Concentrations Of Glibenclamide Research Articles

Lipid and protein oxidation in Cyprinus carpio muscle by environmentally relevant concentrations of glibenclamide

Diabetes mellitus (DM2) was considered a more common comorbidity and was associated with high mortality due to metabolic disease in the COVID-19 pandemic. For this reason, glibenclamide (GLI) was commonly prescribed for its control. However, it has been found in environmentally relevant concentrations in various water reservoirs due to its high consumption. Common carp (Cyprinus carpio ), besides its nutritional properties and economic importance, is also considered a good bioindicator for assessing environmental health and pollutants presence; nonetheless, there are currently insufficient studies on the effect of GLI on the physicochemical and textural properties of the muscle of this bioindicator species. In this study, the effect of this drug at two environmentally relevant concentrations (50 and 1,000 ng/L) at five exposure times (every 24 up to 96 h) on the quality of carp muscle was investigated. Parameters such as carbonylated proteins, lipid peroxidation, total sulfhydryl content, water holding capacity, pH, electrophoretic profile, and texture profile analysis were determined. Regardless of the concentration of GLI used, the evaluated parameters showed significant muscle damage; therefore, it must be emphasized that this emerging pollutant not only damages environment, but also affects edible species present in different water reservoirs.

A dual-emission ratiometric fluorescent biosensor for ultrasensitive detection of glibenclamide using S-CDs/CdS quantum dots

In the present work, sulfide-doped carbon dots (S-CDs)/cadmium sulfide quantum dots (CdS QDs) ratiometric fluorescent nanosensor has been developed for sensitive and selective determination of glibenclamide (GLC) in biological fluids. The method was based on the quenching effect of GLC on the dual-emission intensity of the S-CDs/CdS QDs system at 420 nm and 650 nm, which are related to S-CDs and CdS QDs, respectively. The fluorimetric data analysis indicated that the fluorescence signals of the system were quenched by adding GLC in a concentration-dependent manner. A good linear relationship was observed between GLC concentration and the quenched fluorescence intensity of the S-CDs/CdS QDs in the range of 0.3 nM−10.0 μM. The limit of detection (LOD) value was estimated to be 0.12 nM. Furthermore, under optimum conditions, GLC was detected in spiked human serum sample (as real media) using the developed ratiometric nanosensor with an accuracy of 99.6%. According to the results, the developed dual-emission system can be used as a reliable method for the quantitative detection of GLC in biological samples.

Effect of Ethanol Root Extract of Calliandra Portoricensis on the Pharmacokinetic fate of Glibenclamide in Rats

Background: Concomitant administration of herbs with conventional drugs may cause pharmacokinetic modification of the drugs, leading to drug toxicity or therapeutic failure. The observed use of Calliandra portoricensis (Jacq.) Benth in diabetic patients on glibenclamide has aroused interest and its possible influence on the pharmacokinetics of glibenclamide was investigated. Methods: The dried root of Calliandra portoricensis was pulverized and extracted in 90% ethanol to obtain the crude extract used in this study. After 10 h overnight fast, 30 rats previously acclimatized were given glibencalmide (10 mg/kg) orally. Following a 3-week washout period, the rats were given ethanol root extract of C. portoricensis (500 mg/kg) for 5 days followed by glibenclamide (10 mg/kg). In both studies, blood samples were collected from 0 - 48 h post-dosing and assayed for the concentration of glibenclamide using a well-validated HPLC method and the Pharmacokinetic parameters computed. Results: The Cmax, AUC0-∞ were significantly lowered (P<0.05) in the co-administered group, which resulted in a decrease in bioavailability. The Kel, Vd, Tmax and MCR were higher in the combination group (P<0.05). Thus suggests that concomitant use of C. portoricensis may have influenced the absorption of glibenclamide thus resulting in decreasedAUCan ; also, increase in Vd may have caused increase in the clearance. Conclusion: The interaction between glibenclamide and C. portoricensis may be attributed to the presence of some metal ions in the plant. Thus, caution is advised in the concurrent use of these remedies.

