The bioavailability time of commonly used thymidine analogues after intraperitoneal delivery in mice: labeling kinetics in vivo and clearance from blood serum.

Detection of synthetic thymidine analogues after their incorporation into replicating DNA during the S-phase of the cell cycle is a widely exploited methodology for evaluating proliferative activity, tracing dividing and post-mitotic cells, and determining cell-cycle parameters both in vitro and in vivo. To produce valid quantitative readouts for in vivo experiments with single intraperitoneal delivery of a particular nucleotide, it is necessary to determine the time interval during which a synthetic thymidine analogue can be incorporated into newly synthesized DNA, and the time by which the nucleotide is cleared from the blood serum. To date, using a variety of methods, only the bioavailability time of tritiated thymidine and 5-bromo-2'-deoxyuridine (BrdU) have been evaluated. Recent advances in double- and triple-S-phase labeling using 5-iodo-2'-deoxyuridine (IdU), 5-chloro-2'-deoxyuridine (CldU), and 5-ethynyl-2'-deoxyuridine (EdU) have raised the question of the bioavailability time of these modified nucleotides. Here, we examined their labeling kinetics in vivo and evaluated label clearance from blood serum after single intraperitoneal delivery to mice at doses equimolar to the saturation dose of BrdU (150 mg/kg). We found that under these conditions, all the examined thymidine analogues exhibit similar labeling kinetics and clearance rates from the blood serum. Our results indicate that all thymidine analogues delivered at the indicated doses have similar bioavailability times (approximately 1 h). Our findings are significant for the practical use of multiple S-phase labeling with any combinations of BrdU, IdU, CldU, and EdU and for obtaining valid labeling readouts.

An analog of glibenclamide selectively enhances autophagic degradation of misfolded α1-antitrypsin Z.

The classical form of α1-antitrypsin deficiency (ATD) is characterized by intracellular accumulation of the misfolded variant α1-antitrypsin Z (ATZ) and severe liver disease in some of the affected individuals. In this study, we investigated the possibility of discovering novel therapeutic agents that would reduce ATZ accumulation by interrogating a C. elegans model of ATD with high-content genome-wide RNAi screening and computational systems pharmacology strategies. The RNAi screening was utilized to identify genes that modify the intracellular accumulation of ATZ and a novel computational pipeline was developed to make high confidence predictions on repurposable drugs. This approach identified glibenclamide (GLB), a sulfonylurea drug that has been used broadly in clinical medicine as an oral hypoglycemic agent. Here we show that GLB promotes autophagic degradation of misfolded ATZ in mammalian cell line models of ATD. Furthermore, an analog of GLB reduces hepatic ATZ accumulation and hepatic fibrosis in a mouse model in vivo without affecting blood glucose or insulin levels. These results provide support for a drug discovery strategy using simple organisms as human disease models combined with genetic and computational screening methods. They also show that GLB and/or at least one of its analogs can be immediately tested to arrest the progression of human ATD liver disease.

New Quinazoline-Sulfonylurea Conjugates: Design, Synthesis and Hypoglycemic Activity.

BACKGROUND: Sulphonylureas are the oldest and commonly used to treat diabetic patients, but its efficacy declines by time. It was reported that quinazoline nucleus exhibits a potent hypoglycemic effect in diabetic animal models. OBJECTIVE: The current study aimed to synthesize new quinazoline-sulfonylurea conjugates and evaluate their hypoglycemic effects in alloxan-induced diabetic rats. METHODS: The conjugates were synthesized by bioisosteric replacement of 5-chloro-2-methoxybenzamide moiety in glibenclamide or 1,3-dioxo-3,4-dihydroisoquinoline moiety in gliquidone with 6,7-dimethoxy-4-oxoquinazoline moiety (compounds 4a-4d, 9b-9c and 10b-10d). Diabetes was induced in rats by a single i.p. administration of alloxan, followed by treatment with the synthesized conjugates (5mg/kg Body weight). RESULTS: All conjugates showed hypoglycemic effects with different efficacy indicated by the reduction in blood glucose and elevation of insulin levels. Moreover, these conjugates up-regulated the expression of pancreatic glucose transporter 2, muscle glucose transporter 4, and insulin receptor substrate-1 genes, compared to the diabetic group. A normal pancreatic tissue pattern was noticed in diabetic rats treated with compounds 9b, 9c, and 10c. CONCLUSION: Conjugation of sulfonylurea with quinazoline (especially 9b, 9c, 10c) possessed a significant hypoglycemic effect through improving blood insulin level and insulin action and consequently increased the glucose uptake by the skeletal muscles.

