A loss-of-function mutation in KCNJ11 causing sulfonylurea-sensitive diabetes in early adult life.

AIMS/HYPOTHESIS: The ATP-sensitive potassium (KATP) channel couples beta cell electrical activity to glucose-stimulated insulin secretion. Loss-of-function mutations in either the pore-forming (inwardly rectifying potassium channel 6.2 [Kir6.2], encoded by KCNJ11) or regulatory (sulfonylurea receptor 1, encoded by ABCC8) subunits result in congenital hyperinsulinism, whereas gain-of-function mutations cause neonatal diabetes. Here, we report a novel loss-of-function mutation (Ser118Leu) in the pore helix of Kir6.2 paradoxically associated with sulfonylurea-sensitive diabetes that presents in early adult life. METHODS: A 31-year-old woman was diagnosed with mild hyperglycaemia during an employee screen. After three pregnancies, during which she was diagnosed with gestational diabetes, the patient continued to show elevated blood glucose and was treated with glibenclamide (known as glyburide in the USA and Canada) and metformin. Genetic testing identified a heterozygous mutation (S118L) in the KCNJ11 gene. Neither parent was known to have diabetes. We investigated the functional properties and membrane trafficking of mutant and wild-type KATP channels in Xenopus oocytes and in HEK-293T cells, using patch-clamp, two-electrode voltage-clamp and surface expression assays. RESULTS: Functional analysis showed no changes in the ATP sensitivity or metabolic regulation of the mutant channel. However, the Kir6.2-S118L mutation impaired surface expression of the KATP channel by 40%, categorising this as a loss-of-function mutation. CONCLUSIONS/INTERPRETATION: Our data support the increasing evidence that individuals with mild loss-of-function KATP channel mutations may develop insulin deficiency in early adulthood and even frank diabetes in middle age. In this case, the patient may have had hyperinsulinism that escaped detection in early life. Our results support the importance of functional analysis of KATP channel mutations in cases of atypical diabetes.

Discovery and Characterization of VU0542270, the First Selective Inhibitor of Vascular Kir6.1/SUR2B KATP Channels.

Vascular smooth muscle KATP channels critically regulate blood flow and blood pressure by modulating vascular tone and therefore represent attractive drug targets for treating several cardiovascular disorders. However, the lack of potent inhibitors that can selectively inhibit Kir6.1/SUR2B (vascular KATP) over Kir6.2/SUR1 (pancreatic KATP) has eluded discovery despite decades of intensive research. We therefore screened 47,872 chemically diverse compounds for novel inhibitors of heterologously expressed Kir6.1/SUR2B channels. The most potent inhibitor identified in the screen was an N-aryl-N'-benzyl urea compound termed VU0542270. VU0542270 inhibits Kir6.1/SUR2B with an IC50 of approximately 100 nM but has no apparent activity toward Kir6.2/SUR1 or several other members of the Kir channel family at doses up to 30 µM (>300-fold selectivity). By expressing different combinations of Kir6.1 or Kir6.2 with SUR1, SUR2A, or SUR2B, the VU0542270 binding site was localized to SUR2. Initial structure-activity relationship exploration around VU0542270 revealed basic texture related to structural elements that are required for Kir6.1/SUR2B inhibition. Analysis of the pharmacokinetic properties of VU0542270 showed that it has a short in vivo half-life due to extensive metabolism. In pressure myography experiments on isolated mouse ductus arteriosus vessels, VU0542270 induced ductus arteriosus constriction in a dose-dependent manner similar to that of the nonspecific KATP channel inhibitor glibenclamide. The discovery of VU0542270 provides conceptual proof that SUR2-specific KATP channel inhibitors can be developed using a molecular target-based approach and offers hope for developing cardiovascular therapeutics targeting Kir6.1/SUR2B. SIGNIFICANCE STATEMENT: Small-molecule inhibitors of vascular smooth muscle KATP channels might represent novel therapeutics for patent ductus arteriosus, migraine headache, and sepsis; however, the lack of selective channel inhibitors has slowed progress in these therapeutic areas. Here, this study describes the discovery and characterization of the first vascular-specific KATP channel inhibitor, VU0542270.

Vitamin D supplementation could enhance the effectiveness of glibenclamide in treating type 2 diabetes by improving the function of pancreatic ß-cells through the NF-κB pathway.