Development of a Selective, Sensitive, and Rapid LC‐MS/MS Assay for Studying the Effect of Concurrent Intake of Pepper and Turmeric on Curcumin Level in Healthy Human Urineμ

Hypothesis We hypothesized that co-administration of turmeric with pepper through a meal increases the bioavailability of curcumin (CCM), one of the components of turmeric, which could enhance its beneficial effects. Methods Fasted individuals (n=3) consumed a standardized test meal on two occasions, each separated by one week. Meal 1 consisted of 120 ml egg white (cooked) mixed with 5 g powdered turmeric. Meal 2 contained the same ingredients plus 0.5 g black pepper. At baseline and 10 subsequent time points, participants collected batches of urine for 24 hrs. The volume of urine at each period was measured and a portion of it collected in a 50-mL Falcon tube and stored in -80 oC until analysis. Samples were thawed and aliquoted while protected from light. Then, to 400 μL of the urine sample was added 160 μL of triple-deionized water, and 50 μL of glibenclamide (20 ng/mL) as internal standard (IS) and extracted twice with 500 μL of Ethyl acetate: Methanol (95:5). After centrifugation at 13.2 x103g for 5 min, the organic phase were separated. The extracted samples were dried, reconstituted with 50 μL of methanol:acetonitrile:water:formic acid (23:41:36:1) used for assay. Separation of analytes was achieved by a C18 column and detected by tandem mass-spectrometry. The concentration of CCM in urine samples was calculated using standard curves constructed based on 7 standard solutions ranged from 0.625-40 ng/mL and accordingly, the amount of excreted CCM in individuals 24-hrs urine was estimated. Results A simple liquid chromatography-mass spectrometry (LC-MS/MS) method for quantification of CCM in human urine was successfully developed. The mass spectrometry was more sensitive when negative mode was used. The ion transitions of m/z 367.0 →133.9 and 367.0 →173.0 for CCM, and m/z 494.0 →369.1 and 494 →171.8 for IS were used in multiple reaction mode. The ion spectra of CCM and IS are shown in figure 1. Chromatographic peaks of all analytes were separated with no interference with substances. The retention time of CCM and IS were 3.81, and 3.93 minutes, respectively. The limit of detection and limit of quantification were 0.156 and 0.312 ng/mL respectively. The validated method was then successfully applied to determine the urine concentration of CCM in human. The results of this study show that the total urinary excreted amount of CCM after 24-hrs in individuals concurrently receiving turmeric and pepper (7.53±0.94 μg /24 h vs 23.71 ± 7.84 μg/24 hr) were significantly higher than individual receiving only turmeric. Conclusion A simple, selective, sensitive, and rapid LC-MS/MS method was developed and validated to simultaneously determine the concentration of CCM and glibenclamide in human urine samples. This method has many advantages in terms of simple extraction procedure, higher sensitivity, and shorter run time. Moreover, the total urinary execrated CCM significantly increased in the group receiving CCM concomitantly with pepper compared with the group received only turmeric. The observed higher urine concentrations reveal that pepper could promote a higher rate of absorption of and enhance bioavailability of CCM, and increase its beneficial effects.

The antagonistic atorvastatin-glibenclamide interactions suppressed the atorvastatin-induced Bax/cytochrome c/p53 mRNA expressions and increased Rho A mRNA expression in B16f10 melanoma cell culture

Many studies have shown promising benefits for using statins among cancer patients. Here, we considered the effects of atorvastatin-glibenclamide interactions on pro-apoptotic and metastatic factors of Bax, p53, cytochrome c and Rho A genes in melanoma cell cancer. Melanoma B16F10 cells were treated for 48 h with glibenclamide and atorvastatin alone and glibenclamide-atorvastatin combination followed by quantitative RT- PCR assay. Cells with no treatment were considered as control. Results revealed an increase in the expression of Bax, cytochrome c and p53 mRNAs in atorvastatin group without any changes in Rho A gene expression. In contrast to, glibenclamide increased Rho A gene expression and suppressed cytochrome c expression by decreasing Bax level. Effects of combination therapy on atorvastatin-induced increase in Bax/cytochrome c/p53 mRNA expression depended on glibenclamide concentration. Glibenclamide at low concentrations of 1 μM and 10 μM suppressed atorvastatin-induced increase in Bax/cytochrome c/p53 mRNA and promoted metastatic signal of Rho A in melanoma cells, while glibenclamide at high concentration of 100 μM induced an increase in mRNA expression of cytochrome c and p53 in melanoma cells. Together, our study provides evidence that the anticancer effect of statins on melanoma could be antagonized by associated sulfonylureas use.