Glibenclamide Mimics Metabolic Effects of Metformin in H9c2 Cells.

BACKGROUND: Sulfonylureas, such as glibenclamide, are antidiabetic drugs that stimulate beta-cell insulin secretion by binding to the sulfonylureas receptors (SURs) of adenosine triphosphate-sensitive potassium channels (KATP). Glibenclamide may be also cardiotoxic, this effect being ascribed to interference with the protective function of cardiac KATP channels for which glibenclamide has high affinity. Prompted by recent evidence that glibenclamide impairs energy metabolism of renal cells, we investigated whether this drug also affects the metabolism of cardiac cells. METHODS: The cardiomyoblast cell line H9c2 was treated for 24 h with glibenclamide or metformin, a known inhibitor of the mitochondrial respiratory chain. Cell viability was evaluated by sulforodhamine B assay. ATP and AMP were measured according to the enzyme coupling method and oxygen consumption by using an amperometric electrode, while Fo-F1 ATP synthase activity assay was evaluated by chemiluminescent method. Protein expression was measured by western blot. RESULTS: Glibenclamide deregulated energy balance of H9c2 cardiomyoblasts in a way similar to that of metformin. It inhibited mitochondrial complexes I, II and III with ensuing impairment of oxygen consumption and ATP synthase activity, ATP depletion and increased AMPK phosphorylation. Furthermore, glibenclamide disrupted mitochondrial subcellular organization. The perturbation of mitochondrial energy balance was associated with enhanced anaerobic glycolysis, with increased activity of phosphofructo kinase, pyruvate kinase and lactic dehydrogenase. Interestingly, some additive effects of glibenclamide and metformin were observed. CONCLUSIONS: Glibenclamide deeply alters cell metabolism in cardiac cells by impairing mitochondrial organization and function. This may further explain the risk of cardiovascular events associated with the use of this drug, alone or in combination with metformin.

Pharmacological evidence: a new therapeutic approach to the treatment of chronic heart failure through SUR2B/Kir6.1 channel in endothelial cells.

Both iptakalim (Ipt) and natakalim (Nat) activate the SUR2B/Kir6.1 channel, an ATP-sensitive potassium channel (KATP) subtype, with high selectivity. In this study we investigated the therapeutic effects of Ipt and Nat against isoproterenol-induced chronic heart failure (ISO-CHF) in rats, and demonstrated a new therapeutic approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells. In ISO-CHF rats, oral administration of Nat (1, 3, 9 mg·kg-1·d-1) or Ipt (3 mg·kg-1·d-1) for 60 days significantly improved cardiac dysfunction, reversed cardiac remodeling, significantly attenuated the pathological increases in BNP levels, and improved endothelial dysfunction by adjusting the balance between endothelin and NO systems. The therapeutic effects of Nat were prevented by the selective KATP blocker glibenclamine (Gli, 50 mg·kg-1·d-1), confirming that these effects were mediated through activation of the SUR2B/Kir6.1 channel in endothelial cells. The molecular mechanisms underlying the therapeutic effects of Nat were further addressed using proteomic methods. We identified 724 proteins in the plasma of ISO-CHF rats; 55 proteins were related to Nat. These differentially expressed proteins were mainly involved in single-organism processes and the regulation of biological quality relative to CHF, including proteasome (Psm) and ATP protein clusters. We screened out PRKAR2ß, GAS6/eNOS/NO and NO/PKG/VASP pathways involved in the amelioration of CHF among the 24 enriched pathways. We further confirmed 6 protein candidates, including PRKAR2ß, GAS6 and VASP, which were involved in the endothelial mechanisms, and ATP, TIMP3 and AGT, which contributed to its cardiovascular actions. This study demonstrates a new pharmacological approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells, and that the eNOS/VASP pathways are involved in its signaling mechanisms.

Antihyperglycemic and antihyperlipidemic effects of newly synthesized glibenclamide analogues on streptozotocin-diabetic rats.

In this study, new glibenclamide analogues (5a-d) with substituted pharmacological triethoxysilyl propan, allyl and ethoxyphenyl groups for cyclohexyl moiety have been synthesized by condensing sulfonamide (4) with related isocyanate or isothiocyanate's compounds. The newly synthesized drugs were evaluated for their antihyperglycemic and antihyperlipidemic activities with streptozotocin (STZ)-induced diabetic rats. All showed hypoglycemic and hypolipidemic activities compared to the control animals but 5c and 5d exhibited more and significant lowering blood activities similar to glibenclamide. This was concerned with identical affinities to bind with SUR1 receptor. Moreover, the new drugs displayed high efficiency for reducing serum LDL level which resulted in a high HDL/LDL ratio as a good lipid profile compared to other groups.

