Repurposed drugs in combinations exert additive anti-chikungunya virus activity: an in-vitro study.

Chikungunya virus (CHIKV) infection causes chikungunya, a viral disease that currently has no specific antiviral treatment. Several repurposed drug candidates have been investigated for the treatment of the disease. In order to improve the efficacy of the known drugs, combining drugs for treatment is a promising approach. The current study was undertaken to explore the antiviral activity of a combination of repurposed drugs that were reported to have anti-CHIKV activity. We explored the effect of different combinations of six effective drugs (2-fluoroadenine, emetine, lomibuvir, enalaprilat, metyrapone and resveratrol) at their non-toxic concentrations against CHIKV under post infection treatment conditions in Vero cells. Focus-forming unit assay, real time RT-PCR, immunofluorescence assay, and western blot were used to determine the virus titre. The results revealed that the combination of 2-fluoroadenine with either metyrapone or emetine or enalaprilat exerted inhibitory activity against CHIKV under post-infection treatment conditions. The effect of these drug combinations was additive in nature compared to the effect of the individual drugs. The results suggest an additive anti-viral effect of these drug combinations against CHIKV. The findings could serve as an outline for the development of an innovative therapeutic approach in the future to treat CHIKV-infected patients.

Lack of an Effect of Polysorbate 80 on Intestinal Drug Permeability in Humans.

PURPOSE: Despite no broad, direct evidence in humans, there is a potential concern that surfactants alter active or passive drug intestinal permeation to modulate oral drug absorption. The purpose of this study was to investigate the impact of the surfactant polysorbate 80 on active and passive intestinal drug absorption in humans. METHODS: The human (n = 12) pharmacokinetics (PK) of three probe substrates of intestinal absorption, valacyclovir, chenodeoxycholic acid (CDCA), and enalaprilat, were assessed. Endogenous bile acid levels were assessed as a secondary measure of transporter and microbiota impact. RESULTS: Polysorbate 80 did not inhibit peptide transporter 1 (PepT1)- or apical sodium bile acid transporter (ASBT)-mediated PK of valacyclovir and CDCA, respectively. Polysorbate 80 did not increase enalaprilat absorption. Modest increases in unconjugated secondary bile acid Cmax ratios suggest a potential alteration of the in vivo intestinal microbiota by polysorbate 80. CONCLUSIONS: Polysorbate 80 did not alter intestinal membrane fluidity or cause intestinal membrane disruption. This finding supports regulatory relief of excipient restrictions for Biopharmaceutics Classification System-based biowaivers.

[Enalaprilat Inhibits Zinc-Dependent Oligomerization of Metal-Binding Domain of Amyloid-beta Isoforms and Protects Human Neuroblastoma Cells from Toxic Action of these Isoforms].

Intact amyloid-ß peptides (Aß) may undergo prion-like aggregation when they interact with chemically or structurally modified variants of Aß present in extracellular pathohistological inclusions (amyloid plaques). This aggregation is regarded as one of the key molecular mechanisms of Alzheimer's disease (AD) pathogenesis. Zinc ions are involved in the pathological dimerization and oligomerization of natural Aß isoforms, and zinc-induced oligomers can also initiate the pathological aggregation of Aß. Based on the earlier found molecular mechanism of zinc-dependent oligomerization of Aß, it has been suggested that the targeted inhibition of the 11EVHH14 site in one Aß molecule from zinc-mediated interactions with the same site of another Aß molecule can effectively inhibit the oligomerization and aggregation of Aß. Taking into account the similarity in the structural organization of zinc-binding sites within Aß and angiotensin-converting enzyme (ACE), we hypothesized that inhibitors of the ACE active sites could specifically interact with the 11EVHH14 site of Aß. Using a surface plasmon resonance biosensor and nuclear magnetic resonance spectroscopy, we have found that the ACE inhibitor enalaprilat effectively inhibits zinc-dependent dimerization of the metal-binding domains of intact Aß and Aß with isomerized Asp7 (isoAß). We have also found that enalaprilat protects SH-SY5Y human neuroblastoma cells from the toxic effects of Aß(1-42) and isoAß(1-42), which are among the most common components of amyloid plaques. The results confirm the role of zincdependent oligomerization of Aß in AD pathogenesis and make it possible one to consider enalaprilat as a prototype of antiaggregation agents for treating AD.