PURPOSE: The high morbidity and mortality associated with type 2 diabetes mellitus (T2DM) pose a significant global health challenge, necessitating the development of more efficient anti-diabetic drugs with fewer side effects. This study investigated the intervention of vitamin D3 combined with glibenclamide in rats with T2DM to elucidate its effects on pancreatic ß-cells through the NF-κB pathway. METHODS: Twenty-four healthy male Sprague-Dawley (SD) rats were randomly assigned to four groups: the control group (CG), the model group (MG), the glibenclamide group (GG), and the glibenclamide + vitamin D3 group (GDG). After inducing the T2DM model using high-fat and high-sugar diet and intraperitoneal injection of streptozotocin, the rats in the GG group were administered glibenclamide orally (0.6 mg/kg/day), while those in the GDG group received both glibenclamide (0.6 mg/kg/day) and vitamin D3 (500 IU/kg/day) in corn oil for a duration of 8 weeks. Biochemical indices were measured, and histopathological changes in pancreatic tissue and islet ß cells were observed using hematoxylin and eosin staining. The expression of pancreatic nuclear factor κB (NF-κB), islet ß-cells, and inflammatory cytokines were assessed using the TUNEL method and PCR. RESULTS: According to the data from this current study, the GDG group showed significant positive differences in plasma biochemical indices, as well as in the expression of ß cells, NF-κB p65, TNF-α, IL-1ß, INF-γ, and Fas, compared to the GG and CG groups (P < 0.05). CONCLUSION: The results suggest that vitamin D has beneficial effects on T2DM by improving the functions of islet ß cells through inhibition of the NF-κB signaling pathway. Therefore, it is suggested that vitamin D supplementation, when used alongside antidiabetic drugs, may more effectively prevent and treat T2DM.

Glyburide use is associated with a greater likelihood of mortality or rehospitalization after acute coronary syndrome compared to gliclazide use in adults with type 2 diabetes: A cohort study.

AIM: To examine the likelihood of mortality or rehospitalization following acute coronary syndrome with glyburide versus gliclazide use in adults with type 2 diabetes undergoing cardiac catheterization. RESEARCH DESIGN AND METHODS: This retrospective cohort study used clinical data linked with administrative health data from Alberta, Canada between April 2008 and March 2021. Three methods were used to define exposure to glyburide and gliclazide in the year before catheterization. Multivariable logistic regression was used to compare the likelihood of a composite outcome of 1-year mortality or rehospitalization with use of glyburide versus use of gliclazide. RESULTS: A total of 11 140 individuals with type 2 diabetes had a cardiac catheterization for acute coronary syndrome. Their mean age was 66 years and 31% were female. In the year before catheterization, 5% used glyburide and 19% used gliclazide. Any glyburide or gliclazide exposure in the year before catheterization was associated with a similar likelihood of all-cause mortality or rehospitalization (adjusted odds ratio [aOR] 1.14, 95% confidence interval [CI] 0.93-1.41; p = 0.20). However, current glyburide exposure (aOR 1.37, 95% CI 1.06-1.79; p = 0.018) and long exposure to glyburide (aOR 1.37, 95% CI 1.03-1.83; p = 0.030) were associated with a higher likelihood of the composite outcome compared to current and long exposure to gliclazide, respectively. CONCLUSIONS: Current and long exposure to glyburide was associated with a greater likelihood of mortality or rehospitalization following cardiac catheterization for acute coronary syndrome, when compared to similar gliclazide exposure definitions. This study adds further evidence of the need to avoid using glyburide if a sulphonylurea is required for type 2 diabetes management.

Role of l -arginine/nitric oxide/cyclic GMP/K ATP channel signaling pathway and opioid receptors in the antinociceptive effect of rutin in mice.

The l -arginine ( l -Arg)/nitric oxide/cyclic GMP/potassium channel (K ATP ) pathway and opioid receptors are known to play critical roles in pain perception and the antinociceptive effects of various compounds. While there is evidence suggesting that the analgesic effects of rutin may involve nitric oxide modulation, the direct link between rutin and the l -Arg/nitric oxide/cyclic GMP/K ATP pathway in the context of pain modulation requires further investigation. The antinociceptive effect of rutin was studied in male NMRI mice using the formalin test. To investigate the role of the l -Arg/nitric oxide/cyclic GMP/K ATP pathway and opioid receptors, the mice were pretreated intraperitoneally with different substances. These substances included l -Arg (a precursor of nitric oxide), S-nitroso- N -acetylpenicillamine (SNAP, a nitric oxide donor), N(gamma)-nitro- l -arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase), sildenafil (an inhibitor of phosphodiesterase enzyme), glibenclamide (a K ATP channel blocker), and naloxone (an opioid receptor antagonist). All pretreatments were administered 20 min before the administration of the most effective dose of rutin. Based on our investigation, it was found that rutin exhibited a dose-dependent antinociceptive effect. The administration of SNAP enhanced the analgesic effects of rutin during both the initial and secondary phases. Moreover, L-NAME, naloxone, and glibenclamide reduced the analgesic effects of rutin in both the primary and secondary phases. In conclusion, rutin holds significant value as a flavonoid with analgesic properties, and its analgesic effect is directly mediated through the nitric oxide/cyclic GMP/K ATP channel pathway.

Baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating ATP-sensitive potassium channels.

Dopaminergic neurons in the substantia nigra (SN) expressing SUR1/Kir6.2 type ATP-sensitive potassium channels (K-ATP) are more vulnerable to rotenone or metabolic stress, which may be an important reason for the selective degeneration of neurons in Parkinson's disease (PD). Baicalein has shown neuroprotective effects in PD animal models. In this study, we investigated the effect of baicalein on K-ATP channels and the underlying mechanisms in rotenone-induced apoptosis of SH-SY5Y cells. K-ATP currents were recorded from SH-SY5Y cells using whole-cell voltage-clamp recording. Drugs dissolved in the external solution at the final concentration were directly pipetted onto the cells. We showed that rotenone and baicalein opened K-ATP channels and increased the current amplitudes with EC50 values of 0.438 µM and 6.159 µM, respectively. K-ATP channel blockers glibenclamide (50 µM) or 5-hydroxydecanoate (5-HD, 250 µM) attenuated the protective effects of baicalein in reducing reactive oxygen species (ROS) content and increasing mitochondrial membrane potential and ATP levels in rotenone-injured SH-SY5Y cells, suggesting that baicalein protected against the apoptosis of SH-SY5Y cells by regulating the effect of rotenone on opening K-ATP channels. Administration of baicalein (150, 300 mg·kg-1·d-1, i.g.) significantly inhibited rotenone-induced overexpression of SUR1 in SN and striatum of rats. We conducted surface plasmon resonance assay and molecular docking, and found that baicalein had a higher affinity with SUR1 protein (KD = 10.39 µM) than glibenclamide (KD = 24.32 µM), thus reducing the sensitivity of K-ATP channels to rotenone. Knockdown of SUR1 subunit reduced rotenone-induced apoptosis and damage of SH-SY5Y cells, confirming that SUR1 was an important target for slowing dopaminergic neuronal degeneration in PD. Taken together, we demonstrate for the first time that baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating K-ATP channels.

Nitric oxide and potassium channels but not opioid and cannabinoid receptors mediate tramadol-induced peripheral antinociception in rat model of paw pressure withdrawal.

Tramadol, an analgesic classified as an "atypical opioid", exhibits both opioid and non-opioid mechanisms of action. This study aimed to explore these mechanisms, specifically the opioid-, cannabinoid-, nitric oxide-, and potassium channel-based mechanisms, which contribute to the peripheral antinociception effect of tramadol, in an experimental rat model. The nociceptive threshold was determined using paw pressure withdrawal. To examine the mechanisms of action, several substances were administered intraplantarly: naloxone, a non-selective opioid antagonist (50 µg/paw); AM251 (80 µg/paw) and AM630 (100 µg/paw) as the selective antagonists for types 1 and 2 cannabinoid receptors, respectively; nitric oxide synthase inhibitors L-NOArg, L-NIO, L-NPA, and L-NIL (24 µg/paw); and the enzyme inhibitors of guanylatocyclase and phosphodiesterase of cGMP, ODQ, and zaprinast. Additionally, potassium channel blockers glibenclamide, tetraethylammonium, dequalinium, and paxillin were used. The results showed that opioid and cannabinoid receptor antagonists did not reverse tramadol's effects. L-NOarg, L-NIO, and L-NPA partially reversed antinociception, while ODQ completely reversed, and zaprinast enhanced tramadol's antinociception effect. Notably, glibenclamide blocked tramadol's antinociception in a dose-dependent manner. These findings suggest that tramadol's peripheral antinociception effect is likely mediated by the nitrergic pathway and sensitive ATP potassium channels, rather than the opioid and cannabinoid pathways.

TNFα and IL-8 vitreous concentrations variations with two antidiabetic therapies in patients with proliferative diabetic retinopathy: an observational study.