Glibenclamide-pregnenolone derivative has greater hypoglycemic effects and biodistribution than glibenclamide-OH in alloxan-rats.

AIM: The present study was designed to investigate the activity of two glibenclamide derivatives on glucose concentration. An additional aim was to identify the biodistribution of glibenclamide derivatives in different organs in a diabetic animal model. METHODS: The effects of two glibenclamide derivatives on glucose concentration were evaluated in a diabetic animal model. In addition, glibenclamide derivatives were bound to Tc-99m using radioimmunoassay methods. To evaluate the pharmacokinetics of the glibenclamide derivatives over time (15, 30, 45 and 60 min) the Tc-99m-glibenclamide conjugates were used. RESULTS: The results showed that glibenclamide-pregnenolone had greater hypoglycemic activity than glibenclamide or glibenclamide-OH. The data also showed that the biodistribution of Tc-99m-glibenclamide-OH in all organs was less than that of the Tc-99m-glibenclamide-pregnenolone derivative. CONCLUSIONS: The glibenclamide-pregnenolone derivative had greater hypoglycemic effects and its biodistribution was wider than glibenclamide-OH. The data suggest that the steroid nucleus may be important to the hypoglycemic activity of the glibenclamide-pregnenolone derivative and this could be related to the degree of lipophilicity induced by the steroid nucleus in the chemical structure of glibenclamide-pregnenolone.

Identification and structural characterization of biodegradation products of atenolol and glibenclamide by liquid chromatography coupled to hybrid quadrupole time-of-flight and quadrupole ion trap mass spectrometry.

In this paper we report about the biodegradation of the beta-blocker atenolol and the hypoglycaemic agent glibenclamide. The biodegradation tests were performed in batch reactors under aerobic conditions, using as inocculums sewage sludge from a conventional activated sludge treatment and a laboratory-scale membrane bioreactor. Pharmaceuticals were used as sole carbon sources, spiked at 50ng/L and 10mg/L concentrations. Quadrupole time-of-flight mass spectrometry coupled to ultra-high-pressure liquid chromatograph was used for the screening and the structural elucidation of biodegradation products. A microbial metabolite of atenolol with [M+H](+) at 268 was detected in the positive electrospray ionization mode. This new compound was determined to be a product of microbial hydrolysis of the amide of the parent compound. Biodegradation of glibenclamide by activated sludge proceeded via bacterial hydroxylation of the cyclohexyl ring, which resulted in formation of metabolite with a protonated molecule, [M+H](+)=510. MS(3) experiments performed by hybrid quadrupole linear ion trap (QqLIT) mass spectrometry coupled to high-performance liquid chromatography enabled further structural elucidation of the identified metabolites. Moreover, the highly sensitive QqLIT instrument in the MRM mode enabled the detection of parent compounds and one of the microbial metabolites identified in real wastewater samples. The methodology used in this study permitted for the first time the identification and detection of biodegradation product of beta-blocker atenolol in real wastewater samples.

Validation of a sensitive LC-MS assay for quantification of glyburide and its metabolite 4-transhydroxy glyburide in plasma and urine: an OPRU Network study.

Glyburide (glibenclamide, INN), a second generation sulfonylurea is widely used in the treatment of gestational diabetes mellitus (GDM). None of the previously reported analytical methods provide adequate sensitivity for the expected sub-nanogram/mL maternal and umbilical cord plasma concentrations of glyburide during pregnancy. We developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for simultaneous determination of glyburide (GLY) and its metabolite, 4-transhydroxy glyburide (M1) in human plasma (0.5 mL) or urine (0.1 mL). The limits of quantitation (LOQ) for GLY and M1 in plasma were 0.25 and 0.40 ng/mL, respectively whereas it was 1.06 ng/mL for M1 in urine. As measured by quality control samples, precision (% coefficient of variation) of the assay was <15% whereas the accuracy (% deviation from expected) ranged from -10.1 to 14.3%. We found that the GLY metabolite, M1 is excreted in the urine as the glucuronide-conjugate.

Fast HPLC method for the determination of glimepiride, glibenclamide, and related substances using monolithic column and flow program.

