Nesiritide, also known as Brain Natriuretic Peptide-32 human, is an agonist of the natriuretic peptide receptor (NPR), with Kd values of 7.3 pM for NPR-A and 13 pM for NPR-C [1]. While ProBNP1-108 is 13 times less powerful than Nesiritide (BNP1-32), it nevertheless stimulates guanylate cyclase-A (GC-A) to almost maximal activity. Nesiritide binds to human GC-A 35 times more strongly than ProBNP1-108. Nesiritide and proBNP1-108 do not activate GC-B. Compared to nesiritide, the binding strength of natriuretic peptide scavenging receptors to proBNP1-108 is three times lower. When degraded by human renal membrane, proBNP1-108 has a half-life that is 2.7 times longer than that of nesiritide and a complete degradation period that is 6 times longer. The first-order and second-order exponential decay models are best fitted by nesiritide and proBNP1-108, respectively [2].

Nesiritide, also known as Brain Natriuretic Peptide-32 human, is an agonist of the natriuretic peptide receptor (NPR), with Kd values of 7.3 pM for NPR-A and 13 pM for NPR-C [1]. While ProBNP1-108 is 13 times less powerful than Nesiritide (BNP1-32), it nevertheless stimulates guanylate cyclase-A (GC-A) to almost maximal activity. Nesiritide binds to human GC-A 35 times more strongly than ProBNP1-108. Nesiritide and proBNP1-108 do not activate GC-B. Compared to nesiritide, the binding strength of natriuretic peptide scavenging receptors to proBNP1-108 is three times lower. When degraded by human renal membrane, proBNP1-108 has a half-life that is 2.7 times longer than that of nesiritide and a complete degradation period that is 6 times longer. The first-order and second-order exponential decay models are best fitted by nesiritide and proBNP1-108, respectively [2].

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Nesiritide (BNP₁–₃₂) activated human guanylyl cyclase-A (GC-A) expressed in HEK293 cells in a concentration-dependent manner, with a half-maximal effective concentration (EC₅₀) of 27 nM (95% CI: 16–46 nM). It did not activate human guanylyl cyclase-B (GC-B). [2]
Nesiritide (BNP₁–₃₂) competed with ⁱ²⁵I-ANP for binding to human GC-A expressed in HEK293 cells with a half-maximal inhibitory concentration (IC₅₀) of 8.3 nM (95% CI: 7–10 nM). [2]
Nesiritide (BNP₁–₃₂) competed with ⁱ²⁵I-ANP for binding to the human natriuretic peptide clearance receptor (NPR-C) expressed in HEK293 cells with an IC₅₀ of 2.6 nM (95% CI: 2.1–3.2 nM). [2]
Nesiritide (BNP₁–₃₂) was susceptible to proteolytic degradation by human kidney membranes. Its degradation followed a first-order exponential decay model with a calculated half-life (t₁/₂) of 4.2 minutes. Complete inactivation occurred after approximately 20 minutes of incubation. The degradation was abolished by boiling the membranes or by adding the broad-spectrum protease inhibitors leupeptin or a complete protease inhibitor cocktail, but not by the neutral endopeptidase inhibitor phosphoramidon or the meprin inhibitor actinonin. [2]

Proteolytic Degradation Assay: Human kidney crude membranes were thawed and diluted to 2 g/L with ice-cold buffer. A proteolysis reaction was initiated by adding the membranes to a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, 0.1% BSA, and 1 µM Nesiritide (BNP₁–₃₂). The mixture was incubated at 37°C for various time periods. The reaction was terminated by adding perchloric acid, followed by neutralization with NaOH. After centrifugation, the supernatant was assayed for remaining peptide bioactivity by measuring its ability to stimulate cGMP production in HEK293 cells stably expressing human GC-A. The amount of starting peptide remaining was calculated by comparing the cGMP response to a standard concentration-response curve generated with intact peptide. [2]
Protease Inhibitor Assay: In some experiments, kidney membranes were pre-incubated on ice for 10 minutes with various protease inhibitors (leupeptin, phosphoramidon, actinonin, or a complete protease inhibitor cocktail) before being added to the reaction mixture containing 1 µM peptide. The degradation reaction proceeded at 37°C for 20 minutes before being stopped and analyzed as described above. [2]

cGMP Elevation Assay (GC-A Activation): HEK293 cells stably expressing human GC-A were grown in 48-well plates until 75-90% confluent in serum-free medium. The medium was aspirated and replaced with DMEM containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) and 25 mM HEPES (pH 7.4) for a 10-minute pre-incubation at 37°C. This medium was then replaced with fresh DMEM/IBMX/HEPES containing varying concentrations of Nesiritide (BNP₁–₃₂) for a 3-minute stimulation at 37°C. The reaction was stopped by adding ice-cold 80% ethanol. The cell extracts were dried and intracellular cGMP content was quantified by radioimmunoassay (RIA). [2]
cGMP Elevation Assay (GC-B Activation): The same procedure was performed using HEK293 cells stably expressing human GC-B to test the selectivity of Nesiritide (BNP₁–₃₂). [2]
Whole-Cell Competitive Binding Assay (GC-A and NPR-C): HEK293 cells stably expressing human GC-A or NPR-C were seeded in poly-D-lysine coated 24-well plates. At 75-90% confluence, cells were washed and pre-incubated with DMEM containing 0.2% BSA at 37°C for 1-2 hours. The medium was replaced with binding medium containing 75 pM ⁱ²⁵I-ANP and 1% BSA, in the presence or absence of increasing concentrations of unlabeled Nesiritide (BNP₁–₃₂). Plates were incubated at 4°C for 1 hour. Unbound ligand was removed by washing with ice-cold PBS. Cells were solubilized in NaOH, and bound radioactivity was measured using a gamma counter. Specific binding was plotted against competitor concentration to determine IC₅₀ values. [2]