Ultrasensitive detection of glibenclamide based on its enhancing effect on the fluorescence emission of CdTe quantum dots.

Glibenclamide (GB), as a sulfonylurea-based medication is commonly prescribed for the treatment of type 2 diabetes. Due to its increasing consumption, there is a need to develop a simple, fast, and reliable detection method to follow its concentration in pharmaceutical and biological samples. Herein, a novel fluorometric method is developed for the sensitive measurement of GB. The method is based on the enhancing effect of GB on the fluorescence emission of mercaptopropionic acid (MPA) capped cadmium telluride quantum dots (CdTe QDs). QDs were synthesized in aqueous solution and were characterized by fluorescence spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). Fluorescence intensity of QDs was enhanced by adding GB in a very low concentration. The effect of operative factors such as pH, buffer, contact time and concentration of CdTe QDs were investigated and in the optimized condition, a linear increase was achieved for the emission intensity of QDs by increasing GB concentration in the range 49-345ng mL-1 , with a detection limit of 17.84ng mL-1 . The offered method has an acceptable precision (relative standard deviations were<2.8%) and was satisfactorily applied for the determination of GB in pharmaceutical products and human urine samples.

Binding of sulphonylureas to plasma proteins - A KATP channel perspective.

Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo. KATP currents from mouse pancreatic β-cells and Xenopus oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of KATP currents in pancreatic β-cells and Xenopus oocytes. In contrast, glibenclamide inhibition of recombinant KATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug’s therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close KATP channels and stimulate insulin secretion.

Environmentally relevant concentrations of glibenclamide induce oxidative stress in common carp (Cyprinus carpio)

The hypoglycemic pharmaceutical glibenclamide (GLB) is widely used around the world. This medication is released into the environment by municipal, hospital and industrial wastewater discharges. Although there are reports of its environmental occurrence in the scientific literature, toxicity studies on aquatic species of commercial interest such as the common carp Cyprinus carpio are scarce. The present study aimed to evaluate the oxidative stress induced on C. carpio by environmentally relevant concentrations of GLB. Biomarkers of oxidative damage such as hydroperoxide content, lipid peroxidation and protein carbonyl content were evaluated as well as the activity of the antioxidant enzymes superoxide dismutase and catalase. The concentration of GLB was determined in water as well as in gill, liver, muscle, brain and blood of carp at 12, 24, 48, 72 and 96 h. The findings obtained in the study prove that GLB induces increases in biomarkers of oxidative damage and antioxidant enzyme activity in the teleost C. carpio, that this response is not concentration dependent and that the organs evaluated bioconcentrate this hypoglycemic agent. These findings permit us to conclude that the presence of GLB in water bodies represents a risk for aquatic species.

Effect of Tinospora cordifolia aqua-alcoholic extract on pharmacokinetic of Glibenclamide in rat: An herb-drug interaction study

Tinospora cordifolia (TC) has been used as a complimentary/alternative medicine against diabetes. Considering its potential to modulate metabolic enzymes, Tinospora cordifolia extract (TCE) may influence the metabolism of the antidiabeic drug Glibenclamide following co-administration. Accordingly, this work was undertaken to evaluate impact of TCE on fate of Glibenclamide. Activity of clinically important Cytochrome P450 isoenzymes were inhibited in the order of CYP2C9 > CYP2D6 > CYP2C19 > CYP1A2 > CYP3A4. Formations of metabolites were inhibited with increasing concentration of TCE in both rat and human liver microsomes. TCE was co- administered in three different groups (0, 100 and 400 mg/kg) with Glibenclamide at 1 mg/kg dose to observe the alteration in pharmacokinetic parameters of Glibenclamide. The rats were pretreated with 0 (vehicle), 100 and 400 mg/kg dose of TCE b.i.d for 14 days and on the 14th day all three groups were administered with 1 mg/kg Glibenclamide. Pharmacokinetic parameters were analyzed based on plasma concentrations of Glibenclamide from all the groups by LC–HRMS methods using Glipizide as an internal standard. At 400 mg/kg dose, a marked increase in the bio availability of Glibenclamide was observed with a significant delay of Tmax and suppression of clearance.