A Soluble Guanylate Cyclase Activator Inhibits the Progression of Diabetic Nephropathy in the ZSF1 Rat.

Therapies that restore renal cGMP levels are hypothesized to slow the progression of diabetic nephropathy. We investigated the effect of BI 703704, a soluble guanylate cyclase (sGC) activator, on disease progression in obese ZSF1 rats. BI 703704 was administered at doses of 0.3, 1, 3, and 10 mg/kg/d to male ZSF1 rats for 15 weeks, during which mean arterial pressure (MAP), heart rate (HR), and urinary protein excretion (UPE) were determined. Histologic assessment of glomerular and interstitial lesions was also performed. Renal cGMP levels were quantified as an indicator of target modulation. BI 703704 resulted in sGC activation, as evidenced by dose-dependent increases in renal cGMP levels. After 15 weeks of treatment, sGC activation resulted in dose-dependent decreases in UPE (from 463 ± 58 mg/d in vehicle controls to 328 ± 55, 348 ± 23, 283 ± 45, and 108 ± 23 mg/d in BI 703704-treated rats at 0.3, 1, 3, and 10 mg/kg, respectively). These effects were accompanied by a significant reduction in the incidence of glomerulosclerosis and interstitial lesions. Decreases in MAP and increases in HR were only observed at the high dose of BI 703704. These results are the first demonstration of renal protection with sGC activation in a nephropathy model induced by type 2 diabetes. Importantly, beneficial effects were observed at doses that did not significantly alter MAP and HR.

Blockade of the renin-angiotensin system prevents acute and immunologically relevant colitis in murine models.

BACKGROUND: Blockade of the renin-angiotensin system (RAS) has been shown to alleviate inflammatory processes in the gastrointestinal tract. The aim of this study was to determine if blockade of the RAS would be effective in an immunologically relevant colitis model, and to compare outcome with an acute colitis model. METHODS: A losartan analog, CCG-203025 (C23H26ClN3O5S) containing a highly polar sulfonic acid moiety that we expected would allow localized mucosal antagonism with minimal systemic absorption was selected as an angiotensin II type 1a receptor antagonist (AT1aR-A). Two colitis models were studied: (1) Acute colitis was induced in 8- to 10-week-old C57BL/6J mice by 2.5 % dextran sodium sulfate (DSS, in drinking water) for 7 days. (2) IL10-/-colitis Piroxicam (200 ppm) was administered orally in feed to 5-week-old IL-10-/-mice (C57BL/6J background) for 14 days followed by enalaprilat (ACE-I), CCG-203025 or PBS administered transanally for 14 days. RESULTS: In the DSS model, weight loss and histologic score for CCG-203025 were better than with placebo. In the IL10-/-model, ACE-I suppressed histologic damage better than CCG-203025. Both ACE-I and CCG-203025 reduced pro-inflammatory cytokines and chemokines. CONCLUSIONS: This study demonstrated the therapeutic efficacy of both ACE-I and AT1aR-A for preventing the development of both acute and immunologically relevant colitis.

Upregulation of SERCA2a following short-term ACE inhibition (by enalaprilat) alters contractile performance and arrhythmogenicity of healthy myocardium in rat.

