Racecadotril Versus Loperamide in Acute Radiation Enteritis: A Randomized, Double-Masked, Phase 3, Noninferiority Trial.

PURPOSE: There is currently no gold standard for the management of acute radiation enteritis. We compared the efficacy and safety of Racecadotril, an anti-hypersecretory drug, versus Loperamide, an anti-motility agent, in acute radiation enteritis. METHODS AND MATERIALS: We conducted a randomized, double-masked, non-inferiority trial at a single research institute. Patients receiving curative radiation for pelvic malignancies, who developed grade 2 or 3 diarrhea (as per Common Terminology Criteria for Adverse Events, v 4.0) were included in the study. Patients in the intervention arm received Racecadotril and placebo. Patients in the control arm received Loperamide and placebo. The primary outcome was the resolution of diarrhea, 48 hours after the start of treatment. RESULTS: 162 patients were randomized between 2019 and 2022. On intention-to-treat analysis, 68/81 patients, 84%, (95% CI, 74.1%-91.2%) in the Racecadotril arm and 70/81, 86.4%, (95% CI, 77.0%-93.0%) in the Loperamide arm improved from grade 2 or 3 diarrhea to grade 1 or 0, (P= .66, χ2 test). The difference in proportion was 2.4% (95% CI: -8.5% to 13.4%). Since the upper boundary of the 95% CI crossed our non-inferiority margin of 10% (13.4%) we could not prove the non-inferiority of Racecadotril over Loperamide. Rebound constipation was more in the Loperamide arm compared to Racecadotril (17.3% vs 6.2%; P = .028) CONCLUSIONS: The non-inferiority of Racecadotril to Loperamide in acute radiation enteritis could not be demonstrated. However, Racecadotril can be the preferred drug of choice in acute radiation enteritis because Racecadotril does not affect bowel motility, achieved a high clinical success rate similar to that of Loperamide, and was associated with lesser side effects.

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.

Angiotensin receptor-neprilysin inhibitor (thiorphan/irbesartan) improved cardiac function in a rat model of myocardial ischemic reperfusion injury.

Mitochondria-mediated apoptosis plays a critical role in myocardial ischemia reperfusion (IR) injury and causes a negative impact on cardiac efficiency and function. The combined angiotensin receptor-neprilysin inhibitor (ARNI) is a promising cardioprotective pharmacological agent that could rescue the heart from IR injury. This study investigated the cardioprotective effect of thiorphan (TH) in combination with three different doses of irbesartan (IRB) on myocardial IR injury and detected the most effective dose combination. Male Wistar rats were used and divided into five groups (10 rats/group): (I) Sham, (II) ischemia-reperfusion I/R, (III) TH/IRB + IR (0.1/5 mg/kg), (IV) TH/IRB + IR (0.1/10 mg/kg), and (V) TH/IRB + IR (0.1/15 mg/kg) groups. Thiorphan and irbesartan were injected intraperitoneally 15 min before IR induction. Mean arterial blood pressure, left ventricular end diastolic pressure (LVEDP), left ventricular maximum rate of pressure (LVdp/dtmax ), and cardiac levels of creatine kinase-MB, malondialdehyde, superoxide dismutase, and endothelin-1 were measured. Cardiac mitochondria complexes activities, histopathological examination of myocardial tissues, immunohistochemistry studies for myocardial apoptosis (Bax and Bcl-2), and electron microscopy examination of left ventricle were performed. TH/IRB combination preserved cardiac functions and mitochondria complex activities and mitigated cardiac damage, oxidative stress, and apoptosis following IR. Also, there was an evident improvement in histopathological changes and electron microscopy examination of left ventricle compared with I/R group. TH/IRB in a dose of 0.1/10 mg/kg showed significant improvement compared with the other treated groups. Thiorphan/irbesartan improved cardiac functions following IR injury. This could be explained by the reported improvement of mitochondria complex activities and reduction of oxidative stress, endothelin-1, and apoptosis.

Alpha Ketoglutarate Downregulates the Neutral Endopeptidase and Enhances the Growth Inhibitory Activity of Thiorphan in Highly Aggressive Osteosarcoma Cells.

