Acute Inhibition of Intestinal Neprilysin Enhances Insulin Secretion via GLP-1 Receptor Signaling in Male Mice.

The peptidase neprilysin modulates glucose homeostasis by cleaving and inactivating insulinotropic peptides, including some produced in the intestine such as glucagon-like peptide-1 (GLP-1). Under diabetic conditions, systemic or islet-selective inhibition of neprilysin enhances beta-cell function through GLP-1 receptor (GLP-1R) signaling. While neprilysin is expressed in intestine, its local contribution to modulation of beta-cell function remains unknown. We sought to determine whether acute selective pharmacological inhibition of intestinal neprilysin enhanced glucose-stimulated insulin secretion under physiological conditions, and whether this effect was mediated through GLP-1R. Lean chow-fed Glp1r+/+ and Glp1r-/- mice received a single oral low dose of the neprilysin inhibitor thiorphan or vehicle. To confirm selective intestinal neprilysin inhibition, neprilysin activity in plasma and intestine (ileum and colon) was assessed 40 minutes after thiorphan or vehicle administration. In a separate cohort of mice, an oral glucose tolerance test was performed 30 minutes after thiorphan or vehicle administration to assess glucose-stimulated insulin secretion. Systemic active GLP-1 levels were measured in plasma collected 10 minutes after glucose administration. In both Glp1r+/+ and Glp1r-/- mice, thiorphan inhibited neprilysin activity in ileum and colon without altering plasma neprilysin activity or active GLP-1 levels. Further, thiorphan significantly increased insulin secretion in Glp1r+/+ mice, whereas it did not change insulin secretion in Glp1r-/- mice. In conclusion, under physiological conditions, acute pharmacological inhibition of intestinal neprilysin increases glucose-stimulated insulin secretion in a GLP-1R-dependent manner. Since intestinal neprilysin modulates beta-cell function, strategies to inhibit its activity specifically in the intestine may improve beta-cell dysfunction in type 2 diabetes.

A comparative study between angiotensin receptor neprilysin inhibitor (thiorphan/irbesartan) with each of nitrate and carvedilol in a rat model of myocardial ischemic reperfusion injury.

The combined angiotensin receptor neprilysin inhibitor is a promising cardioprotective pharmacological agent. This study investigated the beneficial effects of thiorphan (TH)/irbesartan (IRB), in myocardial ischemia-reperfusion (IR) injury, compared to each of nitroglycerin and carvedilol. Male Wistar rats were divided into five groups (10 rats/group): Sham, untreated I/R, TH/IRB + IR (0.1/10 mg/kg), nitroglycerin + IR (0.2 mg/kg), and carvedilol + IR (10 mg/kg). Mean arterial blood pressure, cardiac functions and arrhythmia incidence, duration and score were assessed. Cardiac levels of creatine kinase-MB (CK-MB), oxidative stress, endothelin-1, ATP, Na+ /K+ ATPase pump activity and mitochondria complexes activities were measured. Histopathological examination, Bcl/Bax immunohistochemistry studies and electron microscopy examination of left ventricle were performed. TH/IRB preserved the cardiac functions and mitochondrial complexes activities, mitigated cardiac damage, reduced oxidative stress and arrhythmia severity, improved the histopathological changes and decreased cardiac apoptosis. TH/IRB showed a comparable effect to each of nitroglycerin and carvedilol in alleviating the IR injury consequences. TH/IRB showed significant preservation of mitochondrial complexes activity I and II compared to nitroglycerin. TH/IRB significantly increased LVdP/dtmax and decreased oxidative stress, cardiac damage and endothelin-1 along with increasing the ATP content, Na+ /K+ ATPase pump activity and mitochondrial complexes activity when compared to carvedilol. TH/IRB showed a cardioprotective effect in reducing IR injury that is comparable to each of nitroglycerin and carvedilol that could be explained in part by its ability to preserve mitochondrial function, increase ATP, decrease oxidative stress as well as endothelin 1.

Enhancement of Anti-Tumoral Immunity by ß-Casomorphin-7 Inhibits Cancer Development and Metastasis of Colorectal Cancer.