Chronic angiotensin-converting enzyme inhibitor (ACEIs) treatment can suppress arrhythmogenesis. To examine whether the effect is more immediate and independent of suppression of pathological remodelling, we tested the antiarrhythmic effect of short-term ACE inhibition in healthy normotensive rats. Wistar rats were administered with enalaprilat (ENA, i.p., 5 mg/kg every 12 h) or vehicle (CON) for 2 weeks. Intraarterial blood pressure in situ was measured in A. carotis. Cellular shortening was measured in isolated, electrically paced cardiomyocytes. Standard 12-lead electrocardiography was performed, and hearts of anaesthetized open-chest rats were subjected to 6-min ischemia followed by 10-min reperfusion to examine susceptibility to ventricular arrhythmias. Expressions of calcium-regulating proteins (SERCA2a, cardiac sarco/endoplasmic reticulum Ca(2+)-ATPase; CSQ, calsequestrin; TRD, triadin; PLB, phospholamban; Thr(17)-PLB-phosphorylated PLB at threonine-17, FKBP12.6, FK506-binding protein, Cav1.2-voltage-dependent L-type calcium channel alpha 1C subunit) were measured by Western blot; mRNA levels of L-type calcium channel (Cacna1c), ryanodine receptor (Ryr2) and potassium channels Kcnh2 and Kcnq1 were measured by qRT-PCR. ENA decreased intraarterial systolic as well as diastolic blood pressure (by 20%, and by 31%, respectively, for both P < 0.05) but enhanced shortening of cardiomyocytes at basal conditions (by 34%, P < 0.05) and under beta-adrenergic stimulation (by 73%, P < 0.05). Enalaprilat shortened QTc interval duration (CON 78 ± 1 ms vs. ENA 72 ± 2 ms; P < 0.05) and significantly decreased the total duration of ventricular fibrillations (VF) and the number of VF episodes (P < 0.05). Reduction in arrhythmogenesis was associated with a pronounced upregulation of SERCA2a (CON 100 ± 20 vs. ENA 304 ± 13; P < 0.05) and complete absence of basal Ca(2+)/calmodulin-dependent phosphorylation of PLB at Thr(17). Short-term ACEI treatment can provide protection against I/R injury-induced ventricular arrhythmias in healthy myocardium, and this effect is associated with increased SERCA2a expression.

Target Reserve and Turnover Parameters Determine Rightward Shift of Enalaprilat Potency From its Binding Affinity to the Angiotensin Converting Enzyme.

Drug effects are often assumed to be directly proportional to the fraction of occupied targets. However, for a number of antagonists that exhibit target-mediated drug disposition (TMDD), such as angiotensin-converting enzyme (ACE) inhibitors, drug binding to the target at low concentrations may be significant enough to influence pharmacokinetics but insufficient to elicit a drug response (i.e., differences in drug-target binding affinity and potency). In this study, a pharmacokinetic/pharmacodynamic model for enalaprilat was developed in humans to provide a theoretical framework for assessing the relationship between ex vivo drug potency (IC50) and in vivo target-binding affinity (KD). The model includes competitive binding of angiotensin I and enalaprilat to ACE and accounts for the circulating target pool. Data were obtained from the literature, and model fitting and parameter estimation were conducted using maximum likelihood in ADAPT5. The model adequately characterized time-courses of enalaprilat concentrations and four biomarkers in the renin-angiotensin system and provided estimates for in vivo KD (0.646 nM) and system-specific parameters, such as total target density (32.0 nM) and fraction of circulating target (19.8%), which were in agreement with previous reports. Model simulations were used to predict the concentration-effect curve of enalaprilat, revealing a 6.3-fold increase in IC50 from KD. Additional simulations demonstrated that target reserve and degradation parameters are key factors determining the extent of shift of enalaprilat ex vivo potency from its in vivo binding affinity. This may be a common phenomenon for drugs exhibiting TMDD and has implications for translating binding affinity to potency in drug development.

Angiotensin converting enzyme-inhibitor reduces colitis severity in an IL-10 knockout model.

BACKGROUND: We previously demonstrated angiotensin converting enzymes (ACE) over-expression in a dextran-sodium sulfate colitis model; ACE inhibitor (ACE-I) treatment reduced colitis severity in this model. However, ACE-I has not been tested in more immunologically relevant colitis models. AIM: We hypothesized that ACE-I would decrease disease severity in an IL-10 knockout (-/-) colitis model. METHODS: Colitis was induced by giving 10-week old IL-10-/- mice piroxicam (P.O.) for 14 days. The ACE-I enalaprilat was given transanally at a dose of 6.25 mg/kg for 21 days. Prednisolone (PSL) with or without enalaprilat were used as therapeutic, comparative groups. All groups were compared to a placebo treated group. Outcome measures were clinical course, histology, abundance of pro-inflammatory cytokines/chemokines, and epithelial barrier function. RESULTS: Enalaprilat exhibited better survival (91 %) versus other treatment groups (PSL: 85.7 %, PSL + ACE-I: 71.4 %, placebo: 66.6 %). The ACE-I and PSL + ACE-I groups showed significantly better histological scores versus placebo mice. ACE-I and the PSL groups significantly reduced several pro-inflammatory cytokines versus placebo mice. FITC-dextran permeability was reduced in the ACE-I and PSL + ACE-I groups. Blood pressure was not affected in ACE-I treated mice compared to placebo mice. CONCLUSIONS: ACE-I was effective in reducing severity of colitis in an IL-10-/- model. The addition of prednisolone minimally augmented this effect. The findings suggest that appropriately dosed ACE-I with or without steroids may be a new therapeutic agent for colitis.