Since natural substances are widely explored as epigenetic modulators of gene expression and epigenetic abnormalities are important causes of cancerogenesis, factors with pro-tumor activities subjected to epigenetic control, e.g., neutral endopeptidase (NEP, neprilysin), are promising anticancer targets for potential therapies acting via epigenetic regulation of gene expression. Alpha-ketoglutarate (AKG) is a naturally occurring co-substrate for enzymes involved in histone and DNA demethylation with suggested anti-cancer activity. Hence, we investigated a potential effect of AKG on the NEP expression in cells derived from various cancers (cervical, colon, osteosarcoma) and normal epithelial cells and osteoblasts. Moreover, the overall methylation status of histone H3 was explored to establish the molecular target of AKG activity. Additionally, it was investigated whether AKG in combination with thiorphan (NEP specific inhibitor) exhibited enhanced anticancer activity. The results revealed that AKG downregulated the expression of NEP at the protein level only in highly aggressive osteosarcoma HOS cells (flow cytometry and fluorometric assays), and this protease was found to be involved in AKG-induced growth inhibition in osteosarcoma cells (siRNA NEP silencing, BrdU assay, flow cytometry). Unexpectedly, AKG-induced hypermethylation of H3K27 in HOS cells, which was partially dependent on EZH2 activity. However, this effect was not implicated in the AKG-induced NEP downregulation (flow cytometry). Finally, the combined treatment with AKG and thiorphan was shown to significantly enhance the growth inhibitory potential of each one towards HOS cells (BrdU assay). These preliminary studies have shown for the first time that the downregulation of NEP expression is a promising target in therapies of NEP-implicating HOS cells. Moreover, this therapeutic goal can be achieved via AKG-induced downregulation of NEP and synergistic activity of AKG with thiorphan, i.e., a NEP specific inhibitor. Furthermore, this study has reported for the first time that exogenous AKG can influence the activity of histone methyltransferase, EZH2. However, this issue needs further investigation to elucidate the mechanisms of this phenomenon.

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.

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.

Simultaneous inhibition of neprilysin and activation of ACE2 prevented diabetic cardiomyopathy.

BACKGROUND: Neprilysin inhibitors (NEPi) are assisting the renin-angiotensin system (RAS) inhibitors in halting diabetic cardiomyopathy (DCM). Away from conventional tactic, a recent report revealed the renoprotective potential of NEPi and angiotensin-converting enzyme (ACE2) activator combination therapy against diabetic nephropathy. However, this combination so far not evaluated against DCM, thus the present investigation aiming the same. METHODS: Streptozotocin-induced (55 mg/kg, ip) type 1 diabetic (T1D) male Wistar rats were treated with either monotherapy of thiorphan (0.1 mg/kg/day, po) or diminazene aceturate (5 mg/kg/day, po), or their combination therapy, for four weeks. After hemodynamic measurements, all the rats' heart and plasma were collected for biochemistry, ELISA, histopathology, and immunoblotting. RESULTS: Metabolic perturbations and failing cardiac functions associated with diabetes were markedly attenuated by combination therapy. Besides, unfavourable alterations in RAS and natriuretic peptides system (NPS) were corrected by combination therapy. Interestingly, combination therapy significantly increased plasma and heart cGMP levels compared to T1D and monotherapy receiving rats. Moreover, rats receiving combination therapy exhibited significant inhibition of activated NF-κB, TGF-ß and apoptotic signalling, and a notable reduction in cardiac fibrosis when compared to T1D rats. Expressions of posttranslational histone modifications markers; H3K4Me2 and its methyltransferases (SET7/9 and RBBP5) were significantly enhanced in T1D hearts, which were significantly reduced by combination therapy. CONCLUSIONS: The NEPi and ACE2 activator combination therapy effectively prevented DCM by normalising RAS and NPS activities, increasing cGMP, inhibiting inflammatory, pro-fibrotic and apoptotic signalling, and reversing H3K4Me2 and its methyl transferases expressions.

Structures of soluble rabbit neprilysin complexed with phosphoramidon or thiorphan.

Neutral endopeptidase (neprilysin; NEP) is a proteinase that cleaves a wide variety of peptides and has been implicated in Alzheimer's disease, cardiovascular conditions, arthritis and other inflammatory diseases. The structure of the soluble extracellular domain (residues 55-750) of rabbit neprilysin was solved both in its native form at 2.1 Šresolution, and bound to the inhibitors phosphoramidon and thiorphan at 2.8 and 3.0 Šresolution, respectively. Consistent with the extracellular domain of human neprilysin, the structure reveals a large central cavity which contains the active site and the location for inhibitor binding.

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 promotes corneal wound healing.