Population pharmacokinetic modeling of glibenclamide in poorly controlled South African type 2 diabetic subjects.

AimThe aim of this study was to describe the pharmacokinetics (PK) of glibenclamide in poorly controlled South African type 2 diabetic subjects using noncompartmental and model-based methods.MethodsA total of 24 subjects with type 2 diabetes were administered increasing doses (0 mg/d, 2.5 mg/d, 5 mg/d, 10 mg/d, and 20 mg/d) of glibenclamide daily at 2-week intervals. Plasma glibenclamide, glucose, and insulin determinations were performed. Blood sampling times were 0 minute, 30 minutes, 60 minutes, 90 minutes, and 120 minutes (post breakfast sampling) and 240 minutes, 270 minutes, 300 minutes, 330 minutes, 360 minutes, and 420 minutes (post lunch sampling) on days 14, 28, 42, 56, and 70 for doses of 0 mg, 2.5 mg, 5.0 mg, 10 mg, and 20 mg, respectively. Blood sampling was performed after the steady state was reached. A total of 24 individuals in the data set contributed to a total of 841 observation records. The PK was analyzed using noncompartmental analysis methods, which were implemented in WinNonLin®, and population PK analysis using NONMEM®. Glibenclamide concentration data were log transformed prior to fitting.ResultsA two-compartmental disposition model was selected after evaluating one-, two-, and three-compartmental models to describe the time course of glibenclamide plasma concentration data. The one-compartment model adequately described the data; however, the two-compartment model provided a better fit. The three-compartment model failed to achieve successful convergence. A more complex model, to account for enterohepatic recirculation that was observed in the data, was unsuccessful.ConclusionIn South African diabetic subjects, glibenclamide demonstrates linear PK and was best described by a two-compartmental model. Except for the absorption rate constant, the other PK parameters reported in this study are comparable to those reported in the scientific literature. The study is limited by the small study sample size and inclusion of poorly controlled type 2 diabetic subjects.

Controversy about the relationship between sulfonylurea use and cardiovascular events and mortality.

Among a number of classes of oral antihyperglycemic drugs, sulfonylureas (SUs) demonstrating the potential effect to reduce blood glucose level have been used in a majority of patients with type 2 diabetes for many years. The pathophysiological processes by which SUs adversely induce the risk of cardiovascular disease have not been fully clarified. We summarized about the relationship between SU use and cardiovascular events and mortality in patients with type 2 diabetes.

Effects of Thymoquinone on the Pharmacokinetics and Pharmacodynamics of Glibenclamide in a Rat Model

Glibenclamide and thymoquinone plasma concentrations were analysed using a sensitive RP-HPLC method, and non-compartmental model pharmacokinetic parameters were calculated. The maximum reduction in blood glucose level was observed 3 hours following glibenclamide administration, which reached 47.4% of baseline, whereas it was reduced by 53.0% to 56.2% when co-administrated with thymoquinone. Plasma concentration of glibenclamide was increased by 13.4% and 21.8% by the co-administration of thymoquinone as single and multiple doses, respectively (P<0.05). The AUC and TI/2 of glibenclamide were also increased respectively by 32.0% and 17.4% with a thymoquinone single dose, and by 52.5% and 92.8% after chronic treatment. Furthermore, diabetic rats treated with thymoquinone demonstrated a marked decrease in hepatic protein expressions of CYP3A2 and CYP2C 11 enzymes that are responsible for the metabolism of glibenclamide. The current data suggest that thymoquinone exhibits a synergistic effect with glibenclamide on glucose level, which could be explained by reducing CYP450 activity at the protein level.

