NLRP3 inhibition improves maternal hypertension, inflammation, and vascular dysfunction in response to placental ischemia.

Preeclampsia (PE) is a pregnancy-specific hypertensive disorder with end-organ damage that presents after 20 wk of gestation. PE pathophysiology often includes vascular dysfunction and increased inflammation that continues to damage patient health even after PE resolves. Currently, there is no cure for PE beyond delivery of the fetal-placental unit. Previous clinical studies have identified elevated placental NLRP3 expression in patients with PE and suggest NLRP3 as a potential therapeutic target. In this study, we examined the effect of NLRP3 inhibition on PE pathophysiology in the reduced uterine perfusion pressure (RUPP) model rat using MCC950 (20 mg/kg/day) or esomeprazole (3.5 mg/kg/day). We hypothesized that increased NLRP3 in response to placental ischemia impairs anti-inflammatory IL-33 signaling to induce T-helper 17 cell (TH17) and cytolytic NK cell (cNK) activation, which is known to mediate oxidative stress and vascular dysfunction leading to maternal HTN and intrauterine growth restriction. RUPP rats had significantly higher placental NLRP3 expression, maternal blood pressure, fetal reabsorption rate, vascular resistance, oxidative stress, cNKs and TH17s, and decreased IL-33 compared with normal pregnant (NP) rats. NLRP3 inhibition, with either treatment, significantly reduced placental NLRP3 expression, maternal blood pressure, fetal reabsorption rates, vascular resistance, oxidative stress, cNK, and TH17 populations in RUPP rats. Based on our findings, NLRP3 inhibition reduces PE pathophysiology and esomeprazole may be a potential therapeutic for PE treatment.

Esomeprazole induces structural changes and apoptosis and alters function of in vitro canine neoplastic mast cells.

Histamine-2 receptor antagonists such as famotidine and proton pump inhibitors such as esomeprazole are commonly used in canine MCT disease, but direct effects on dog MCs have not been evaluated. Omeprazole is a proton pump inhibitor which has been demonstrated to cause structural and functional changes to in vitro murine mast cells (MCs). It has not yet been determined if esomeprazole, the commercially available and commonly prescribed S-isomer of omeprazole, has similar effects. Our primary study objective was to evaluate and compare the effects of acid suppressants (esomeprazole and famotidine) on MC ultrastructure, viability, and function in vitro using both healthy and neoplastic MCs. Murine bone marrow derived mast cells (BMMC), human LAD2, and canine C2 and BR cells, were used for these studies, representing a single healthy (i.e., BMMCs) MC model and multiple neoplastic MC models (i.e., LAD2, C2, BR), respectively. The rat basophilic leukemic (RBL-2H3) and canine B cell lymphoma 17-71 cell lines served as granulocytic and agranulocytic control lines for experiments, respectively. The treatment effect of acid suppressants on MC ultrastructure was assessed via both light and transmission electron microscopy. Differences in MC viability was assessed between groups via MTS-based, colorimetric assays and flow cytometry. Degranulation was assessed by quantification of ß-hexosaminidase (i.e., LAD2 and RBL-2H3). Esomeprazole-treated MCs of all lines exhibited dramatic time and concentration-dependent alterations in ultrastructure (i.e., increased vacuolization, compromise of cell membrane), increased apoptosis, and altered degranulation responses in comparison to famotidine and vehicle-treated cells. The canine B cell lymphoma cells consistently exhibited either no significant (i.e., cytotoxicity assays) or greatly diminished treatment responses (i.e., apoptosis) compared to MCs. Esomeprazole, but not famotidine, induces significant cytotoxicity, as well as alterations to cell structure and function to multiple lines of in vitro neoplastic MCs. Continued in vitro work investigating the specific mechanisms by which proton pump inhibitors induce these effects, as well as prospective, in vivo work comparing the treatment effects of acid suppressants on canine MCTs, are warranted.

Assessment of the Proton Pump Inhibitor, Esomeprazole Magnesium Hydrate and Trihydrate, on Pathophysiological Markers of Preeclampsia in Preclinical Human Models of Disease.