BACKGROUND: Antidiabetic therapies are effective, but could indirectly modify the inflammatory response in the ocular microenvironment; therefore, a study was developed to evaluate the inflammatory cytokine profile in the vitreous humor of diabetic patients with retinopathy under treatment with antidiabetic drugs. METHODS: Observational, comparative, retrospective, cross-sectional study. Interleukins 1ß, 6, 8, 10, and tumor necrosis factor-alpha (TNFα) were evaluated in the vitreous humor obtained from patients with type 2 diabetes mellitus, proliferative diabetic retinopathy, and concomitant retinal detachment or vitreous hemorrhage, and who were already on antidiabetic treatment with insulin or metformin + glibenclamide. The quantification analysis of each cytokine was performed by the cytometric bead array (CBA) technique; medians and interquartile ranges were obtained, and the results were compared between groups using the Mann-Whitney U test, where a p-value < 0.05 was considered significant. RESULTS: Thirty-eight samples; quantification of TNFα concentrations was higher in the group of patients administered insulin, while interleukin-8 was lower; in the metformin + glibenclamide combination therapy group, it occurred inversely. In the stratified analysis, the highest concentrations of interleukin-8 and TNFα occurred in patients with vitreous hemorrhage; however, the only statistical difference existed in patients with retinal detachment, whose TNFα concentration in the combined therapy group was the lowest value found (53.50 (33.03-86.66), p = 0.03). Interleukins 1ß, 6, and 10 were not detected. CONCLUSION: Interleukin-8 and TNFα concentrations are opposite between treatment groups; this change is more accentuated in patients with proliferative diabetic retinopathy and vitreous hemorrhage, where the highest concentrations of both cytokines are found, although only TNFα have statistical difference.

Spectroscopic characterization and pharmacokinetic evaluation of amorphous solid dispersions of glibenclamide for bioavailability enhancement in Wistar rats.

Oral bioavailability of glibenclamide (Glb) was appreciably improved by the formation of an amorphous solid dispersion with Poloxamer-188 (P-188). Poloxamer-188 substantially enhanced the solubility and thereby the dissolution rate of the biopharmaceutics classification system (BCS) class II drug Glb and simultaneously exhibited a better stabilizing effect of the amorphous solid dispersion prepared by the solvent evaporation method. The physical state of the dispersed Glb in the polymeric matrix was characterized by differential scanning calorimetry, X-ray diffraction, scanning electron microscope and Fourier transform infrared studies. In vitro drug release in buffer (pH 7.2) revealed that the amorphous solid dispersion at a Glb-P-188 ratio of 1:6 (SDE4) improved the dissolution of Glb by 90% within 3 h. A pharmacokinetic study of the solid dispersion formulation SDE4 in Wistar rats showed that the oral bioavailability of the drug was greatly increased as compared with the market tablet formulation, Daonil®. The formulation SDE4 resulted in an AUC0-24h ~2-fold higher. The SDE4 formulation was found to be stable during the study period of 6 months.

Enhanced dissolution rates of glibenclamide through solid dispersions on microcrystalline cellulose and mannitol, combined with phosphatidylcholine.

OBJECTIVE: This study aimed to investigate the impact of physical solid dispersions of spray-dried glibenclamide (SG) on the surface of microcrystalline cellulose (MC) and mannitol (M) surfaces, as well as their combination with phosphatidylcholine (P), on enhancing the dissolution rate of glibenclamide (G). METHODS: Solid dispersions were prepared using varying proportions of 1:1, 1:4, and 1:10 for SG on the surface of MC (SGA) and M (SGM), and then combined with P, in a proportion of 1:4:0.02 using spray drying. The particle size, specific surface area, scanning electron microscopy (SEM), X-ray diffraction (XRD), and dissolution rate of SGA and SGM were characterized. RESULTS: SEM analysis revealed successful adhesion of SG onto the surface of the carrier surfaces. XRD showed reduced crystalline characteristic peaks for SGA, while SGM exhibited a sharp peaks pattern. Both SGA and SGM demonstrated higher dissolution rates compared to SG and G alone. Furthermore, the dissolution rates of the solid dispersions of SG, MC and P (SGAP), and SG, M, and P (SGMP) were sequentially higher than that of SGA and SGM. CONCLUSIONS: The study suggests that physical solid dispersions of SG on MC and M, along with their combination with P, can effectively enhance the dissolution rate of G. These findings may be valuable in developing of oral solid drug dosage forms utilizing SGA, SGM, SGAP, and SGMP.

Rapid Characterization of the Functional and Pharmacological Consequences of Cantú Syndrome KATP Channel Mutations in Intact Cells.