This work presents a fast method for the simultaneous separation and determination of glimepiride, glibenclamide, and two related substances by RP LC. The separation was performed on a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column. As mobile phase, a mixture of phosphate buffer pH 3, 7.4 mM, and ACN (55:45 v/v) was used. Column oven temperature was set to 30 degrees C. The total chromatographic run time was 80 s. This was achieved using a flow program from 5 to 9.9 mL/min. Precisions of the interday and the intraday assay for both retention times and peak areas for the four analyzed compounds were less than 1.2%. The method showed good linearity and recovery. The short analysis time makes the method very valuable for quality control and stability testing of drugs and their pharmaceutical preparations.

Synthesis and characterization of glibenclamide complexes of magnesium, chromium, cobalt, nickel, zinc and cadmium salts.

Glibenclamide is the commonly used hypoglycemic agent in NIDDM. Metal complexes of glibenclamide have been synthesized by reaction with different metals such as magnesium, chromium, cobalt, nickel, zinc and cadmium in the form of their chlorides. These complexes were characterized by their physical characteristics, 1H-NMR, IR and Atomic absorption studies.

Probing of specific binding of synthetic sulfonylurea with the insulinoma cell line MIN6.

To overcome the limitation of conventional sulfonylurea (SU) for investigation of biological mechanisms related to KATP channels, a hypoglycemic sulfonylurea (SU) was conjugated with a non-reducing glucose bearing polystyrene (PS) derivative to provide enhanced interaction with an insulinoma cell line (MIN6). The specific interaction between the SU (K+ channel closer)-conjugated copolymer and MIN6 cells was confirmed by confocal laser microscopic images using rhodamine B isothiocyanate (RITC)-labeled SU-conjugated polymer, which revealed the specific interaction between SU-conjugated polymer and MIN6 cells. Moreover, the location of labeled polymer and the site of Ca2+ ion mobilization obtained from the same MIN6 cells were identical. Based on the specificity and insulinotropic activity, the SU-conjugated polymer is expected to be useful tool for the study of biological mechanisms of KATP channels.

In vitro and in vivo evaluation of novel glibenclamide derivatives as imaging agents for the non-invasive assessment of the pancreatic islet cell mass in animals and humans.

Pancreatic islet cell mass (PICM) is a major determinant of the insulin secretory capacity in humans. Currently, the only method for accurate assessment of the PICM is an autopsy study. Thus, development of a technique allowing the non-invasive quantification of PICM is of great interest. The aim of this study was to develop such a non-invasive technique featuring novel fluorine- and (99m)Tc-labelled glibenclamide derivatives. Despite the structural modifications necessary to introduce fluorine into the glibenclamide molecule, all derivatives retained insulin stimulating capacity as well as high affinity binding to human SUR1 when compared to the original glibenclamide. Contrastingly, the lipophilicity of the fluorine-labelled derivatives was altered depending on the particular modification. In the human PET-study a constant but weak radioactive signal could be detected in the pancreas using a fluorine-labelled glibenclamide derivative. However, a reliable assessment and visualisation of the PICM could not be obtained. It can be assumed that the high uptake of the fluorine-labelled tracer e.g. into the the liver and the high plasma protein binding leads to a relatively low signal-to-noise ratio. In case of the presented fluorine-labelled glibenclamide based compounds this could be the result of their invariably high lipophilicity. The development of a (99 m)Tc-labelled glibenclamide derivative with a lower lipophilicity and differing in vivo behaviour, glibenclamide based compounds for non-invasive imaging of the pancreatic islet cell mass may be possible.

Investigation of structure-activity relationships in a series of glibenclamide analogues.

In this study, the synthesis of 15 new glibenclamide analogues is described. The conformational trends of these analogues were investigated using Monte Carlo conformational analysis. The conformational analysis results resolved the discrepancy between previous molecular modelling simulations of glibenclamide and allowed rationalizing the effect of aqueous environment on the overall conformation. The 3D-QSAR study was carried out with respect to the compounds' ability to antagonize the [(3)H]-glibenclamide binging in rat cerebral cortex. Superimposition of the antagonists was performed using the conformations derived from atom-by-atom fit to the glibenclamide crystal structure and this alignment was used to develop CoMFA models. CoMFA provided a good predictability: number of PLS components = 2, q(2) = 0.876, R(2) = 0.921, SEE = 0.455 and F = 70. Best CoMFA models showed the steric and lipophilic properties as the major interacting forces whilst the electrostatic property contribution was a minor factor.

Synthesis and evaluation of fluorine-18 labeled glyburide analogs as beta-cell imaging agents.