ß-Casomorphin-7 (BCM) is a degradation product of ß-casein, a milk component, and has been suggested to affect the immune system. However, its effect on mucosal immunity, especially anti-tumor immunity, in cancer-bearing individuals is not clear. We investigated the effects of BCM on lymphocytes using an in vitro system comprising mouse splenocytes, a mouse colorectal carcinogenesis model, and a mouse orthotopic colorectal cancer model. Treatment of mouse splenocytes with BCM in vitro reduced numbers of cluster of differentiation (CD) 20+ B cells, CD4+ T cells, and regulatory T cells (Tregs), and increased CD8+ T cells. Administration of BCM and the CD10 inhibitor thiorphan (TOP) to mice resulted in similar alterations in the lymphocyte subsets in the spleen and intestinal mucosa. BCM was degraded in a concentration- and time-dependent manner by the neutral endopeptidase CD10, and the formed BCM degradation product did not affect the lymphocyte counts. Furthermore, degradation was completely suppressed by TOP. In the azoxymethane mouse colorectal carcinogenesis model, the incidence of aberrant crypt foci, adenoma, and adenocarcinoma was reduced by co-treatment with BCM and TOP. Furthermore, when CT26 mouse colon cancer cells were inoculated into the cecum of syngeneic BALB/c mice and concurrently treated with BCM and TOP, infiltration of CD8+ T cells was promoted, and tumor growth and liver metastasis were suppressed. These results suggest that by suppressing the BCM degradation system, the anti-tumor effect of BCM is enhanced and it can suppress the development and progression of colorectal cancer.

Development, In Vitro and In Vivo Evaluation of Racecadotril Orodispersible Films for Pediatric Use.

The present study endeavored to develop orodispersible films (ODFs) containing 30 mg racecadotril for pediatric use, which focuses on improving the compliance of pediatric patients and reducing risk of choking. The challenge of this study is to prepare high drug loading ODFs with successful mechanical and physicochemical properties. Compatibilities between drug and different polymers (hydroxypropyl methylcellulose, HPMC; polyvinyl alcohol, PVA; low-substituted hydroxypropyl cellulose, L-HPC; pullulan, PU) were investigated to select stable and safe film-forming polymers. Afterwards, the study explored the maximum amount of racecadotril incorporated into PVA films and PU films. Subsequently, disintegrant (Lycoat RS720, 4-10%, w/w) and plasticizers (glycerol, 2-6%, w/w) were investigated to reduce disintegration time of PVA films and enhance the flexibility of PU films, respectively. Formulation characteristics (appearance, tensile strength, percent elongation, disintegration time, drug content, weight, thickness, pH value, moisture content, moisture uptake, and Q5min) of prepared ODFs were examined to obtain the optimal compositions of racecadotril ODFs. Differential scanning calorimetry (DSC) study, powder X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) study, comparative in vitro dissolution study, and pharmacokinetic study in Beagle dogs of optimized racecadotril ODFs were then conducted. Eventually, ODFs containing 50% racecadotril, 38% PVA, 7% Lycoat RS720, 2% sucralose, 2% apricot, and 1% titanium dioxide could achieve desirable mechanical properties, disintegrating within a few seconds and releasing more than 85% drug within 5 min in four dissolution media. An in vivo study showed optimized racecadotril ODF and Hidrasec were bioequivalent in Beagle dogs. In summary, ODFs containing 30 mg racecadotril were successfully prepared by solvent casting method, and it was suitable for the administration to the pediatric patients.

Neprilysin Controls the Synaptic Activity of Neuropeptides in the Intercalated Cells of the Amygdala.