Systemic Administration of Glibenclamide Fails to Achieve Therapeutic Levels in the Brain and Cerebrospinal Fluid of Rodents.

Activating mutations in the Kir6.2 (KCNJ11) subunit of the ATP-sensitive potassium channel cause neonatal diabetes (ND). Patients with severe mutations also suffer from neurological complications. Glibenclamide blocks the open KATP channels and is the treatment of choice for ND. However, although glibenclamide successfully restores normoglycaemia, it has a far more limited effect on the neurological problems. To assess the extent to which glibenclamide crosses the blood-brain barrier (BBB) in vivo, we quantified glibenclamide concentrations in plasma, cerebrospinal fluid (CSF), and brain tissue of rats, control mice, and mice expressing a human neonatal diabetes mutation (Kir6.2-V59M) selectively in neurones (nV59M mice). As only small sample volumes can be obtained from rodents, we developed a highly sensitive method of analysis, using liquid chromatography tandem mass spectrometry acquisition with pseudo-selected reaction monitoring, achieving a quantification limit of 10ng/ml (20nM) glibenclamide in a 30μl sample. Glibenclamide was not detectable in the CSF or brain of rats after implantation with subcutaneous glibenclamide pellets, despite high plasma concentrations. Further, one hour after a suprapharmacological glibenclamide dose was administered directly into the lateral ventricle of the brain, the plasma concentration was twice that of the CSF. This suggests the drug is rapidly exported from the CSF. Elacridar, an inhibitor of P-glycoprotein and breast cancer resistance protein (major multidrug resistance transporters at the BBB), did not affect glibenclamide levels in CSF and brain tissue. We also identified a reduced sensitivity to volatile anaesthetics in nV59M mice and showed this was not reversed by systemic delivery of glibenclamide. Our results therefore suggest that little glibenclamide reaches the central nervous system when given systemically, that glibenclamide is rapidly removed across the BBB when given intracranioventricularly, and that any glibenclamide that does enter (and is below our detection limit) is insufficient to influence neuronal function as assessed by anaesthesia sensitivity.

Pharmacokinetic interaction of curcumin and glibenclamide in diabetic rats.

Aim:The aim was to assess the pharmacokinetic (PK) interaction of curcumin and glibenclamide (GL) in diabetic rats.Materials and Methods:Sprague-Dawley rats induced with diabetes were divided into 2 groups of six rats in each. Group I: GL (6 mg/kg po once daily) treatment in diabetic rats and group 2: Curcumin (50 mg/Kg po once daily) + GL (dose as above) in diabetic rats. Blood samples were collected at pre-determined time intervals for kinetic analysis after the first and last oral dosing of GL for single and multiple dose studies, respectively. Plasma samples were assayed for GL concentration by high-performance liquid chromatography and PK parameters were analyzed.Results:The half-life (t1/2) and mean residence time (MRT) of GL were significantly increased in curcumin pre-treated rats as compared to GL alone in single and multiple dose studies. Similarly, the Vdss was significantly increased in curcumin pre-treated rats in single dose study as compared to GL alone treated group, but no significant difference was observed in multiple dose kinetics.Conclusion:The study revealed higher values (t1/2, MRT and Vdss) of GL in curcumin pre-treated group due to the inhibitory effect of curcumin on intestinal CYP3A4.

Toxic effects of glibenclamide in fetuses of normoglycemic rats: an alternative therapy for gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is defined as glucose intolerance first diagnosed during the second or third trimester of pregnancy. The treatment aims at glycemic control through changes in the patient\\'s diet with or without exercise, but some patients need insulin therapy. An alternative would be to use oral hypoglycemic agents such as glibenclamide (GLIB). The present study aims to analyze the toxic effects of GLIB in fetuses of pregnant rats which received 5 or 20mg/kg doses of GLIB. Glycemic dosage reveals no significant difference between control (deionized water) and treated groups, showing that these concentrations of GLIB were not effective to cause hypoglycemia in rats. The vitality of the fetuses in all groups was 100%. GLIB administration promoted increase in weight and significant changes in measures of external morphological parameters of treated fetuses. Histological analysis revealed that liver lobes, lobules and central lobular veins were well defined for all treatments. However, GLIB animals presented a light brownish precipitate into the center-lobular veins and in the liver parenchyma among the hepatocytes. These results indicated a possible passage of the drug through the blood-placental membrane, without serious changes that impair the development of neither bone tissue, nor the liver of these animals.