Glyburide is a prescribed hypoglycemic drug for the treatment of type 2 diabetic patients. We have synthesized two of its analogs, namely N-[4-[beta-(2-(2'-fluoroethoxy)-5-chlorobenzenecarboxamido)ethyl]benzenesulfonyl]-N'-cyclohexylurea (2-fluoroethoxyglyburide, 8b) and N-[4-[beta-(2-(2'-fluoroethoxy)-5-iodobenzenecarboxamido)ethyl]benzenesulfonyl]-N'-cyclohexylurea (2-fluoroethoxy-5-deschloro-5-iodoglyburide, 8a), and their fluorine-18 labeled analogs as beta-cell imaging agents. Both F-18 labeled compound 8a and compound 8b were synthesized by alkylation of the corresponding multistep synthesized hydroxy precursor 4a and 4b with 2-[(18)F]fluoroethyl tosylate in DMSO at 120 degrees C for 20 minutes followed by HPLC purification in an overall radiochemical yield of 5-10% with a synthesis time of 100 minutes from EOB. The octanol/water partition coefficients of compounds 8a and 8b were 141.21 +/- 27.77 (n = 8) and 124.33 +/- 21.61 (n = 8), respectively. Insulin secretion experiments of compounds 8a and 8b on rat islets showed that both compounds have a similar stimulating effect on insulin secretion as that of glyburide. In vitro binding studies showed that approximately 2% of compounds 8a and 8b bound to beta TC3 and Min6 cells and that the binding was saturable. Preliminary biodistribution studies in mice showed that the uptake of both compounds 8a and 8b in liver and small intestine were high, whereas the uptake in other organs studied including pancreas were low. Additionally, the uptake of compound 8b in vivo was nonsaturable. These results tend to suggest that compounds 8a and 8b may not be the ideal beta-cell imaging agents.

Synthesis and characterization of water-soluble hydroxybutenyl cyclomaltooligosaccharides (cyclodextrins).

We have examined the synthesis of hydroxybutenyl cyclomaltooligosaccharides (cyclodextrins) and the ability of these cyclodextrin ethers to form guest-host complexes with guest molecules. The hydroxybutenyl cyclodextrin ethers were prepared by a base-catalyzed reaction of 3,4-epoxy-1-butene with the parent cyclodextrins in an aqueous medium. Reaction byproducts were removed by nanofiltration before the hydroxybutenyl cyclodextrins were isolated by co-evaporation of water-EtOH. Hydroxybutenyl cyclodextrins containing no unsubstituted parent cyclodextrin typically have a degree of substitution of 2-4 and a molar substitution of 4-7. These hydroxybutenyl cyclodextrins are randomly substituted, amorphous solids. The hydroxybutenyl cyclodextrin ethers were found to be highly water soluble. Complexes of HBen-beta-CD with glibenclamide and ibuprofen were prepared and isolated. In both cases, the guest content of the complexes was large, and a significant increase in the solubility of the free drug was observed. Dissolution of the complexes in pH 1.4 water was very rapid, and significant increases in the solubility of the free drugs were observed. Significantly, after reaching equilibrium concentration, a decrease in the drug concentration over time was not observed.

In vitro and in vivo effects of new insulin releasing agents.

The present study aimed at characterizing in vitro and in vivo the effects of BM 208 (N-[4-(5-chloro-2-methoxybenzamidoethyl)benzenesulfonyl]-N'-cyano-N"-cyclohexylguanidine) and BM 225 (1-[4-(5-chloro-2-methoxybenzamidoethyl)benzene sulfonamido]-1-cyclohexylamino-2-nitroethylene); two new isosteres of the hypoglycemic sulfonylurea glibenclamide. In rat pancreatic islets perifused at close to normal (8.3mM) D-glucose concentration, both BM 208 and BM 225 (10 and 25 microM) increased 45Ca outflow and insulin release. The compounds did not affect the 45Ca outflow rate from islets exposed to Ca(2+)-free media. In single pancreatic islet cells loaded with the fluorescent Ca(2+) indicator fura-2 and incubated in the presence of 8.3mM glucose, BM 208 and BM 225 raised the [Ca(2+)](i). All these findings indicate that, in islet cells exposed to a physiological concentration of D-glucose, the secretory capacity of the new glibenclamide isosteres is related to a facilitation of Ca(2+) entry. The potency and duration of action of BM 225 was, however, more pronounced than that of BM 208. Successive additions of BM 208 provoked repeated increments in 45Ca outflow and insulin release, without evidence of tachyphylaxis. Lastly, intraperitoneal injection of BM 208 and BM 225 to fed rats lowered plasma glucose concentration in a dose-dependent manner. BM 225 was more potent and acting faster than BM 208. Our results indicate that appropriate structural modification can generate isosteres of glibenclamide with different features and activity profiles.