Endogenous opioid peptides in the amygdala regulate many of our behaviors and emotional responses. In particular, the endogenous opioid enkephalin plays a significant role in regulating amygdala activity, but its action is strongly limited by peptidases, which degrade enkephalin into inactive fragments. Inhibiting peptidases may be an attractive method to enhance endogenous opioid signaling; however, we do not know which specific peptidase(s) to target. Using inhibition of glutamate release onto the intercalated cells of the amygdala as an assay for enkephalin activity, we applied specific peptidase inhibitors to determine which peptidase(s) regulate enkephalin signaling in this region. Thiorphan (10 µM), captopril (1 µM), or bestatin (10 µM) were used to inhibit the activity of neprilysin, angiotensin-converting enzyme, or aminopeptidase N, respectively. In rat brain slices containing the intercalated cells, we found that inhibition of glutamate release by a submaximal concentration of enkephalin was doubled by application of all three peptidase inhibitors combined. Then, we tested inhibitors individually and found that inhibition of neprilysin alone could enhance enkephalin responses to the same extent as inhibitors of all three peptidases combined. This indicates neprilysin is the predominant peptidase responsible for degrading enkephalins in the intercalated cells of the amygdala. This differs from the striatum, locus coeruleus, and spinal cord, where multiple peptidases metabolize enkephalin. These data highlight the importance of knowing which specific peptidase(s) control opioid actions in the relevant neural circuit and how they change in disease states to allow rational choices of drugs targeting the specific peptidase of interest. SIGNIFICANCE STATEMENT: Endogenous opioids modulate many of our emotional and behavioral responses. In the amygdala, they modulate our pain, fear, and addictive behaviors. Their actions are terminated when they are catabolized into inactive fragments by at least three different peptidases. In this study, we found that neprilysin selectively controls endogenous opioid concentrations at synapses in the intercalated cells of the amygdala. This peptidase may be a target for regulation of endogenous opioid modulation of amygdala-mediated emotional and behavioral responses.

Angiotensin receptor-neprilysin inhibitior (thiorphan/irbesartan) decreased ischemia-reperfusion induced ventricular arrhythmias in rat; in vivo study.

Ventricular arrhythmias are considered as a major risk of sudden cardiac death. This study was designed to investigate the potential effects of angiotensin receptor neprilysin inhibitor; thiorphan/irbesartan (TH/IRB) combination therapy on myocardial ischemic-reperfusion (I/R)-induced arrhythmia. Fifty male Wistar rats were divided into 5 groups; (I, II): Sham, I/R both received DMSO intraperitoneally before the procedure. (III, IV, V): TH/IRB + IR (0.1/5 mg/kg, 0.1/10 mg/kg and 0.1/15 mg/kg). The drugs were injected intraperitoneally 15 min before I/R induction. Electrocardiograms changes, mean arterial blood pressure, incidence of ventricular tachycardia (VT), incidence of ventricular fibrillation (VF) and arrhythmia score were assessed. Cardiac levels of creatinine kinase-MB (CK-MB), Malondialdehyde (MDA), superoxide dismutase (SOD), endothelin-1 (ET-1), ATP content, and Na+/K+-ATPase pump activity were measured. TH (0.1 mg/kg) in combination with IRB (5, 10 and 15 mg/kg) produced significant decrease in QTc interval duration, ST height, incidence of VT and VF, duration of VT + VF, and arrhythmia score compared to I/R group. All treated groups showed significant decrease in the cardiac levels of: CK-MB, MDA and ET-1 and significant increase in SOD, ATP content, and Na+/K+-ATPase pump activity compared to I/R. TH/IRB + IR (0.1/10 mg/kg) group produced significant decrease in CK-MB, MDA and ET-1 and a significant increase in SOD, ATP content, and Na+/K+-ATPase pump activity compared to other treated groups. In conclusion, angiotensin receptor neprilysin inhibitor (thiorphan/irbesartan) decreased arrhythmia score and decreased cardiac damage. These could be explained in part by its ability to decrease oxidative stress and ET-1, increase ATP, and Na+/K+-ATPase pump activity in this rat model of I/R-induced arrhythmia.

Neutral endopeptidase (NEP) inhibitors - thiorphan, sialorphin, and its derivatives exert anti-proliferative activity towards colorectal cancer cells in vitro.