Neprilysin (NEP), an ectoenzyme that modulates inflammation by degrading neuropeptides, was recently identified in the human corneal epithelium. The cornea expresses many NEP substrates, but the function of NEP in homeostatic maintenance and wound healing of the cornea is unknown. We therefore investigated the role of this enzyme under naive and injured conditions using NEP-deficient (NEP-/-) and wild type (WT) control mice. In vivo ocular surface imaging and histological analysis of corneal tissue showed no differences in limbal vasculature or corneal anatomy between naive NEP-/- and WT mice. Histological examination revealed increased corneal innervation in NEP-/- mice. In an alkali burn model of corneal injury, corneal wound healing was significantly accelerated in NEP-/- mice compared to WT controls 3 days after injury. Daily intraperitoneal administration of the NEP inhibitor thiorphan also accelerated corneal wound healing after alkali injury in WT mice. Collectively, our data identify a previously unknown role of NEP in the cornea, in which pharmacologic inhibition of its activity may provide a novel therapeutic option for patients with corneal injury.

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.

Discovery of Potent ALK Inhibitors Using Pharmacophore-Informatics Strategy.

Anaplastic lymphoma kinase is a tyrosine kinase receptor protein belonging to insulin receptor superfamily. Gene fusions in anaplastic lymphoma kinase are associated with non-small cell lung cancer development. Hence, they are of immense importance in targeted therapies. Thus, for the treatment of non-small cell lung cancer, effective anaplastic lymphoma kinase inhibitors are of great significance. Therefore, our objective is to find hit compounds that could have better inhibitory activity than the existing anaplastic lymphoma kinase inhibitors. Keeping this in mind, in the present study pharmacophore based virtual screening was performed to identify possible anaplastic lymphoma kinase inhibitors. Initially, a five-point common pharmacophore hypothesis was generated based on twelve anaplastic lymphoma kinase inhibitors using PHASE module of Schrödinger. Subsequently, common pharmacophore hypothesis-based screening was conducted against in-trials subset of ZINC database and a total of 1000 hits were identified. The molecules obtained were further screened by three stages of docking using GLIDE software. The docking results reveal that six hit molecules showed higher glide score in comparison with the reference molecules. Finally, pharmacokinetic properties of the hit molecules were also analysed using QikProp programme. The results indicate that molecules namely videx, dexecadotril, chloramphenicol, naficillin were found to have good pharmacokinetic properties and human oral absorption. Moreover, videx, naficillin and chloramphenicol were found to have significant inhibitory activity for mutant (F1174L) anaplastic lymphoma kinase. It was also found that videx exhibited crucial interactions with the Met1199 residue of the native and mutant anaplastic lymphoma kinase protein. Furthermore, PASS algorithm predicted anti-neoplastic activity for all the four molecules. Thus these hits are found to be promising leads for anaplastic lymphoma kinase inhibitors. We believe that this study will be useful for the discovery and designing of more potent anaplastic lymphoma kinase inhibitors in the near future.

Enantioseparation of racecadotril using polysaccharide-type chiral stationary phases in polar organic mode.

Enantioseparation of the antidiarrheal drug, racecadotril, was investigated by liquid chromatography using polysaccharide-type chiral stationary phases in polar organic mode. The enantiodiscrimininating properties of 4 different chiral columns (Chiralpak AD, Chiralcel OD, Chiralpak AS, Chiralcel OJ) with 5 different solvents (methanol, ethanol, 1-propanol, 2-propanol, and acetonitrile) at 5 different temperatures (5-40 °C) were investigated. Apart from Chiralpak AS column the other 3 columns showed significant enantioseparation capabilities. Among the tested mobile phases, alcohol type solvents were superior over acetonitrile, and significant differences in enantioselective performance of the selector were observed depending on the type of alcohol employed. Van't Hoff analysis was used for calculation of thermodynamic parameters which revealed that enantioseparation is mainly enthalpy controlled; however, enthropic control was also observed. Enantiopure standard was used to determine the enantiomer elution order, revealing chiral selector-and mobile-phase dependent reversal of enantiomer elution order. Using the optimized method (Chiralcel OJ stationary phase, thermostated at 10 °C, 100% methanol, flow rate: 0.6 mL/min) baseline separation of racecadotril enantiomers (resolution = 3.00 ± 0.02) was achieved, with the R-enantiomer eluting first. The method was validated according to the ICH guidelines, and its application was tested on capsule and granules containing the racemic mixture of the drug.

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.

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.