Interaction of sulfonylurea-conjugated polymer with insulinoma cell line of MIN6 and its effect on insulin secretion.

A carboxylated derivative of sulfonylurea (SU), an insulinotropic agent, was synthesized and grafted onto a water-soluble polymer as a biospecific and stimulating polymer for insulin secretion. To evaluate the effect of the SU-conjugated polymer on insulin secretion, its solution in dimethyl sulfoxide was added to the culture of insulinoma cell line of MIN6 cells to make 10 nM of SU units in the medium and incubated for 3 h at 37 degrees C. The culture medium was conditioned with glucose concentration of 3.3 or 25 mM. To verify the specific interaction between the SU (K+ channel closer)-conjugated polymer and MIN6 cells, the cells were pretreated with diazoxide, an agonist of adenosine triphosphate-sensitive K+ channel (K+ channel opener), before adding the SU-conjugated polymer to the cell culture medium. This treatment suppressed the action of SUs on MIN6 cells. Fluorescence-labeled polymer with rodamine-B isothiocyanate was used to visualize the interactions, and we found that the labeled polymer strongly absorbed to MIN6 cells, probably owing to its specific interaction mediated by SU receptors on the cell membrane. The fluorescence intensity on the cells significantly increased with an increase in incubation time and polymer concentration. A confocal laser microscopic study further confirmed this interaction. The results from this study provided evidence that SU-conjugated copolymer stimulates insulin secretion by specific interactions of SU moieties in the polymer with MIN6 cells.

Potent inhibition of the CFTR chloride channel by suramin.

After phosphorylation by protein kinase A and in the presence of ATP, the cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel. In this study we have examined the effects of suramin on the CFTR Cl- current (I(CFTR)) in excised inside-out macropatches from Xenopus oocytes expressing human CFTR; glibenclamide, the standard inhibitor of I(CFTR), and some congeners were tested in comparison. I(CFTR) was activated by addition of the catalytic subunit of protein kinase A and MgATP to the bath. Suramin inhibited I(CFTR) with an IC50 value of 1 microM and a Hill coefficient close to 1; the inhibition showed little voltage dependence and was easily reversed upon washout of the drug. In comparison, glibenclamide inhibited I(CFTR) with an IC50 value of approximately 20 microM. When tested against I(CFTR) in whole oocytes, bath application of suramin was ineffective whereas glibenclamide was about four times weaker than in the inside-out patch configuration. The data show that suramin is the most potent inhibitor of CFTR yet described and suggest that the compound approaches its site of action from the cytosol.

Insulinotropic effect of new glibenclamide isosteres.

The aim of the present study was to characterize the effects of BM 208 (N-[4-(5-chloro-2-methoxybenzamidoethyl)benzenesulfonyl]-N'-cyano- N"- cyclohexylguanidine) and BM 225 (1-[4-(5-chloro-2-methoxybenzamidoethyl)benzene sulfonamido]-1-cyclohexylamino-2-nitroethylene), two newly synthesized isosteres of glibenclamide, on ionic and secretory events in rat pancreatic islet cells. Both compounds inhibited 86Rb (42K substitute) outflow from rat pancreatic islets perifused throughout at low (2.8 mM) D-glucose concentration. In excised inside-out membrane patches, BM 208 and BM 225 reduced the frequency of KATP+ channel openings. The inhibition of 86Rb outflow induced by BM 208 and BM 225 coincided with an increase in 45Ca outflow. The latter phenomenon was abolished in islets exposed to Ca2+-free media. Both isosteres of glibenclamide increased the [Ca2+]i in single pancreatic islet cells. This effect was counteracted by verapamil, a Ca2+ entry blocker. In islets exposed to 2.8 mM glucose and extracellular Ca2+, BM 208 and BM 225 stimulated insulin output. The secretory capacity of BM 225 was more marked than that of BM 208, but the time courses of the cationic and secretory responses exhibited obvious dissociations. These data suggest that the secretory capacity of BM 208 and BM 225 results, at least in part, from the inhibition of ATP-sensitive K+ channels with subsequent increase in Ca2+ inflow. The dissociation between cationic and secretory variables further suggests that the modifications in Ca2+ handling are not solely attributable to a primary inhibition of the ATP-sensitive K+ channels.