Neutral endopeptidase (NEP) is an enzyme implicated in development of different tumors, e.g. colorectal cancer (CRC). In this study, the anti-cancer effects of NEP inhibitors, thiorphan (synthetic compound) and sialorphin (naturally occurring pentapeptide) on CRC cells were investigated. Moreover, we synthesized some derivatives of sialorphin (alanine scan analogues: AHNPR, QANPR, QHAPR, QHNAR; N-acetylated sialorphin; C-amidated sialorphin, and C-amidated alanine scan analogues) to examine the biological activity of these inhibitors on CRC cells. The cytotoxic activity of the NEP inhibitors against CRC cell lines (SW620 and LS180) and normal human fibroblasts (HSF) was evaluated. Additionally, the influence of NEP inhibitors on proliferation, cell cycle progression, induction of apoptosis, and the level of phosphorylation of MAP kinases and mTORC1 signaling pathway proteins in CRC cells were examined. The NEP inhibitors were non-cytotoxic to HSF cells; however, most of them slightly decreased the viability and inhibited proliferation of CRC cells. The N-acetylation or C-amidation of sialorphin or its alanine scan analogues resulted in decreased or abolished anti-proliferative activity of the NEP inhibitors towards the CRC cells. Additionally, thiorphan and sialorphin enhanced the anti-proliferative activity of other CRC-cell growth inhibitors (atrial natriuretic peptide-ANP and melphalan-MEL). The mechanisms involved in the anti-proliferative effects of the tested inhibitors were mediated via NEP and associated with induction of cell cycle arrest in the G0/G1 phase, increased activity of ERK1/2, and a reduced level of phosphorylation of mTOR (Ser2448), 4E-BP1, and p70S6K. However, the NEP inhibitors did not induce apoptosis in the CRC cells. These results have indicated that thiorphan and sialorphin or its derivatives AHNPR, QANPR, QHAPR, and QHNAR have the potential to be used as agents in treatment of patients with CRC.

Concurrent neprilysin inhibition and renin-angiotensin system modulations prevented diabetic nephropathy.

AIMS: Renin-angiotensin system (RAS) and natriuretic peptides system (NPS) perturbations govern the development of diabetic nephropathy (DN). Hence, in search of a novel therapy against DN, present study targeted both, NPS and RAS simultaneously using a neprilysin inhibitor (NEPi) in combination with either angiotensin receptor blocker (ARB) or angiotensin-converting enzyme 2 (ACE2) activator. METHODS: We induced diabetes in male Wistar rats by a single dose of streptozotocin (55 mg/kg, i.p.). After four weeks, we treated diabetic rats with thiorphan, telmisartan or diminazene aceturate (Dize) 0.1, 10, 5 mg/kg/day, p.o. alone as monotherapy, or both thiorphan/telmisartan or thiorphan/Dize as combination therapy, for four weeks. Then, plasma and urine biochemistry were performed, and kidneys from all the groups were collected and processed separately for histopathology, ELISA and Western blotting. KEY FINDINGS: Proposed combination therapies attenuated metabolic perturbations, prevented renal functional decline, and normalised adverse alterations in renal ACE, ACE2, Ang-II, Ang-(1-7), neprilysin and cGMP levels in diabetic rats. Histopathological evaluation revealed a significant reduction in glomerular and tubulointerstitial fibrosis by combination therapies. Importantly, combination therapies inhibited inflammatory, profibrotic and apoptotic signalling, way better than respective monotherapies, in preventing DN. CONCLUSION: Renoprotective potential of thiorphan (NEPi)/telmisartan (ARB) and thiorphan/Dize (ACE2 activator) combination therapies against the development of DN is primarily attributed to normalisation of RAS and NPS components and inhibition of pathological signalling related to inflammation, fibrosis, and apoptosis. Hence, we can conclude that NEPi/ARB and NEPi/ACE2 activator combination therapies might be new therapeutic strategies in preventing DN.

Telmisartan and thiorphan combination treatment attenuates fibrosis and apoptosis in preventing diabetic cardiomyopathy.