Previously, we demonstrated that the proton pump inhibitor, esomeprazole magnesium hydrate (MH), could have potential as a repurposed treatment against preeclampsia, a serious obstetric condition. In this study we investigate the difference in the preclinical effectiveness between 100 µM of esomeprazole MH and its hydration isomer, esomeprazole magnesium trihydrate (MTH). Here, we found that both treatments reduced secretion of sFLT-1 (anti-angiogenic factor) from primary cytotrophoblast, but only esomeprazole MH reduced sFLT-1 secretion from primary human umbilical vein endothelial cells (assessed via ELISA). Both drugs could mitigate expression of the endothelial dysfunction markers, vascular cell adhesion molecule-1 and endothelin-1 (via qPCR). Neither esomeprazole MH nor MTH quenched cytotrophoblast reactive oxygen species production in response to sodium azide (ROS assay). Finally, using wire myography, we demonstrated that both compounds were able to induce vasodilation of human omental arteries at 100 µM. Esomeprazole is safe to use in pregnancy and a candidate treatment for preeclampsia. Using primary human tissues and cells, we validated that esomeprazole is effective in enhancing vascular relaxation, and can reduce key factors associated with preeclampsia, including sFLT-1 and endothelial dysfunction. However, esomeprazole MH was more efficacious than esomeprazole MTH in our in vitro studies.

"Nonpolarity paving" in substrate tunnel of a Limnobacter sp. Phenylacetone monooxygenase for efficient single whole-cell synthesis of esomeprazole.

Baeyer-Villiger monooxygenase (BVMO) mediated sulfoxidation is a sustainable approach for the synthesis of esomeprazole. In this work, a novel phenylacetone monooxygenase from Limnobacter sp. (LnPAMO) was found to have trace activity for synthesis of enantiopure esomeprazole. Through engineering in the substrate tunnel using a mutagenesis strategy called "nonpolarity paving" and some modifications in cofactor binding domains, a mutant harboring 15 mutations (LnPAMO Mu15) was obtained with 6.6 × 103-fold higher activity to convert omeprazole sulfide into esomeprazole. The activities of the mutant for synthesis of (S)-methyl phenyl sulfoxide and (S)-pantoprazole also increased much, indicating the versatility of the mutant for sulfoxide synthesis. Importantly, no over-oxidation byproduct omeprazole sulfone was detected in the sulfoxidation products by both mass spectrometry and HPLC analysis. Then NADP-dependent Burkholderia stabili formate dehydrogenase was ligated behind Mu15 along with a ribosome binding site sequence in pET-28a for co-expression. By single whole-cell of recombinant Escherichia coli BL21 coexpressing Mu15 and formate dehydrogenase, omeprazole sulfide was efficiently converted into esomeprazole without production of sulfone (16 g/L substrate, enantiomeric excess > 99.9% (S) and > 99% conversion) and the space-time-yield reached 1.67 g product/L/h.

Esomeprazole inhibits hypoxia/endothelial dysfunction-induced autophagy in preeclampsia.

Preeclampsia (PE) affects 3 to 5% of pregnant women worldwide and is associated with fetal and maternal morbidity and mortality. Although a complete understanding of PE remains elusive, it has been widely accepted that a dysfunction of the placenta plays a key role in the pathogenesis of PE. In this study, we investigated the role of excessive placental autophagy during PE pathogenesis and explored whether esomeprazole ameliorates PE by inhibiting the autophagy in the placenta. The PE cellular model was established by treating the cells' L-NAME and hypoxia. The PE mice model was established by L-NAME administration and was confirmed by the increased systolic blood pressure (SBP) and urinary protein detected. The autophagy and key proteins were detected in human placental tissue, in cells, and in the mice model by Western blot and immunofluorescence staining. Results showed that excessive autophagy could be detected in human PE placental tissue, in the PE cellular model, and in the PE mice model. Hypoxia induces autophagy by activating AMPKα and inhibiting mTOR in vivo and in vitro. Esomeprazole inhibits L-NAME-induced autophagy in mice by inhibiting AMPKα and activating mTOR. In conclusion, this study demonstrates that the excessive autophagy induced by the SIRT1/AMPKα-mTOR pathway plays a significant role in the pathogenesis of PE. However, esomeprazole treatment inhibits AMPKα but activates mTOR, resulting in the inhibition of autophagy in the placenta and, therefore, mitigates PE symptoms.