Gain-of-function of KATP channels, resulting from mutations in either KCNJ8 (encoding inward rectifier sub-family 6 [Kir6.1]) or ABCC9 (encoding sulphonylurea receptor [SUR2]), cause Cantú syndrome (CS), a channelopathy characterized by excess hair growth, coarse facial appearance, cardiomegaly, and lymphedema. Here, we established a pipeline for rapid analysis of CS mutation consequences in Landing pad HEK 293 cell lines stably expressing wild type (WT) and mutant human Kir6.1 and SUR2B. Thallium-influx and cell membrane potential, reported by fluorescent Tl-sensitive Fluozin-2 and voltage-sensitive bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) dyes, respectively, were used to assess channel activity. In the Tl-influx assay, CS-associated Kir6.1 mutations increased sensitivity to the ATP-sensitive potassium (KATP) channel activator, pinacidil, but there was strikingly little effect of pinacidil for any SUR2B mutations, reflecting unexpected differences in the molecular mechanisms of Kir6.1 versus SUR2B mutations. Compared with the Tl-influx assay, the DiBAC4(3) assay presents more significant signal changes in response to subtle KATP channel activity changes, and all CS mutants (both Kir6.1 and SUR2B), but not WT channels, caused marked hyperpolarization, demonstrating that all mutants were activated under ambient conditions in intact cells. Most SUR2 CS mutations were markedly inhibited by <100 nM glibenclamide, but sensitivity to inhibition by glibenclamide, repaglinide, and PNU37883A was markedly reduced for Kir6.1 CS mutations. Understanding functional consequences of mutations can help with disease diagnosis and treatment. The analysis pipeline we have developed has the potential to rapidly identify mutational consequences, aiding future CS diagnosis, drug discovery, and individualization of treatment. SIGNIFICANCE STATEMENT: We have developed new fluorescence-based assays of channel activities and drug sensitivities of Cantú syndrome (CS) mutations in human Kir6.1/SUR2B-dependent KATP channels, showing that Kir6.1 mutations increase sensitivity to potassium channel openers, while SUR2B mutations markedly reduce K channel opener (KCO) sensitivity. However, both Kir6.1 and SUR2B CS mutations are both more hyperpolarized than WT cells under basal conditions, confirming pathophysiologically relevant gain-of-function, validating DiBAC4(3) fluorescence to characterize hyperpolarization induced by KATP channel activity under basal, non KCO-activated conditions.

Precision Effects of Glibenclamide on MRI Endophenotypes in Clinically Relevant Murine Traumatic Brain Injury.

OBJECTIVES: Addressing traumatic brain injury (TBI) heterogeneity is increasingly recognized as essential for therapy translation given the long history of failed clinical trials. We evaluated differential effects of a promising treatment (glibenclamide) based on dose, TBI type (patient selection), and imaging endophenotype (outcome selection). Our goal to inform TBI precision medicine is contextually timely given ongoing phase 2/planned phase 3 trials of glibenclamide in brain contusion. DESIGN: Blinded randomized controlled preclinical trial of glibenclamide on MRI endophenotypes in two established severe TBI models: controlled cortical impact (CCI, isolated brain contusion) and CCI+hemorrhagic shock (HS, clinically common second insult). SETTING: Preclinical laboratory. SUBJECTS: Adult male C57BL/6J mice (n = 54). INTERVENTIONS: Mice were randomized to naïve, CCI±HS with vehicle/low-dose (20 µg/kg)/high-dose glibenclamide (10 µg/mouse). Seven-day subcutaneous infusions (0.4 µg/hr) were continued. MEASUREMENTS AND MAIN RESULTS: Serial MRI (3 hr, 6 hr, 24 hr, and 7 d) measured hematoma and edema volumes, T2 relaxation (vasogenic edema), apparent diffusion coefficient (ADC, cellular/cytotoxic edema), and 7-day T1-post gadolinium values (blood-brain-barrier [BBB] integrity). Linear mixed models assessed temporal changes. Marked heterogeneity was observed between CCI versus CCI+HS in terms of different MRI edema endophenotypes generated (all p < 0.05). Glibenclamide had variable impact. High-dose glibenclamide reduced hematoma volume ~60% after CCI (p = 0.0001) and ~48% after CCI+HS (p = 4.1 × 10-6) versus vehicle. Antiedema benefits were primarily in CCI: high-dose glibenclamide normalized several MRI endophenotypes in ipsilateral cortex (all p < 0.05, hematoma volume, T2, ADC, and T1-post contrast). Acute effects (3 hr) were specific to hematoma (p = 0.001) and cytotoxic edema reduction (p = 0.0045). High-dose glibenclamide reduced hematoma volume after TBI with concomitant HS, but antiedema effects were not robust. Low-dose glibenclamide was not beneficial. CONCLUSIONS: High-dose glibenclamide benefitted hematoma volume, vasogenic edema, cytotoxic edema, and BBB integrity after isolated brain contusion. Hematoma and cytotoxic edema effects were acute; longer treatment windows may be possible for vasogenic edema. Our findings provide new insights to inform interpretation of ongoing trials as well as precision design (dose, sample size estimation, patient selection, outcome selection, and Bayesian analysis) of future TBI trials of glibenclamide.

siGCD: a web server to explore survival interaction of genes, cells and drugs in human cancers.