Aims: LCZ696, a first-generation dual angiotensin receptor-neprilysin inhibitor (ARNi), is effective in treating heart failure patients. However, the role of ARNis in treating diabetic cardiomyopathy is poorly understood. This study evaluates the efficacy of a novel combination of telmisartan [angiotensin receptor blocker (ARB)] and thiorphan [neprilysin inhibitor (NEPi)] in ameliorating diabetic cardiomyopathy while, at the same time, exploring the relevant underlying molecular mechanism(s). Methods and results: Diabetes was induced by administration of streptozotocin (55 mg/kg, i.p.) in male Wistar rats. After 4 weeks, diabetic rats were subjected to either thiorphan (0.1 mg/kg/day, p.o.) or telmisartan (10 mg/kg/day, p.o.) monotherapy, or their combination, for a period of 4 weeks. Metabolic and morphometric alterations, failing ventricular functions, and diminished baroreflex indicated development of diabetic cardiac complications. Apart from morphometric alterations, all pathological consequences were prevented by telmisartan and thiorphan combination therapy. Diabetic rats exhibited significant modulation of the natriuretic peptide system, a key haemodynamic regulator; this was normalized by combination therapy. Histopathological studies showed augmented myocardial fibrosis, demonstrated by increased % PSR-positive area, with combination therapy giving the best improvement in these indices. More importantly, the combination of thiorphan and telmisartan was superior in attenuating inflammatory (NF-κB/MCP-1), profibrotic (TGF-ß/Smad7) and apoptotic (PARP/Caspase-3) cascades compared to respective monotherapies when treating rats with diabetic cardiomyopathy. In addition, diabetic heart chromatin was in a state of active transcription, indicated by increased histone acetylation (H2AK5Ac, H2BK5Ac, H3K9Ac, and H4K8Ac) and histone acetyltransferase (PCAF and Ac-CBP) levels. Interestingly, combination treatment was sufficiently potent to normalize these alterations. Conclusion: The protective effect of novel ARB and NEPi combination against diabetic cardiomyopathy can be attributed to inhibition of inflammatory, profibrotic, and apoptotic cascades. Moreover, reversal of histone acetylation assists its protective effect.

Neprilysin inhibition and endothelin-1 elevation: Focus on the kidney.

Increasing the degree of renin-angiotensin system (RAS) blockade by combining ≥2 RAS blockers marginally increases efficacy, but results in more side effects. Hence, interference with other systems is currently being investigated, like potentiation of natriuretic peptides with neprilysin inhibitors. However, the neprilysin inhibitor thiorphan was recently found to increase endothelin-1 when administered to TGR(mREN2)27 (Ren2) rats on top of RAS blockade. Here we investigated whether this effect is thiorphan-specific, by comparing the neprilysin inhibitors thiorphan and sacubitril, administered by osmotic minipumps at a low or high dose for 7 days, in Ren2 rats. Plasma and urinary levels of endothelin-1, atrial and brain natriuretic peptide (ANP, BNP) and their second messenger cyclic guanosine 3'5' monophosphate (cGMP) were monitored. No significant differences were found in the plasma concentrations of endothelin-1, cGMP, ANP and BNP after treatment, although plasma ANP tended to be higher in the high-dose thiorphan treatment group and the low- and high-dose sacubitril treatment groups, compared with vehicle. Urinary endothelin-1 increased in the low-dose thiorphan and high-dose sacubitril groups, compared with baseline, although significance was reached for the former only. Urinary cGMP rose significantly in the high-dose sacubitril treatment group compared with baseline. Both urinary endothelin-1 and cGMP were significantly higher in the high-dose sacubitril group compared with the low-dose sacubitril group. In conclusion, endothelin-1 upregulation occurs with both thiorphan and sacubitril, and is particularly apparent in neprilysin-rich organs like the kidney. High renal neprilysin levels most likely also explain why sacubitril increased cGMP in urine only.

Beneficial Effects of Combined AT1 Receptor/Neprilysin Inhibition (ARNI) Versus AT1 Receptor Blockade Alone in the Diabetic Eye.