Esomeprazole reduces sperm motility index by targeting the spermic cholinergic machinery: A mechanistic study for the association between use of proton pump inhibitors and reduced sperm motility index.

Recent studies have linked prolonged use of the most commonly prescribed proton pump inhibitors (PPIs) with declined human sperm function and infertility. Here, we report for the first time the most plausible underlying mechanism for this unwarranted secondary mode of action. We followed up on a recent serendipitous discovery in our laboratory regarding PPIs' off-target action and performed detailed pharmacodynamic analyses by combining in silico and in vitro studies to determine the off-target effect of one of the most commonly used PPI, esomeprazole, on the key human acetylcholine biosynthesizing enzyme, choline acetyltransferase (ChAT; EC 2.3.1.6). A pivotal enzyme in the spermic cholinergic system that governs the sperm motility, concentration and quality. Our results were conclusive and showed that both the racemic form, omeprazole and its pure S-enantiomer, esomeprazole, acted as potent mixed-competitive inhibitor of human ChAT with a global inhibition constant (Ki) of 88 nM (95%CI: 10-167 nM) for esomeprazole and 178 nM (95%CI: 140-230 nM) for the racemic drug omeprazole. Most importantly, esomeprazole substantially reduces both total number of motile sperm (by 36%, p < 0.001; and 21% p < 0.0001, at 10 and 100 nM, respectively) as well as the total number of sperm with progressive motility (by 42% p < 0.0016 and by 26% p < 0.0001, respectively) after 60 min relative to 20 min incubation in our ex vivo functional assay performed on ejaculated human sperm. In conclusion, this study presents a completely new perspective regarding PPIs secondary mode of action/unwarranted side effects and calls for further mechanistic and larger clinical studies to elucidate the role of PPIs in infertility.

Predictive Performance of Physiologically Based Pharmacokinetic (PBPK) Modeling of Drugs Extensively Metabolized by Major Cytochrome P450s in Children.

The accuracy of physiologically based pharmacokinetic (PBPK) model prediction in children, especially those younger than 2 years old, has not been systematically evaluated. The aim of this study was to characterize the pediatric predictive performance of the PBPK approach for 10 drugs extensively metabolized by CYP1A2 (theophylline), CYP2C8 (desloratidine, montelukast), CYP2C9 (diclofenac), CYP2C19 (esomeprazole, lansoprazole), CYP2D6 (tramadol), and CYP3A4 (itraconazole, ondansetron, sufentanil). Model performance in children was evaluated by comparing simulated plasma concentration-time profiles with observed clinical results for each drug and age group. PBPK models reasonably predicted the pharmacokinetics of desloratadine, diclofenac, itraconazole, lansoprazole, montelukast, ondansetron, sufentanil, theophylline, and tramadol across all age groups. Collectively, 58 out of 67 predictions were within 2-fold and 43 out of 67 predictions within 1.5-fold of observed values. Developed PBPK models can reasonably predict exposure in children age 1 month and older for an array of predominantly CYP metabolized drugs.

Esomeprazole immediate release tablets: Gastric mucosa ex vivo permeation, absorption and antisecretory activity in conscious rats.

The aim of this work was to study the esomeprazole activity on the control of gastric secretion after administration of a novel immediate release tablet. The ex vivo permeation of esomeprazole across porcine gastric mucosa from immediate release tablets, containing sodium carbonate or magnesium oxide as alkalinizing agents, was firstly assessed. Pharmacokinetics and pharmacodynamics studies in conscious rats following the administration of immediate release tablets with sodium carbonate, in comparison with delayed-release tablets having the same formula, were also conducted. The results showed an important effect of sodium carbonate and magnesium oxide on the drug release, on the ex vivo trans-mucosal transport and the stability in acid environment. In particular, the presence of sodium carbonate in esomeprazole tablet formulation provided the maximum increase of the drug in vitro transport across the mucosa. Then, the absorption and the antisecretory activity of this proton pump inhibitor orally administered in rats as immediate release tablets containing Na2CO3, was superior but not significantly different compared to delayed-release tablets having the same formula. In the adopted animal model, an activity of esomeprazole from immediate release alkaline formulation was seen also in presence of partial gastric absorption allowing inhibition of proton pumps reached via systemic circulation. This esomeprazole immediate release formulation could be used for the on-demand treatment of acid-related disorders such as gastro-esophageal reflux disease.