Absorption, Distribution and Excretion
Following administration, nesiritide exhibits a biphasic distribution in plasma. Human brain natriuretic peptide (BNP) is cleared from circulation primarily through three independent mechanisms, listed in descending order of importance: 1) binding to cell surface clearance receptors, followed by intracellular endocytosis and lysosomal proteolysis; 2) proteolytic cleavage of the peptide by endopeptidases (e.g., neutral endopeptidases present on the vascular lumen surface); 3) renal filtration. 0.19 L/kg 9.2 mL/min/kg [Patients with congestive heart failure receiving intravenous infusion] Metabolism/Metabolites nesiritide is hydrolyzed on the vascular lumen surface by endopeptidases (e.g., neutral endopeptidases). Biological Half-Life Approximately 18 minutes.

[1]. Molecular biology of the natriuretic peptides and their receptors. Circulation. 1992 Oct;86(4):1081-8.

[2]. ProBNP(1-108) is resistant to degradation and activates guanylyl cyclase-A with reduced potency. Clin Chem. 2011 Sep;57(9):1272-8.

Nesiriside is a polypeptide. It is a medication used to treat acute decompensated congestive heart failure, a condition that causes shortness of breath at rest or during mild activity (such as talking, eating, or bathing). Nesiriside is a recombinant human B-type natriuretic peptide composed of 32 amino acids. Nesiriside is a recombinant version of the cardiac neurohormone, human B-type natriuretic peptide (hBNP), produced by the ventricular myocardium. Nesiriside binds to natriuretic peptide receptors on vascular smooth muscle and endothelial cells, triggering guanylate cyclase-dependent signaling transduction via these receptors, leading to an increase in intracellular cGMP concentration. This causes smooth muscle cells to relax, resulting in arterial and venous dilation. It is a peptide secreted by the brain and atria, primarily stored in the ventricular myocardium. It can induce natriuresis; diuresis; vasodilation; and inhibition of renin and aldosterone secretion. It improves cardiac function. It contains 32 amino acids. Drug Indications For intravenous treatment of patients with acute decompensated congestive heart failure who experience dyspnea at rest or with minimal activity. FDA Label Mechanism of Action Human brain natriuretic peptide (BNP) binds to granular guanylate cyclase receptors on vascular smooth muscle and endothelial cells, leading to an increase in intracellular guanosine 3',5'-cyclic phosphate (cGMP) concentration, thereby relaxing smooth muscle cells. CcGMP acts as a second messenger, dilating veins and arteries. Studies have shown that nesiritide can relax isolated human arterial and venous tissue specimens pre-constricted with endothelin-1 or the alpha-adrenergic agonist phenylephrine. In human studies, nesiritide dose-dependently reduced pulmonary capillary wedge pressure (PCWP) and systemic arterial pressure in patients with heart failure. In animal studies, nesiritide had no effect on myocardial contractility or cardiac electrophysiological parameters such as atrial and ventricular effective refractory periods or atrioventricular nodal conduction. Naturally occurring atrial natriuretic peptide (ANP) is a related peptide that increases vascular permeability in animals and humans and may reduce intravascular volume. The effect of nesiritide on vascular permeability has not been studied.

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nesiritide (BNP₁–₃₂) is a 32-amino acid C-terminal active peptide produced by proteolytic cleavage of the 108-amino acid precursor hormone proBNP₁–₁₀₈. [2]
During heart failure, the concentration of the precursor hormone (proBNP₁–₁₀₈) in serum may exceed the concentration of the active nesiritide (BNP₁–₃₂). One reason may be the difference in their degradation rates, as nesiritide (BNP₁–₃₂) is more readily degraded by proteases present on the cell membranes of human kidneys compared to the precursor hormone. [2]
This study indicates that although the potency of proBNP₁–₁₀₈ is reduced, the overall increase in circulating levels of immune-reactive BNP (including both forms) in patients with advanced heart failure may still exert significant biological activity through GC-A activation. [2]

C143H244N50O42S4

3464.05

3461.737

124584-08-3

71308561

White to off-white solid powder

1.5±0.1 g/cm3

1.679

-16.34

56

55

91

239

7750

28

CC[C@@H]([C@H]1C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(NCC(N[C@H](C(NCC(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(O)=O)CC2=CN=CN2)=O)CCCNC(N)=N)=O)CCCNC(N)=N)=O)CC(C)C)=O)C(C)C)=O)CCCCN)=O)CSSC[C@H](NC(CNC([C@@H](NC(CNC([C@@H](NC([C@@H](NC([C@@H](NC([C@@H](NC([C@@H]3CCCN3C([C@@H](N)CO)=O)=O)CCCCN)=O)CCSC)=O)C(C)C)=O)CCC(N)=O)=O)=O)CO)=O)=O)C(N[C@H](C(NCC(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N1)=O)CCCNC(N)=N)=O)CC(O)=O)=O)CCSC)=O)CCCCN)=O)CCCNC(N)=N)=O)=O)CC4=CC=CC=C4)=O)=O)=O)CC(C)C)=O)=O)CO)=O)CO)=O)CO)=O)CO)=O)C

Nesiritide Brain Natriuretic Peptide-32 human

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 40 mg/mL (~11.55 mM)

Solubility in Formulation 1: 100 mg/mL (28.87 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)