Purpose: Dysfunction of the renin-angiotensin system (RAS) contributes to pathogenesis of diabetic retinopathy (DR). Yet RAS blockers have only limited beneficial effects on progression of DR in clinical trials. The natriuretic peptide system offsets RAS, so that enhancing the activity of this system on top of RAS blockade might be beneficial. Neprilysin has an important role in the degradation of natriuretic peptides. Therefore, we hypothesize that dual angiotensin receptor-neprilysin inhibition (ARNI) may outperform angiotensin receptor blocker (ARB) in protection against DR. We tested this hypothesis in streptozotocin-induced diabetic transgenic (mRen2)27 rats. Methods: Adult male diabetic (mRen2)27 rats were followed for 5 or 12 weeks. Treatment with vehicle, irbesartan (ARB), or ARB combined with the neprilysin inhibitor thiorphan (irbesartan+thiorphan [ARNI]) occurred during the final 3 weeks. Retinal cell death, gliosis, and capillary loss were evaluated. Real-time polymerase chain reaction (RT-PCR) analyses were performed to quantify the retinal level of inflammatory cell markers. Results: Both ARB- and ARNI-treated groups showed similarly reduced retinal apoptotic cell death, gliosis, and capillary loss compared to the vehicle-treated group in the 5-week study. Treatment with ARNI reduced the expression of inflammatory markers more than ARB treatment in the 5-week study. In the 12-week study, ARNI treatment showed significantly more reduction in apoptotic cell death (51% vs. 25% reduction), and capillary loss (68% vs. 43% reduction) than ARB treatment. Conclusions: Treatment with ARNI provides better protection against DR in diabetic (mRen2)27 transgenic rats, compared to ARB alone. This approach may be a promising treatment option for patients with DR.

Exogenous Angiotensin I Metabolism in Aorta Isolated from Streptozotocin Treated Diabetic Rats.

Purpose. Products of angiotensin (ANG) I metabolism may predispose to vascular complications of diabetes mellitus. Methods. Diabetes was induced with streptozotocin (75 mg/kg i.p.). Rat aorta fragments, isolated 4 weeks later, were pretreated with perindoprilat (3 µM), thiorphan (3 µM), or vehicle and incubated for 15 minutes with ANG I (1 µM). Products of ANG I metabolism through classical (ANG II, ANG III, and ANG IV) and alternative (ANG (1-9), ANG (1-7), and ANG (1-5)) pathways were measured in the buffer, using liquid chromatography-mass spectrometry. Results. Incubation with ANG I resulted in higher concentration of ANG II (P = 0.02, vehicle pretreatment) and lower of ANG (1-9) (P = 0.048, perindoprilat pretreatment) in diabetes. Preference for the classical pathway is suggested by higher ANG III/ANG (1-7) ratios in vehicle (P = 0.03), perindoprilat (P = 0.02), and thiorphan pretreated (P = 0.02) diabetic rat. Within the classical pathway, ratios of ANG IV/ANG II (P = 0.01) and of ANG IV/ANG III (P = 0.049), but not of ANG III/ANG II are lower in diabetes. Conclusions. Diabetes in rats led to preference toward deleterious (ANG II, ANG III) over protective (ANG IV, ANG (1-9), and ANG (1-7)) ANG I metabolites.

Is racecadotril effective for acute diarrhea in children? -First update. / ¿Es efectivo el racecadotrilo como tratamiento para la diarrea aguda en niños? -Primera actualización.

This article updates the December 2015 Living FRISBEE (Living FRISBEE: Living FRIendly Summary of the Body of Evidence using Epistemonikos), based on the detection of two systematic reviews not identified in the previous version. Gastroenteritis or acute watery diarrhea is usually a self-limited disease, but it is still associated to substantial healthcare costs and remains a frequent demand for medical care. Racecadotril, an intestinal enkephalinase inhibitor, has been used as treatment because it would decrease the duration of acute diarrhea and fluid loss. However there is still no evidence supporting its routine use. Searching in Epistemonikos database, which is maintained by screening 30 databases, we identified five systematic reviews including nine randomized trials relevant for our question. We combined the evidence using meta-analysis and generated a summary of findings table following the GRADE approach. We concluded racecadotril probably reduces the duration of acute diarrhea in pediatric patients, without increasing adverse effects.