It is pivotal and remains challenge for cancer precision treatment to identify the survival outcome interactions between genes, cells and drugs. Here, we present siGCD, a web-based tool for analysis and visualization of the survival interaction of Genes, Cells and Drugs in human cancers. siGCD utilizes the cancer heterogeneity to simulate the manipulated gene expression, cell infiltration and drug treatment, which overcomes the data and experimental limitations. To illustrate the performance of siGCD, we identified the survival interaction partners of EGFR (gene level), T cells (cell level) and sorafenib (drug level), and our prediction was consistent with previous reports. Moreover, we validate the synergistic effect of regorafenib and glyburide, and found that glyburide could significantly improve the regorafenib response. These results demonstrate that siGCD could benefit cancer precision medicine in a wide range of advantageous application scenarios including gene regulatory network construction, immune cell regulatory gene identification, drug (especially multiple target drugs) response biomarker screening and combination therapeutic design.

Co-administration of metformin and/or glibenclamide with losartan reverse NG-nitro-l-arginine-methyl ester-streptozotocin-induced hypertensive diabetes and haemodynamic sequelae in rats.

Over the years, there have been opinions on whether to reduced blood pressure (BP) to a different levels in patients with diabetes mellitus. Hence, this study investigated the efficacy of the co-administration of losartan (angiotensin receptor blocking antihypertensive agent) with metformin and/or glibenclamide (antidiabetic agents) on hypertensive-diabetic experimental rats induced by NG-nitro-l-arginine-methyl-ester hydrochloride (l-NAME), and streptozotocin (STZ). STZ (45 mg/kg, i.p.)-induced diabetic rats combined with l-NAME (40 mg/kg, p.o.)-induced hypertension were allotted into different groups. Group 1 received distilled water (10 mL/kg) and served as normal control, group 2 comprised hypertensive diabetic rats with distilled water, groups 3-5 were hypertensive-diabetic rats but received combination treatments of losartan + metformin, losartan + glibenclamide, and losartan + metformin + glibenclamide daily for 8 weeks, respectively. Our finding revealed no changes in the body weights, but there was a significant increase in fasting blood sugar levels in l-NAME - STZ-induced hypertensive-diabetes, which were lowered by losartan + metformin, losartan + glibenclamide, and losartan + metformin + glibenclamide treatments. Moreover, the increased systolic-BP, mean arterial pressure but not diastolic-BP and heart rate by l-NAME + STZ were attenuated more significantly by losartan + metformin + glibenclamide between weeks 2-8 relative to hypertensive-diabetic control. l-NAME + STZ-induced elevated levels of lactate dehydrogenase and creatinine kinase, were differentially reversed by losartan + metformin, losartan + glibenclamide, and losartan + metformin + glibenclamide. However, l-NAME + STZ-induced decreased nitrite level was significantly restored by all treatments, suggesting increased nitrergic transmission. Additionally, l-NAME + STZ-induced degeneration of pancreatic islet and myocardial cells were dramatically alleviated by losartan + metformin + glibenclamide treatments. Our findings suggest hyperglycemia with raised systolic-BP should be managed with losartan combined with both metformin and glibenclamide than single combination of losartan with antidiabetics.

The Rat Continuous Intestine Model Predicts the Impact of Particle Size and Transporters on the Oral Absorption of Glyburide.

Oral drug absorption is known to be impacted by the physicochemical properties of drugs, properties of oral formulations, and physiological characteristics of the intestine. The goal of the present study was to develop a mathematical model to predict the impact of particle size, feeding time, and intestinal transporter activity on oral absorption. A previously published rat continuous intestine absorption model was extended for solid drug absorption. The impact of active pharmaceutical ingredient particle size was evaluated with glyburide (GLY) as a model drug. Two particle size suspensions of glyburide were prepared with average particle sizes of 42.7 and 4.1 µm. Each suspension was dosed as a single oral gavage to male Sprague Dawley rats, and concentration-time (C-t) profiles of glyburide were measured with liquid chromatography coupled with tandem mass spectrometry. A continuous rat intestine absorption model was extended to include drug dissolution and was used to predict the absorption kinetics of GLY depending on particle size. Additional literature datasets of rat GLY formulations with particle sizes ranging from 0.25 to 4.0 µm were used for model predictions. The model predicted reasonably well the absorption profiles of GLY based on varying particle size and varying feeding time. The model predicted inhibition of intestinal uptake or efflux transporters depending on the datasets. The three datasets used formulations with different excipients, which may impact the transporter activity. Model simulations indicated that the model provides a facile framework to predict the impact of transporter inhibition on drug C-t profiles. Model simulations can also be conducted to evaluate the impact of an altered intestinal lumen environment. In conclusion, the rat continuous intestine absorption model may provide a useful tool to predict the impact of varying drug formulations on rat oral absorption profiles.