Comprehensive evaluation of the effects of enalapril on matrix metalloproteinases levels in hypertension.

PURPOSE: Angiotensin-converting enzyme inhibitors (ACEi) may downregulate matrix metalloproteinases (MMPs). We examined whether enalapril affects MMP-2, MMP-8, and MMP-9 levels and activity, and their endogenous inhibitors (tissue inhibitors of MMPs, TIMP-1 and TIMP-2) levels in hypertensive patients. Moreover, we assessed the effects of enalaprilat on MMP-9 and TIMP-1 secretion by human endothelial cells (HUVECs). METHODS: Thirty-eight hypertensive patients received enalapril for 8 weeks and were compared with thirty-eight normotensive controls. Blood samples were collected at baseline and after treatment. Plasma ACE activity was determined by a fluorimetric assay. Plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 were measured by ELISA and gelatin zymography. A fluorogenic peptide cleavage assay was used to measure MMP activity. HUVECs cells were stimulated by phorbol-12-myristate-13-acetate (PMA) and the effects of enalaprilat (10(-10) to 10(-6) M) on MMP-9 and TIMP-1 levels were determined. RESULTS: Enalapril decreased blood pressure and ACE activity in hypertensive patients (P < 0.05), but had no effects on plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 levels, or MMP activity. Enalaprilat had no effects on PMA-induced increases in MMP-9 and TIMP-1 secretion by HUVECs or on MMP activity. CONCLUSIONS: We show consistent evidence, both in vivo and in vitro, that enalapril does not affect MMPs and TIMPs levels in hypertensive patients.

The pharmacological mechanism of angiotensin-converting enzyme inhibition by green tea, Rooibos and enalaprilat - a study on enzyme kinetics.

Green tea (Camellia sinensis L.) and Rooibos (Aspalathus linearis Dahlg.) inhibit angiotensin-converting enzyme (ACE) in vitro and in vivo. The ACE inhibitor enalaprilat has been described previously as a competitive inhibitor and sometimes as a non-competitive inhibitor. The aim of this study was to investigate the pharmacological mechanism of ACE inhibition of green tea and Rooibos by enzyme kinetics, and to compare this with enalaprilat. A Michaelis-Menten kinetics and Lineweaver-Burk graph showed mean values of V(max) = 3.73 µM and K(m) = 0.71 µM for green tea, of V(max) = 6.76 µM and K(m) = 0.78 µM for Rooibos, of V(max) = 12.54 µM and K(m) = 2.77 µM for enalaprilat, and of V(max) = 51.33 µM and K(m) = 9.22 µM for the PBS control. Incubating serum with green tea or Rooibos saturated with zinc chloride did not change the inhibitory effect. Enalaprilat preincubated with zinc chloride showed a decrease in the inhibitory effect. In conclusion, green tea, Rooibos and enalaprilat seem to inhibit ACE activity using a mixed inhibitor mechanism.

Inhibition of ACE activity contributes to the intestinal structural compensation in a massive intestinal resection rat model.