Clinicopharmacological evaluation of drug interaction between Rifampicin and gliburide in Tuberculosis patients with Type II diabetes.

In our community Diabetes Mellitus and pulmonary Tuberculosis are still very common. The % of Pulmonary Tuberculosis among diabetic patients is nearly 27% by radiological diagnosis (377J IMA vol 100NO.6 june 2002). The % of diabetes among pulmonary tuberculosis patients is 6.5%. Both diseases are chronic and co-exist not commonly. It is said that if tuberculosis is uncontrolled inspite of meticulous anti TB treatment of tuberculosis patients suspect latent diabetes. When both diseases coexist they are mutually aggravating. If they are simultaneously treated effectively the effect is good. Unfortunately TB requires polypharmacy. Some of the recent anti TB drugs like Rifampicin have enzyme inducing capacity producing a low blood level concentration of certain drugs like glibenclamide ( sulphonylurea) with which it interacts. In maturity onset diabetic (NIDDM)patients with TB taking glibenclamide for the control of diabetes and Rifampicin containing regimens., simultaneously for TB it was found that the requirements of the oral hypoglycaemic agents are increased to control the diabetes 1 . It was proved that Rifampicin by inducing the cytchrome P450 enzymes in the liver interacts with glibenclamide resulting in low blood level concentration of glibenclamide with less stimulation of Beta cells of pancreas with lowered endogenous insulin production, with little hypoglycaemic effect resulting in uncontrolled hyperglycaemia. This was an established fact. So to evaluate and assess the drug interaction of Rifampicin with glibenclamide in Type II diabetes with PT patients, this study was under taken. Similar study was not done previously in this hospital.

Low glibenclamide concentrations affect endoplasmic reticulum stress in INS-1 cells under glucotoxic or glucolipotoxic conditions

Background/Aimsβ-Cell apoptosis caused by increased endoplasmic reticulum (ER) stress is an important pathogenic component of type 2 diabetes mellitus. In theory, sulfonylureas, used for the treatment of diabetes, can contribute to ER stress. We assessed changes in ER stress in pancreatic β-cells under glucotoxic or glucolipotoxic conditions using low concentrations of the sulfonylurea, glibenclamide (GB).MethodsLow concentrations of GB (10 or 100 nM) were added to INS-1 cells cultured under glucotoxic or glucolipotoxic conditions. The degree of viability, level of apoptosis and levels of markers associated with ER stress were measured.ResultsApoptosis decreased in response to low concentrations of GB under glucolipotoxic but not glucotoxic conditions. Most ER stress markers decreased upon the addition of GB. Under glucotoxic conditions, changes in the levels of ER stress markers were not consistent. However, all decreased significantly under glucolipotoxic conditions.ConclusionsLow concentrations of GB exerted antiapoptotic effects through the attenuation of ER stress under glucolipotoxic conditions.

A Single Point Mutation in the Distal C-Terminal of the Pore Forming Kir6.1 Subunit Modifies ATP-Sensitive Potassium (KATP) Channel Regulation

