasty or stenting. This study was designed to investigate the effect of nesiritide, a recombinant B-type natriuretic peptide (BNP), on vascular remodeling following balloon-induced endothelial injuries. Twenty-eight male New Zealand rabbits were divided into nesiritide-treated (0.1 mg/kg/day, sc, for 4 weeks, n = 10), saline-treated control (n = 10) and sham-operated groups (n = 8). In the nesiritide and control groups, a balloon catheter was inserted to the right iliac artery to induce injuries. Vascular endothelial growth factor (VEGF) was measured by immunohistochemistry. The area under internal elastic membrane of the arterial wall (643.2 ± 134.1 vs 493.7 ± 139.3 µm(2), p < 0.05) and the area under external elastic membrane (1495.1 ± 204.9 vs 1265.9 ± 232.6 µm(2), p < 0.05) in the nesiritide group were greater than those in the control group, but were smaller than those in the sham-operated group (p < 0.05). The stenosis ratio was lower in the nesiritide group than in the control group (18.7 ± 7.7% vs 38.0 ± 8.3%, p < 0.01). Importantly, the VEGF expression rate was significantly lower in the nesiritide group than in the control group (42.2 ± 8.8% vs 56.1 ± 13.1%, p < 0.05), while there were no signs of VEGF expression in the non-injured arteries of the three groups. In conclusion, nesiritide treatment reduces the stenosis of the rabbit iliac artery following balloon-induced endothelial injuries probably by decreasing VEGF expression.

Chronic actions of a novel oral B-type natriuretic peptide conjugate in normal dogs and acute actions in angiotensin II-mediated hypertension.

BACKGROUND: We previously reported the feasibility of an acute, orally delivered, newly developed, conjugated form of human B-type natriuretic peptide (hBNP) in normal animals. The objective of the present study was to extend our findings and to define the chronic actions of an advanced oral conjugated hBNP (hBNP-054) administered for 6 days on sodium excretion and blood pressure. We also sought to establish the ability of this new conjugate to acutely activate cGMP and to reduce blood pressure in an experimental model of angiotensin II (ANG II) -mediated hypertension. METHODS AND RESULTS: First, we developed additional novel conjugated forms of oral hBNP that were superior to our previously reported hBNP-021 in reducing blood pressure in 6 normal dogs. We then tested the new conjugate, hBNP-054, chronically in 2 normal dogs to assess its biological actions as a blood pressure-lowering agent and as a natriuretic factor. Second, we investigated the effects of acute oral hBNP-054 or vehicle in 6 dogs that received continuous infusion of ANG II to induce hypertension. After baseline determination of mean blood pressure (MAP) and blood collection for plasma hBNP and cGMP, all dogs received continuous ANG II infusion (20 ng . kg(-1) . min(-1), 1 mL/min) for 4 hours. After 30 minutes of ANG II, dogs received oral hBNP-054 (400 microg/kg) or vehicle in a random crossover fashion with a 1-week interval between dosing. Blood sampling and MAP measurements were repeated 30 minutes after ANG II administration and 10, 30, 60, 120, 180, and 240 minutes after oral administration of hBNP-054 or vehicle. In the chronic study in normal dogs, oral hBNP-054 effectively reduced MAP for 6 days and induced a significant increase in 24-hour sodium excretion. hBNP was not present in the plasma at baseline in any dogs, and it was not detected at any time in the vehicle group. However, hBNP was detected throughout the duration of the study after oral hBNP-054, with a peak concentration at 30 minutes of 1060+/-818 pg/mL. In the acute study, after ANG II administration, plasma cGMP was not activated after vehicle, whereas it was significantly increased after oral hBNP-054 (P=0.01 between the 2 groups). Importantly, MAP was significantly increased after ANG II throughout the acute study protocol. However, although no changes occurred in MAP after vehicle administration, oral hBNP-054 reduced MAP for >2 hours (from 138+/-1 mm Hg after ANG II to 124+/-2 mm Hg at 30 minutes, 124+/-2 mm Hg at 1 hour, and 130+/-5 mm Hg at 2 hours after oral hBNP-054; P<0.001). CONCLUSIONS: This study reports for the first time that a novel conjugated oral hBNP possesses blood pressure-lowering and natriuretic actions over a 6-day period in normal dogs. Furthermore, hBNP-054 activates cGMP and reduces MAP in a model of acute hypertension. These findings advance the concept that orally administered chronic BNP is a potential therapeutic strategy for cardiovascular diseases such as hypertension.