BACKGROUND: Intestinal adaptation in short bowel syndrome (SBS) consists of increased epithelial cells (ECs) proliferation as well as apoptosis. Angiotensin-converting enzyme (ACE) has been shown to regulate ECs apoptosis. In this study, we investigated the effect of ACE inhibition on intestinal adaptation after small bowel resection (SBR) in a rat model. METHODS: Sprague-Dawley rats were used and were divided into four groups: (1) Sham group received an ileum transection (n = 6); (2) Sham + ACE-I group received an ileum transaction and lavage with ACE inhibitor (ACE-I, enalaprilat, 2 mg/kg/day) (n = 6); (3) SBS group received a 70 % mid-intestinal resection (n = 6); (4) SBS + ACE-I group received a 70 % mid-intestinal resection and lavage with enalaprilat (2 mg/kg/day) (n = 6). Sampling was done 10 days after surgery. ECs apoptosis was studied by TUNEL staining. ACE, angiotensin II (ANGII) receptor type 1 (AT1R) and receptor type 2 (AT2R) expressions were detected with RT-PCR and immunofluorescent confocal microscopy. RESULTS: SBR leads to significant intestinal hypertrophy. The addition of ACE-I to SBS rat resulted in a significant decline in ECs apoptosis. ACE mRNA expression was significantly elevated after SBS creation (0.24 ± 0.07 vs. 0.42 ± 0.11), and ACE-I administration further increased mucosal ACE mRNA expression (0.54 ± 0.12). Interestingly, AT1R mRNA expression showed a significant decline in the SBS group compared to Sham levels, and ACE-I administration increased AT1R mRNA expression to Sham levels. No significant difference in AT2R mRNA expression was found between Sham and SBS group. CONCLUSION: These results offer further insight into the role of ACE on intestinal mucosal remolding after massive bowel resection. ACE-I may be beneficial to SBS patients via a reduction of the apoptotic rate, thus facilitating the degree of adaptation.

Inhibitory effects of enalaprilat on rat cardiac fibroblast proliferation via ROS/P38MAPK/TGF-ß1 signaling pathway.

Enalaprilat (Ena.), an angiotensin II (Ang II) converting enzyme inhibitor (ACEI), can produce some therapeutic effects on hypertension, ventricular hypertrophy and myocardial remodeling in clinic, but its precise mechanism, especially its signaling pathways remain elusive. In this study, cardiac fibroblasts (CFb) was isolated by the trypsin digestion method; a BrdU proliferation assay was adopted to determine cell proliferation; an immunofluorescence assay was used to measure intracellular reactive oxygen species (ROS); immunocytochemistry staining and Western blotting assay were used to detect phosphorylated p38 mitogen activated protein kinase (p-p38MAPK) and transforming growth factor-ß(1) (TGF-ß(1)) protein expression, respectively. The results showed that Ang II (10(-7) M) stimulated the cardiac fibroblast proliferation which was inhibited by NAC (an antioxidant), SB203580 (a p38MAPK inhibitor) or enalaprilat; Ang II caused an burst of intracellular ROS level within thirty minutes, an increase in p-p38MAPK (3.6-fold of that in the control group), as well as an elevation of TGF-ß(1) meantime; NAC, an antioxidant, and enalaprilat treatment attenuated cardiac fibroblast proliferation induced by Ang II and decreased ROS and p-p38MAPK protein levels in rat cardiac fibroblast; SB203580 lowered TGF-ß(1) protein expression in rats' CFb in a dose-dependent manner. It could be concluded that enalaprilat can inhibit the cardiac fibroblast proliferation induced by Ang II via blocking ROS/P38MAPK/TGF-ß(1) signaling pathways and the study provides a theoretical proof for the application of ACEIs in treating myocardial fibrosis and discovering the primary mechanism through which ACEIs inhibit CFb proliferation.

A Transcriptomics-Based Bioinformatics Approach for Identification and In Vitro Screening of FDA-Approved Drugs for Repurposing against Dengue Virus-2.

The rising incidence of dengue virus (DENV) infections in the tropical and sub-tropical regions of the world emphasizes the need to identify effective therapeutic drugs against the disease. Repurposing of drugs has emerged as a novel concept to combat pathogens. In this study, we employed a transcriptomics-based bioinformatics approach for drug identification against DENV. Gene expression omnibus datasets from patients with different grades of dengue disease severity and healthy controls were used to identify differentially expressed genes in dengue cases, which were then applied to the query tool of Connectivity Map to identify the inverse gene-disease-drug relationship. A total of sixteen identified drugs were investigated for their prophylactic, virucidal, and therapeutic effects against DENV. Focus-forming unit assay and quantitative RT-PCR were used to evaluate the antiviral activity. Results revealed that five compounds, viz., resveratrol, doxorubicin, lomibuvir, elvitegravir, and enalaprilat, have significant anti-DENV activity. Further, molecular docking studies showed that these drugs can interact with a variety of protein targets of DENV, including the glycoprotein, the NS5 RdRp, NS2B-NS3 protease, and NS5 methyltransferase The in vitro and in silico results, therefore, reveal that these drugs have the ability to decrease DENV-2 production, suggesting that these drugs or their derivatives could be attempted as therapeutic agents against DENV infections.