Este resumen Epistemonikos (Living FRISBEE: Living FRIendly Summary of the Body of Evidence using Epistemonikos) es una actualización del resumen publicado en Diciembre de 2015, basado en la detección de dos nuevas revisiones sistemáticas que no habían sido identificadas en la versión anterior. La gastroenteritis o diarrea aguda es una enfermedad habitualmente autolimitada, pero que consume recursos sanitarios y constituye un motivo de consulta frecuente en pediatría. El racecadotrilo, un inhibidor de la encefalinasa intestinal, se ha usado como tratamiento porque disminuiría la duración de la diarrea y la pérdida de líquidos, pero no hay evidencia que justifique su uso rutinario. Utilizando la base de datos Epistemonikos, la cual es mantenida mediante búsquedas en 30 bases de datos, identificamos cinco revisiones sistemáticas que en conjunto incluyen nueve estudios aleatorizados relevantes para nuestra pregunta. Realizamos un metanálisis y tablas de resumen de los resultados utilizando el método GRADE. Concluimos que el uso de racecadotrilo probablemente disminuye la duración del cuadro de diarrea aguda en población pediátrica y que no se asociaría a mayor tasa de eventos adversos.

In silico based investigation of dynamic variations in neprilysin (NEP and NEP2) proteins for extracting the point of specificity.

Neprilysin-2 (NEP2) in the central nervous system controls Alzheimer's protein (amyloid-ß) deposition, and prevents its occurrence. However, in the peripheral system, its closest homolog, neutral endopeptidase (NEP), regulates hypertension and heart related diseases. Inhibitors of NEP with a lesser degree of specificity can treat hypertension with an increased risk of cerebral deposition of amyloid-ß. In order to rationalize the point of selectivity, the dynamic behavior of human NEP and NEP2 proteins was monitored by conducting molecular dynamics (MD) simulations. A computationally reliable model of NEP2 was achieved with 79.9%, 19.1% and 0.2% residues in the allowed, additionally allowed and disallowed regions respectively, using as a reference protein. Additionally, molecular docking studies were carried out for a set of five already known inhibitors of NEP and modeled NEP2 to obtain the comparative behaviors of the complexes. MD results highlighted their different responses along with important residues having a part in ligand-protein binding. For substrate and inhibitor binding, Arg664/661 and Zn697/694 were identified as the most conserved residues. High degree flexible transitions during the MD simulations were also observed in loop areas along with active site residues. Energy calculations, hydrogen bonds and their occupancy rates helped to conclude each ligand's potency towards a particular target. In most complexes of hNEP2, the ligands showed weak interactions which might be due to its larger pocket size or huge conformational variations in active site residues upon complexation. In the case of inhibitors of a small size like thiorphan, Arg49 and Arg664 are found to be acting to support the ligand binding in NEP while only Arg661 is acting in NEP2.

[des-Arg(1)]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom.

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

Optimum AT1 receptor-neprilysin inhibition has superior cardioprotective effects compared with AT1 receptor blockade alone in hypertensive rats.

Neprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium-hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor-mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension.

Augmentation of the effects of vasoactive intestinal peptide aerosol on pulmonary hypertension via coapplication of a neutral endopeptidase 24.11 inhibitor.

A deficiency of the pulmonary vasodilative vasoactive intestinal peptide (VIP) has been suggested to be involved in the pathophysiology of pulmonary hypertension (PH). Supplementation of VIP as an aerosol is hampered by the fact that it is rapidly inactivated by neutral endopeptidases (NEP) located on the lung surface. Coapplication of thiorphan, an NEP 24.11 inhibitor, could augment the biological effects of inhaled VIP alone. A stable pulmonary vasoconstriction with a threefold increase of pulmonary artery pressure was established by application the thromboxane mimetic U46619 in the isolated rabbit lung model. VIP and thiorphan were either applied intravascularly or as an aerosol. VIP caused a significant pulmonary vasodilation either during intravascular application or inhalation. These effects were of short duration. Thiorphan application had no effects on pulmonary vasoconstriction per se but significantly augmented the effects of VIP aerosol. Thiorphan, not only augmented the maximum hemodynamic effects of VIP aerosol, but also led to a significant prolongation of these effects. VIP causes pulmonary vasodilation in a model of acute experimental PH. The hemodynamic effects of VIP aerosol can be significantly augmented via coapplication of an NEP inhibitor.

Targets and candidate agents for type 2 diabetes treatment with computational bioinformatics approach.