Levofloxacin, bismuth, amoxicillin and esomeprazole as second-line Helicobacter pylori therapy after failure of non-bismuth quadruple therapy.

BACKGROUND: The best rescue therapy for Helicobacter pylori (H. pylori) infection following failure of non-bismuth quadruple therapy (NBQT) remains unanswered. AIMS: To determine the efficacy, safety and compliance of levofloxacin, bismuth, amoxicillin and esomeprazole (LBAE) regimen following failure of NBQT. METHODS: 132 patients with H. pylori infection refractory to first-line NBQT received LBAE regimen (levofloxacin 500mg once/day, bismuth potassium citrate 220mg twice/day, amoxicillin 1000mg twice/day and esomeprazole 20mg twice/day for 14 days). Gastric mucosal biopsy was obtained for H. pylori culture, antimicrobial sensitivity test and cytochrome P450 isoenzyme 2C19 polymorphism analysis. RESULTS: LBAE therapy achieved eradication rates of 73.5% [95% confidence intervals (CI) 65.9-81.1%] in intention-to-treat and 78.5% (71.1-85.9%) in per-protocol analyses in patients with high antibiotic resistance (amoxicillin 8.3%, clarithromycin 55.6%, metronidazole 73.6% and levofloxacin 36.1%). Adverse effects were found in 19.2% and compliance in 96.1% of the treated patients. Multivariate analyses identified levofloxacin resistance [odds ratio (OR) 7.183, 95% CI 1.616-31.914, P=0.010] and history of quinolone intake (4.844, 1.174-19.983, P=0.029) as independent predictors of treatment failure. The eradication rate of patients with dual amoxicillin and levofloxacin resistance was significantly decreased (33.3%, P=0.006). CONCLUSIONS: In populations with high levofloxacin resistance, 14-day second-line LBAE regimen resulted in an unsatisfactory efficacy in patients resistant to NBQT despite good safety and compliance.

Novel naproxen/esomeprazole magnesium compound pellets based on acid-independent mechanism: in vitro and in vivo evaluation.

The purpose of this study was to develop the novel naproxen/esomeprazole magnesium compound pellets (novel-NAP/EMZ) depending on EMZ acid-independent mechanism which has been proved to be predominate in the mechanism of co-therapy with nonsteroidal anti-inflammatory drug. The novel-NAP/EMZ compound pellets, composed of NAP colon-specific pellets (NAP-CSPs) and EMZ modified-release pellets (EMZ-MRPs), were prepared by fluid-bed coating technology with desired in vitro release profiles. The resulting pellets were filled into hard gelatin capsules for in vivo evaluation in rats and compared with the reference compound pellets, consisted of NAP enteric-coated pellets (NAP-ECPs) and EMZ immediate-release pellets (EMZ-IRPs). The reference compound pellets were prepared simulating the drug delivery system of VIMOVO(®). In vivo pharmacokinetics, EMZ-MRPs had significantly larger AUC0-t (p < 0.01), 1.67 times more than that of EMZ-IRPs, and prolonged mean residence time (7.55 ± 0.12 h) than that of IRPs (1.46 ± 0.39 h). NAP-CSPs and NAP-ECPs showed similar AUC0-t. Compared to the reference compound pellets, the novel-NAP/EMZ compound pellets did not show distinct differences in histological mucosal morphology. However, biochemical tests exhibited enhanced total antioxidant capacity, increased nitric oxide content and reduced malondialdehyde level for novel-NAP/EMZ compound pellets, indicating that the acid-independent action took effect. The gastric pH values of novel-NAP/EMZ compound pellets were at a low and stable level, which could ensure normal physiological range of human gastric pH. As a result, the novel-NAP/EMZ compound pellets may be a more suitable formulation with potential advantages by improving bioavailability of drug and further reducing undesirable gastrointestinal damages.