KATP channels are hetero-octamers composed of two subunits; a pore forming Kir6 and a sulphonylurea receptor (SUR). SUR subunits regulate KATP channel gating allosterically, in response to nucleotides and pharmacological agents.We have previously shown that residue D323 of Kir6.2 is central to interaction with the nucleotide-binding fold 2 of SUR2A (Rubaiy et al, Biophysical J. 2011: 100; P432a). The aim of this study was to assess whether E332, the corresponding residue in Kir6.1, plays an equally important role in Kir6.1/SUR2A complexes. Wild type Kir6.1/SUR2A channels expressed in HEK293 cells required activation by potassium channel opener pinacidil (EC50 = 43.90 ± 1.28 µM), in the presence of UDP (10 mM) before passing current. Introduction of the single point mutation E332K into full length Kir6.1 caused constitutive opening of Kir6.1/SUR2A channels in the absence of pinacidil. Reinstatement of putative inter-subunit salt bridges by expressing Kir6.1-E332K with charge reversal mutants SUR2A-Q1336E or SUR2A-K1322D failed to restore regulated opening. Channels containing the Kir6.1E332K mutant were also insensitive to block by high concentrations of glibenclamide (100 µM). However, co-expression of Kir6.1-E332K with SUR2A-K1322D restored glibenclamide sensitivity to wild type levels ((IC50 = 9.12±1.12 nM), p < 0.15 versus wild type Kir6.1/SUR2A channel (IC50 = 6.14±1.13 nM). Together, these data suggest a key functional role for inter-subunit salt bridges involving Kir6.1-E332K. Constitutive channel opening on mutation of this residue suggests that Kir6.1E332 and salt bridge(s) formed between it and the SUR subunit are crucial for stabilizing closed states of Kir6.1-containing KATP channels.

Small-molecule inhibitors of the cystic fibrosis transmembrane conductance regulator increase pancreatic endocrine cell development in rat and mouse

Aims/hypothesisThe main objective of this work was to discover new drugs that can activate the differentiation of multipotent pancreatic progenitors into endocrine cells.MethodsIn vitro experiments were performed using fetal pancreatic explants from rats and mice. In this assay, we examined the actions on pancreatic cell development of glibenclamide, a sulfonylurea derivative, and glycine hydrazide (GlyH-101), a small-molecule inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR). We next tested the actions of GlyH-101 on in vivo pancreatic cell development.ResultsGlibenclamide (10 nmol/l–100 μmol/l) did not alter the morphology or growth of the developing pancreas and exerted no deleterious effects on exocrine cell development in the pancreas. Unexpectedly, glibenclamide at its highest concentration promoted endocrine differentiation. This glibenclamide-induced promotion of the endocrine pathway could not be reproduced when other sulfonylureas were used, suggesting that glibenclamide had an off-target action. This high concentration of glibenclamide had previously been reported to inhibit CFTR. We found that the effects of glibenclamide on the developing pancreas could be mimicked both in vitro and in vivo by GlyH-101.Conclusions/interpretationCollectively, we demonstrate that two small-molecule inhibitors of the CFTR, glibenclamide and GlyH-101, increase the number of pancreatic endocrine cells by increasing the size of the pool of neurogenin 3-positive endocrine progenitors in the developing pancreas.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2778-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Determination of glibenclamide and puerarin in rat plasma by UPLC–MS/MS: Application to their pharmacokinetic interaction study

In the treatment of diabetes mellitus, glibenclamide and puerarin may be co-administered unwittingly or wittingly. An ultra performance liquid chromatography–tandem mass spectrometry method was developed to determine the concentrations of glibenclamide and puerarin in rat plasma for the study of pharmacokinetic interaction between them. Analytes were extracted using liquid–liquid extraction. The separation was achieved on a Waters BEH C18 column using 5mmol/L ammonium acetate solution (containing 0.1% formic acid) and methanol as mobile phase with a linear gradient program. Electrospray ionization source was applied and operated in the multiple reaction monitoring positive mode. The proposed method was proved simple, specific and reliable. Glibenclamide, Pueraria lobata extract and glibenclamide in combination with P. lobata extract were orally administered to rats, respectively. Pharmacokinetic parameters were estimated by Microsoft Excel software and analyzed by SPSS 12.0 software. Compared with glibenclamide group, pharmacokinetic parameters of glibenclamide in the co-administration group such as area under the curve and mean residence time were increased while clearance was decreased. Pharmacokinetic parameters of puerarin in the co-administration group such as peak concentration and area under the curve were enlarged while clearance and apparent volume of distribution were reduced compared with P. lobata extract group. These changes could enhance drug efficacy, but could also make drug accumulation to increase adverse effects, so it was suggested that the dosage should be adjusted or the drug concentration in plasma should be monitored if glibenclamide and puerarin were co-administered.