化合物信息

Description

Cardiovascular Disease

Description

Enalaprilat is an angiotensin-converting enzyme (ACE) inhibitor with IC50 of 1.94 nM.

Targets

IC₅₀

In Vitro

Enalaprilat has high affinity for human endothelial ACE with IC50 of 1.94 nM in vitro binding assay by displacing a saturating concentration of [^125I]351A, a radiolabeled lisinopril analogue from ACE binding sites, and shows bradykinin/angiotensin I selectivity ratio of 1.00 calculated from double displacement experiments. ^[1] Enalaprilat has the strong inhibitory effect on Aβ42-to-Aβ40-converting activity found in the N-domain of ACE, exhibiting a 10-fold lower IC50 (0.003~0.01 μM) than captopril (0.03~0.1 μM). ^[2] Enalaprilat (100 nM) blocks protein kinase C epsilon by directly activating bradykinin B1 receptor at the canonical Zn²⁺ binding site, leading to prolonged nitric oxide (NO) production in cytokine-treated human lung microvascular endothelial cells. ^[3] Enalaprilat attenuates the IGF-I induced neonatal rat cardiac fibroblast growth (30% reduction) in a concentration-dependent fashion, with IC50 of 90 mM. ^[4]

In Vivo

Enalaprilat has unfavourable ionisation characteristics to allow sufficient potency for oral administration, thus Enalaprilat is only suitable for intravenous administration, which is overcome by the esterification with ethanol to produce Enalapril. Administration of Enalaprilat induces a significant reduction of MAP at 70 minutes compared with the placebo group during haemorrhagic shock in rats, and results in a 50% reduction of CO, a general tendency of EB extravasation which is significant in the kidney and lungs, and a significant increase in ileal EB extravasation (53%). ^[5] Enalaprilat has no effect in nonhypertrophied hearts, but significantly attenuates the greater increase in left ventricular end-diastolic pressure in hypertrophied hearts compared with no drug (65±7 versus 50±5 mm Hg, p<0.01).>^[6]

Clinical Trials

Phase III has been completed in the extension study of comparing the long-term safety of Valsartan versus Enalapril, and the effectiveness of the combination of Valsartan and Enalapril versus Enalapril alone in children with hypertension.

Features

Enalaprilat is the first dicarboxylate-containing ACE inhibitor developed partly to overcome limitations of captopril.

Single displacement binding assay

The binding assay is based on the competitive displacement of [^125I]351A by Enalaprilat performed on whole endothelial cells. Subconfluent HUVECs in 6-well plates are rinsed with 2 mL binding buffer (140 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1.03 mM MgCl₂, 0.42 mM NaH₂PO₄, 10 mM HEPES, 2 mM sodium pyruvate and 5 mM glucose, pH 7.4), and the culture medium is replaced with 2.5 mL fresh binding buffer containing 5% fetal bovine serum (FBS). The Enalaprilat (2.5-12.5 μL, 0.1-50 nM) or equivalent volumes of diluent are added to the binding buffer. A saturating amount of [^125I]351A (10 μL, typically 10⁶ cpm) is then added to each sample and the plates are incubated at 37 °C for 2 hours in a thermostatic bath. The cells are then rinsed twice with 1.5 mL binding buffer. Finally, the cells are extracted with 0.5 mL NaOH 1 N, incubated for 5 minutes, and the radioactivity is counted with a gamma counter. The ratio of specific [^125I]351A bound to total bound activity (B/B0) is calculated, and the inhibitory potency of Enalaprilat expressed as the concentration of ACE inhibitors able to displace 50% of the bound radioligand, i.e. the IC50.

Cell Lines

Rat cardiac fibroblasts cell lines

Concentrations

Dissolved in DMSO, final concentrations 1 nM - 10 μM

Incubation Time

24 hours

Methods

After 24 hours incubation in serum-free medium (DMEM), cells are stimulated with IGF-I (1-100 nM) and coincubated with Enalaprilat (1 nM-10 μM) for 24 hours. Cellular proliferation is assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation during the last 4 hours of the 24 hours incubation period using a colorimetric immunoassay. The extinctions are measured at 450 nm in an ELISA plate reader. All values consist of an n=9.

Animal Models

Male Sprague–Dawley rats

Formulation

Dissolved in a vehicle solution (1 mg in 950 μL of phosphate buffered saline and 50 μL 1M Na₂CO₃).

Doses

1 mg/kg

Administration

I.V. bolus