We sought to explore the molecular mechanism of type 2 diabetes (T2D) and identify potential drug targets and candidate agents for T2D treatment. The differentially expressed genes (DEGs) were assessed between human pancreatic islets with T2D and normal islets. The dysfunctional pathways, the potential transcription factor, and microRNA targets were analyzed by bioinformatics methods. Moreover, a group of bioactive small molecules were identified based on the connectivity map database. The pathways of Eicosanoid Synthesis, TGF-beta signaling pathway, Prostaglandin Synthesis and Regulation, and Integrated Pancreatic Cancer Pathway were found to be significantly dysregulated in the progression of T2D. The genes of ZADH2 (zinc binding alcohol dehydrogenase domain containing 2), BTBD3 (BTB (POZ) domain containing 3), Cul3-based ligases, LTBP1 (latent-transforming growth factor beta binding protein 1), PDGFRA (alpha-type platelet-derived growth factor receptor), and FST (follistatin) were determined to be significant nodes regulated by potential transcription factors and microRNAs. Besides, two small molecules (sanguinarine and DL-thiorphan) were identified to be capable of reverse T2D. In the present study, a systematic understanding for the mechanism underlying T2D development was provided with biological informatics methods. The significant nodes and bioactive small molecules may be drug targets and candidate agents for T2D treatment.

Granulocyte-colony stimulating factor attenuates oligomeric amyloid ß neurotoxicity by activation of neprilysin.

Soluble oligomeric amyloid ß (oAß) causes synaptic dysfunction and neuronal cell death, which are involved in the pathogenesis of Alzheimer's disease (AD). The hematopoietic growth factor granulocyte-colony stimulating factor (G-CSF) is expressed in the central nervous system (CNS) and drives neurogenesis. Here we show that G-CSF attenuated oAß neurotoxicity through the enhancement of the enzymatic activity of Aß-degrading enzyme neprilysin (NEP) in neurons, while the NEP inhibitor thiorphan abolished the neuroprotection. Inhibition of MEK5/ERK5, a major downstream effector of G-CSF signaling, also ablated neuroprotective effect of G-CSF. Furthermore, intracerebroventricular administration of G-CSF enhanced NEP enzymatic activity and clearance of Aß in APP/PS1 transgenic mice. Thus, we propose that G-CSF may be a possible therapeutic strategy against AD.

Involvement of the Apoer2 and Lrp1 receptors in mediating the pathological effects of ApoE4 in vivo.

This study investigated the possible role of the ApoE receptors Lrp1 and Apoer2 in mediating the pathological effects of ApoE4 in ApoE-targeted-replacement mice expressing either the human ApoE3 or ApoE4 allele. In this study we show that activation of the amyloid cascade by inhibition of the Aß-degrading enzyme neprilysin results in upregulation of the ApoE receptor Lrp1 in the CA1 hippocampal neurons of 4-month-old ApoE4 mice, but not in the corresponding ApoE3 or ApoE-deficient (KO) mice. These results are in accordance with the previous findings that activation of the amyloid cascade induces Aß accumulation in the CA1 neurons of ApoE4 mice, but not in ApoE3 or ApoE-KO mice. This suggests that the apoE4-driven elevation of Lrp1 is mediated via a gain of function mechanism and may play a role in mediating the effects of ApoE4 on Aß. In contrast, no changes were observed in the levels of the corresponding Apoer2 receptor following the neprilysin inhibition. The ApoE receptors of naive ApoE4 mice were also affected differentially and isoform specifically by ApoE4. However, under these conditions, the effect was an ApoE4-driven reduction in the levels of Apoer2 in CA1 and CA3 pyramidal neurons, whereas the levels of Lrp1 were not affected. RT-PCR measurements revealed that the levels of Apoer2 and Lrp1 mRNA in the hippocampus of naïve and neprilysin-inhibited mice were not affected by ApoE4, suggesting that the observed effects of ApoE4 on the levels of these receptors is posttranscriptional. In conclusion, this study shows that the levels of hippocampal ApoE receptors Lrp1 and Apoer2 in vivo are affected isoform specifically by ApoE4 and that the type of receptor affected is context dependent.