Evaluation of the impact of polypeptide-p on diabetic rats upon its cloning, expression, and secretion in Saccharomyces boulardii.

This study was designed to evaluate the effectiveness of recombinant polypeptide-p derived from Momordica charantia on diabetic rats. In this research, the optimized sequence of polypeptide-p gene fused to a secretion signal tag was cloned into the expression vector and transformed into probiotic Saccharomyces boulardii. The production of recombinant secretion protein was verified by western blotting, HPLC, and mass spectrometry. To assay recombinant yeast bioactivity in the gut, diabetic rats were orally fed wild-type and recombinant S. boulardii, in short SB and rSB, respectively, at two low and high doses as well as glibenclamide as a reference drug. In untreated diabetic and treated diabetic + SB rats (low and high doses), the blood glucose increased from 461, 481, and 455 (mg/dl), respectively, to higher than 600 mg/dl on the 21st day. Whereas glibenclamide and rSB treatments showed a significant reduction in the blood glucose level. The result of this study promised a safe plant-source supplement for diabetes through probiotic orchestration.

Repositioning Glibenclamide in cardiac fibrosis by targeting TGF-ß1-pSmad2/3-NLRP3 cascade.

The proposed objective of this study is to attenuate cardiac fibrosis by inhibiting NLRP3 inflammasome and related genes in uninephrectomized-DOCA fed rat model. Cardiac fibrosis was induced in male Sprague Dawley rats by uninephrectomy and by subsequent administration of deoxycorticosterone acetate (DOCA) every 4th day till 28 days along with 1% NaCl in drinking water. Further, the animals in treatment groups were treated with Glibenclamide (10, 20 and 40 mg/kg) for 28 days which was selected based on docking study. Interim analysis was carried out on the 14th day to assess the hemodynamic parameters. On the 28th day, anthropometric, hemodynamic, biochemical and oxidative stress parameters, gene expression (TGF-ß1, pSmad 2/3, NLRP3, IL-1ß and MMP-9), ex vivo Langendorff studies and Masson's trichrome staining of heart was carried out. Results were interpreted using ANOVA followed by post hoc Bonferroni test. Glibenclamide treatment significantly reduced the increase in blood pressure. Furthermore, the ECG patterns of the treatment groups displayed a lower frequency of the slow repolarizing events seen in the model animals. Moreover, Glibenclamide treatment demonstrated normal LV function as evidenced by a significant decrease in LVEDP. Besides, this intervention improved the anthropometric parameters and less collagen deposition in Masson's trichrome staining. The cascade of TGF-ß1-pSmad2/3-NLRP3 was downregulated along with suppression of IL-1ß. Our study repositioned anti-diabetic drug Glibenclamide to treat cardiac fibrosis by inhibiting the TGF-ß1-pSmad2/3-NLRP3 cascade.

The shortcoming of using glibenclamide in exploratory clinical headache provocation studies.

OBJECTIVE: Preclinical and clinical studies implicate the vascular ATP-sensitive potassium (KATP) channel in the signaling cascades underlying headache and migraine. However, attempts to demonstrate that the KATP channel inhibitor glibenclamide would attenuate triggered headache in healthy volunteers have proven unsuccessful. It is questionable, however, whether target engagement was achieved in these clinical studies. METHODS: Literature data for human glibenclamide pharmacokinetics, plasma protein binding and functional IC50 values were used to predict the KATP receptor occupancy (RO) levels obtained after glibenclamide dosing in the published exploratory clinical headache provocation studies. RO vs. time profiles of glibenclamide were simulated for the pancreatic KATP channel subtype Kir6.2/SUR1 and the vascular subtype Kir6.1/SUR2B. RESULTS: At the clinical dose of 10 mg of glibenclamide used in the headache provocation studies, predicted maximal occupancy levels of up to 90% and up to 26% were found for Kir6.2/SUR1 and Kir6.1/SUR2B, respectively. CONCLUSIONS: The findings of the present study indicate that effective Kir6.1/SUR2B target engagement was not achieved in the clinical headache provocation studies using glibenclamide. Therefore, development of novel selective Kir6.1/SUR2B inhibitors, with good bioavailability and low plasma protein binding, is required to reveal the potential of KATP channel inhibition in the treatment of migraine.