Effects of soluble guanylate cyclase stimulator on renal function in ZSF-1 model of diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is associated with endothelial dysfunction and oxidative stress, in which the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway is impaired. We hypothesize that sGC stimulator Compound 1 can enhance NO signaling, reduce proteinuria in a diabetic nephropathy preclinical model with diminished NO bioavailability and increased oxidized sGC. Therefore, we evaluated the effect of sGC stimulator Compound 1 on the renal effect in obese ZSF1 (ZSF1 OB) rats. MATERIALS AND METHODS: The sGC stimulator Compound 1, the standard of care agent Enalapril, and a combination of Compound 1 and Enalapril were administered chronically to obese ZSF1 rats for 6 months. Mean arterial pressure, heart rate, creatinine clearance for glomerular filtration rate (eGFR), urinary protein excretion to creatinine ratio (UPCR), and urinary albumin excretion ratio (UACR) were determined during the study. The histopathology of glomerular and interstitial lesions was assessed at the completion of the study. RESULTS: While both Compound 1 and Enalapril significantly reduced blood pressure, the combination of Compound 1 and Enalapril normalized blood pressure levels. Compound 1 improved eGFR and reduced UPCR and UACR. A combination of Enalapril and Compound 1 resulted in a marked reduction in UPCR and UACR and improved GFR. CONCLUSION: The sGC stimulator Compound 1 as a monotherapy slowed renal disease progression, and a combination of the sGC stimulator with Enalapril provided greater renal protection in a rodent model of diabetic nephropathy.

A novel pathway for receptor-mediated post-translational activation of inducible nitric oxide synthase.

Inducible nitric oxide synthase (iNOS) is a major source of nitric oxide during inflammation whose activity is thought to be controlled primarily at the expression level. The B1 kinin receptor (B1R) post-translationally activates iNOS beyond its basal activity via extracellular signal regulated kinase (ERK)-mediated phosphorylation of Ser(745) . Here we identified the signalling pathway causing iNOS activation in cytokine-treated endothelial cells or HEK293 cells transfected with iNOS and B1R. To allow kinetic measurements of nitric oxide release, we used a sensitive porphyrinic microsensor (response time = 10 msec.; 1 nM detection limit). B1Rs signalled through Gαi coupling as ERK and iNOS activation were inhibited by pertussis toxin. Furthermore, transfection of constitutively active mutant Gαi Q204L but not Gαq Q209L resulted in high basal iNOS-derived nitric oxide. G-ßγ subunits were also necessary as transfection with the ß-adrenergic receptor kinase C-terminus inhibited the response. B1R-dependent iNOS activation was also inhibited by Src family kinase inhibitor PP2 and trans-fection with dominant negative Src. Other ERK-MAP kinase members were involved as the response was inhibited by dominant negative H-Ras, Raf kinase inhibitor, ERK activation inhibitor and MEK inhibitor PD98059. In contrast, PI3 kinase inhibitor LY94002, calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), protein kinase C inhibitor calphostin C and protein kinase C activator PMA had no effect. Angiotensin converting enzyme inhibitor enalaprilat also directly activated B1Rs to generate high output nitric oxide via the same pathway. These studies reveal a new mechanism for generating receptor-regulated high output nitric oxide in inflamed endothelium that may play an important role in the development of vascular inflammation.

Modulation of nitric oxide affects myocardial perfusion-contraction matching in anaesthetized dogs with recurrent no-flow ischaemia.

Myocardial perfusion and contraction are closely coupled; however, the effect of recurrent no-flow ischaemia on perfusion-contraction matching remains to be established. In the present studies, we examined the influence of modulating nitric oxide availability on perfusion-contraction matching after recurrent no-flow ischaemia in acute open-chest, anaesthetized dogs. The following three groups were studied: (1) saline; (2) L-NAME (10 mg kg(-1) I.V.); and (3) enalaprilat (1.5 mg kg(-1) I.V.). Regional myocardial blood flow was measured with microspheres and contractile function with piezoelectric crystals to determine systolic wall thickening. Dogs underwent four cycles of 5 min acute ischaemia and 5 min coronary reperfusion; area at risk was similar for all groups. In all dogs, ischaemic zone contractile function was depressed after recurrent no-flow ischaemia despite increased myocardial blood flow during reperfusion; contractile function was further depressed during L-NAME and was partly restored with enalaprilat. Within the ischaemic region, blood flow in subendocardial and subepicardial layers increased significantly compared with baseline during each reperfusion period independently of treatment. Our findings suggest that reduced NO availability can significantly impair myocardial perfusion-contraction matching, which is partly restored by administration of an NO donor.