Preparation and characterization of pH-sensitive methyl methacrylate-g-starch/hydroxypropylated starch hydrogels: in vitro and in vivo study on release of esomeprazole magnesium.

In the present study, novel hydrogels were prepared through graft copolymerization of methyl methacrylate onto starch and hydroxypropylated starch for intestinal drug delivery. The successful grafting has been confirmed by FTIR, NMR spectroscopy, and elemental analysis. Morphological examination of copolymeric hydrogels by scanning electron microscopy (SEM) confirms the macroporous nature of the copolymers. The high decomposition temperature was observed in thermograms indicating the thermal stability of the hydrogels. To attain a hydrogel with maximum percent graft yield, the impact of reaction variables like concentration of ceric ammonium nitrate as initiator and methyl methacrylate as monomer were consistently optimized. X-ray powder diffraction and differential scanning calorimetric analysis supported the successful entrapment of the drug moiety (esomeprazole magnesium; proton pump inhibitor) within the hydrogel network. Drug encapsulation efficiency of optimized hydrogels was found to be >78%. Furthermore, swelling capacity of copolymeric hydrogels exhibited a pH-responsive behavior which makes the synthesized hydrogels potential candidates for controlled delivery of medicinal agents. In vitro drug release was found to be sustained up to 14 h with 80-90% drug release in pH 6.8 solution; however, the cumulative release was 40-45% in pH 1.2. The gastrointestinal transit behavior of optimized hydrogel was determined by gamma scintigraphy, using (99m)Tc as marker. The amount of radioactive tracer released from the labeled hydrogel was minimal when the hydrogel was in the stomach, whereas it increased as hydrogel reached in intestine. Well-correlated results of in vitro and in vivo analysis proved their controlled release behavior with preferential delivery into alkaline pH environment.

Human P450-like oxidation of diverse proton pump inhibitor drugs by 'gatekeeper' mutants of flavocytochrome P450 BM3.

Production of drug metabolites is one area where enzymatic conversion has significant advantages over synthetic chemistry. These high value products are complex to synthesize, but are increasingly important in drug safety testing. The vast majority of drugs are metabolized by cytochromes P450 (P450s), with oxidative transformations usually being highly regio- and stereo-selective. The PPIs (proton pump inhibitors) are drugs that are extensively metabolized by human P450s, producing diverse metabolites dependent on the specific substrate. In the present paper we show that single mutations (A82F and F87V) in the biotechnologically important Bacillus megaterium P450 BM3 enzyme cause major alterations in its substrate selectivity such that a set of PPI molecules become good substrates in these point mutants and in the F87V/A82F double mutant. The substrate specificity switch is analysed by drug binding, enzyme kinetics and organic product analysis to confirm new activities, and X-ray crystallography provides a structural basis for the binding of esomeprazole to the F87V/A82F enzyme. These studies confirm that such 'gatekeeper' mutations in P450 BM3 produce major perturbations to its conformation and substrate selectivity, enabling novel P450 BM3 reactions typical of those performed by human P450s. Efficient transformation of several PPI drugs to human-like products by BM3 variants provides new routes to production of these metabolites.

Induction of testosterone metabolism by esomeprazole in a CYP2C19*2 heterozygote.

The authors describe a 42-year-old woman with previously normal sexual function who gradually developed loss of libido during treatment with esomeprazole. While taking esomeprazole, the patient's loss of libido improved with oral testosterone supplementation and deteriorated after testosterone withdrawal. There was steady improvement in both sexual function and serum free testosterone concentration after discontinuation of esomeprazole. Due to the temporal relationship between esomeprazole intake and sexual dysfunction, the authors postulate that esomeprazole causes induction of testosterone metabolism. The authors believe this to be the first case of female sexual dysfunction associated with esomeprazole described in the literature. They discuss a number of possible mechanisms for this effect.