Elabela relaxes rat pulmonary artery and trachea via BKCa, KV, and KATP channels.

OBJECTIVE: Elabela is a newly discovered peptide hormone. This study aimed to determine the functional effects and mechanisms of action of elabela in rat pulmonary artery and trachea. MATERIALS AND METHODS: Vascular rings isolated from the pulmonary arteries of male Wistar Albino rats were placed in chambers in the isolated tissue bath system. The resting tension was set to 1 g. After the equilibration period, the pulmonary artery rings were contracted with 10-6 M phenylephrine. Once a stable contraction was achieved, elabela was applied cumulatively (10-10-10-6 M) to the vascular rings. To determine the vasoactive effect mechanisms of elabela, the specified experimental protocol was repeated after the incubation of signaling pathway inhibitors and potassium channel blockers. The effect and mechanisms of action of elabela on tracheal smooth muscle were also determined by a similar protocol. RESULTS: Elabela exhibited a concentration-dependent relaxation in the precontracted rat pulmonary artery rings (p < .001). Maximal relaxation level was 83% (pEC50: 7.947 CI95(7.824-8.069)). Removal of the endothelium, indomethacin incubation, and dideoxyadenosine incubation significantly decreased the vasorelaxant effect levels of elabela (p < .001). Elabela-induced vasorelaxation levels were significantly reduced after iberiotoxin, glyburide, and 4-Aminopyridine administrations (p < .001). L-NAME, methylene blue, apamin, TRAM-34, anandamide, and BaCl2 administrations did not cause a significant change in the vasorelaxant effect level of elabela (p = 1.000). Elabela showed a relaxing effect on precontracted tracheal rings (p < .001). Maximal relaxation level was 73% (pEC50: 6.978 CI95(6.791-7.153)). The relaxant effect of elabela on tracheal smooth muscle was decreased significantly after indomethacin, dideoxyadenosine, iberiotoxin, glyburide, and 4-Aminopyridine incubations (p < .001). CONCLUSIONS: Elabela exerted a prominent relaxant effect in the rat pulmonary artery and trachea. Intact endothelium, prostaglandins, cAMP signaling pathway, and potassium channels (BKCa, KV, and KATP channels) are involved in the vasorelaxant effect of elabela. Prostaglandins, cAMP signaling pathway, BKCa channels, KV channels, and KATP channels also contribute to elabela-induced tracheal smooth muscle relaxant effect.

The role of l -arginine/NO/cGMP/K ATP channel pathway in the local antinociceptive effect of berberine in the rat formalin test.

Berberine is an isoquinoline alkaloid naturally produced by several types of plants. Berberine has extensive pharmacological effects, such as anti-diabetic, anti-inflammatory, and antioxidant effects. In the current study, we assess the antinociceptive effects of berberine and its association with the l -arginine ( l -Arg)/NO/cGMP/K ATP channel pathway via intraplantar administration in rats. To examine the antinociceptive properties of berberine, the formalin test was conducted. The number of rat paw flinches was counted for an h. l -Arg (precursor of nitric oxide, 3-30 µ g/paw), l -NAME (NO synthase inhibitor, 10 and 100 µ g/paw), methylene blue (guanylyl cyclase inhibitor, 100 and 200 µ g/paw), and glibenclamide (ATP-sensitive potassium channel blocker, 10 and 30 µ g/paw) were locally injected, respectively, into the right hind paws of rats as a pre-treatment before berberine injection to understand how the l -Arg/NO/cGMP/K ATP pathway plays a role in the antinociceptive effect of berberine. The ipsilateral injection of berberine into the right paw (0.1-10 0 µ g/paw) showed a dose-dependent antinociceptive effect in both the first and second phases of the formalin test, almost similar to morphine (25 µ g/paw). Intraplantar injection of l -Arg (30 µg/paw) increased the antinociceptive effect of berberine in the second phase. In addition, injection of l -NAME, methylene blue, and glibenclamide caused a reduction in the antinociceptive effect of berberine throughout the second phase in a dose-dependent manner. However, the antinociceptive effects of berberine in the first phase of the rat formalin test were not affected by this pathway. As a novel local antinociceptive agent, berberine can exert a peripheral antinociceptive effect via the l -Arg/NO/cGMP/K ATP channel pathway.