Enalaprilat-mediated activation of kinin b(1) receptors and vasodilation in the rat isolated perfused kidney.

The present study evaluated whether enalaprilat (the active form of enalapril, an angiotensin-converting enzyme inhibitor) activates B(1) receptors. We observed that the levels of B(1) receptor mRNA and protein expression were upregulated in the kidneys of diabetic rats. Bradykinin (BK)-induced renal vasodilation decreased in isolated perfused kidneys of diabetic rats, but des-Arg(9)-BK-induced renal vasodilation increased. Enalaprilat also produced vasodilation in the isolated perfused kidneys of control and diabetic rats. The response to des-Arg(9)-BK or enalaprilat was blocked by Lys-(des-Arg(9), Leu(8))-BK (a B(1) receptor antagonist) and N-nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase). These results suggest that enalaprilat activates B(1) receptors and stimulates the production of nitric oxide in the kidneys of both control and diabetic rats.

Diallyl sulfide protects against dilated cardiomyopathy via inhibition of oxidative stress and apoptosis in mice.

Cytochrome P450 family 2 subfamily E member 1 (CYP2E1) is a member of the cytochrome P450 enzyme family and catalyzes the metabolism of various substrates. CYP2E1 is upregulated in multiple heart diseases and causes damage mainly via the production of reactive oxygen species (ROS). In mice, increased CYP2E1 expression induces cardiac myocyte apoptosis, and knockdown of endogenous CYP2E1 can attenuate the pathological development of dilated cardiomyopathy (DCM). Nevertheless, targeted inhibition of CYP2E1 via the administration of drugs for the treatment of DCM remains elusive. Therefore, the present study aimed to investigate whether diallyl sulfide (DAS), a competitive inhibitor of CYP2E1, can be used to inhibit the development of the pathological process of DCM and identify its possible mechanism. Here, cTnTR141W transgenic mice, which developed typical DCM phenotypes, were used. Following treatment with DAS for 6 weeks, echocardiography, histological analysis and molecular marker detection were conducted to investigate the DAS­induced improvement on myocardial function and morphology. Biochemical analysis, western blotting and TUNEL assays were used to detected ROS production and myocyte apoptosis. It was found that DAS improved the typical DCM phenotypes, including chamber dilation, wall thinning, fibrosis, poor myofibril organization and decreased ventricular blood ejection, as determined using echocardiographic and histopathological analyses. Furthermore, the regulatory mechanisms, including inhibition both of the oxidative stress levels and the mitochondria­dependent apoptosis pathways, were involved in the effects of DAS. In particular, DAS showed advantages in terms of improved chamber dilation and dysfunction in model mice, and the improvement occurred in the early stage of the treatment compared with enalaprilat, an angiotensin­converting enzyme inhibitor that has been widely used in the clinical treatment of DCM and HF. The current results demonstrated that DAS could protect against DCM via inhibition of oxidative stress and apoptosis. These findings also suggest that inhibition of CYP2E1 may be a valuable therapeutic strategy to control the development of heart diseases, especially those associated with CYP2E1 upregulation. Moreover, the development of DAS analogues with lower cytotoxicity and metabolic rate for CYP2E1 may be beneficial.

Abeta42-to-Abeta40- and angiotensin-converting activities in different domains of angiotensin-converting enzyme.

Amyloid beta-protein 1-42 (Abeta42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Abeta. In contrast, Abeta40 is the predominant secreted form of Abeta and recent studies have suggested that Abeta40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Abeta42 to Abeta40, and its inhibition enhances brain Abeta42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628-8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Abeta42 to Abeta40. Interestingly, Abeta42-to-Abeta40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Abeta42-to-Abeta40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Abeta42-to-Abeta40-converting activity of ACE or increasing neurotoxic Abeta42.