Simultaneous determination of atorvastatin,metformin and glimepiride in human plasma by LC-MS/MS and its application to a human pharmacokinetic study

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric （LC MS/ MS） assay method has been developed and fully validated for the simultaneous quantification of atorvastatin, metformin and g.）imepiride in human plasma. Carbamazepine was used as internal standard （IS）. The analytes were extracted from 200 μL aliquots of human plasma via protein precipitation using acetonitrile, The reconstituted samples were chromatographed on a Alltima HP C18 column by using a 60：40 （v/v） mixture of acetonitrile and 10 mM ammonium acetate （pH 3.0） as the mobile phase at a flow rate of 1.1 mL/min. The calibration curves obtained were linear （r2〉0.99） over the concentration range of 0.50-150.03 ng/mL for atorvastatin, 12.14-1207.50 ng/mL for metformin and 4.98-494.29 ng/mL for glimepiride. The API-4000 LC-MS/MS in multiple reaction monitoring （MRM） mode was used for detection. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. All the analytes were found to be stable in a battery of stability studies. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 rain for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers.

Simultaneous Determination of Atorvastatin and Glimepiride by LC-MS/MS in Human Plasma and Its Application to a Pharmacokinetic Study

The aim of the proposed research work was to develop and validate a simple, selective high sensitive and high-throughput assay for the simultaneous estimation of Atorvastatin and Glimepiride in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). Atorvastatin–Glimepiride combines a competitive inhibitor of HMG-CoA reductase and a sulfonylurea anti-diabetic drug. The purpose of this study was to develop single method for Atorvastatin and Glimepiride in plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) that would result into a simultaneous estimation of Atorvastatin and Glimepiride avoiding acid –lactone inter conversions right from sample collections to analysis on the LC-MS/MS. Sample collection procedure optimized for Atorvastatin holds good for Glimepiride, hence resulting into a simultaneous estimation of Atorvastatin and Glimepiride. Liquid-liquid extraction and liquid chromatography coupled to positive ion mode tandem mass spectrometry was used to develop the method and was validated according to US FDA guidelines. The calibration curves for two analytes were linear (R2 ≥ 0.9950, n = 4) over the concentration range of 0.2 - 30 ng/mL for Atorvastatin and 1 - 250 ng/mL for Glimepiride. Mean extraction recoveries 80.34 ± 9.43 for Atorvastatin and 88.19 ± 7.13 for Glimepiride. Intra- and inter-run mean percent accuracy was between 85% - 115% and percent imprecision was ≤15%. Stability studies revealed that Atorvastatin and Glimepiride were stable in plasma during bench top (10.5 h at room temperature), in Injector (47.5 h), at the end of three successive freeze and thaw cycles and long term at -65℃ ± 15℃ for 114 days. The method was successfully applied to the study of pharmacokinetics of Atorvastatin and Glimepiride in healthy volunteers. Simultaneous estimation of Atorvastatin and Glimepiride is cost effective, reduces analysis cycle time, enables effective utilization of resources and reduces bleeding burden on human volunteers.

Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry for Simultaneous Determination of Metformin and Glimepiride in Beagle Dog Plasma and Bioequivalence Study

A sensitive and selective liquid chromatography-electrospray ionization tandem mass spectrometry（LC- ES1-MS/MS） was used for the simultaneous determination of metformin and glimepiride in beagle dog plasma with glipizide as internal standard（IS）. After simplified protein precipitation with methanol, both the analytes and IS were chromatographed on a Zorbax CN column via gradient elution with methanol（containing 5 mmol/L ammonium ace- tate） and 5 mmol/L aqueous ammonium acetate as the mobile phase. Detection was performed by multiple reaction monitoring（MRM） scanning via ESI source operated in positive ionization mode. Specificity, linearity, accuracy, pre- cision, recovery, matrix effect and stability were validated for metformin and glimepiride in beagle dog plasma. The calibration curves were linear in a concentration range of 10-10000 ng/mL for metformin and 4-4000 ng/mL for glimepiride with both correlation coefficients higher than 0.99. The recoveries obtained for the analytes and IS were all between 82.7% and 101.2%. The method exhibited excellent performance in terms of selectivity, robustness, short analytical time and simplicity of sample preparation. Finally, the proposed method was applied to a bioequivalence study of self-made bilayer tablet and commercial formulation containing 500 mg of metformin and 1 mg of glimepi- ride in beagle dogs.

Effects of long-term monotherapy with glimepiride vs glibenclamide on glycemic control and macrovascular events in Japanese Type 2 diabetic patients

We investigated whether long-term glimepiride (GP) monotherapy improves insulin resistance and exerts a beneficial effect on beta cell function, as compared with glibenclamide (GC). One hundred Japanese Type 2 diabetic patients were randomly assigned to the GP (n = 50) or the GC (n = 50) group. During a 5-year monitoring period, patients received the indicated SU monotherapy, while changes in SU doses were allowed as needed to maintain HbA1C below 7.0%. The GC group, in parallel with fasting insulin, showed a rapid homeostatic model assessment (HOMA)-R increase and maintained a high HOMA-R level. In contrast, HOMA-R in the GP group decreased continuously, from 2.9 at baseline to 1.8 at study completion. In the GC group, HOMA-b was markedly increased in the first 6 months, then gradually decreased through 18 months. While the HOMA-β elevation in the GP group was more moderate than that in the GC group, HOMA-β levels were maintained with a slight decrease. The cumulative macrovascular disease outcome was 1 for the GP and 7 for the GC group, showing a significant difference. These results suggest that glimepiride monotherapy markedly improved HOMA-R with moderate insulin stimulation, which may account for the difference in macrovascular disease development as compared with the group receiving glibenclamide.

Determination of Glimepiride in Rat Serum by Rp-Hplc Method

A simple and sensitive reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of glimepiride in rat serum. The assay involves one step liquid-liquid extraction with methanol. Gliclazide was used as an internal standard. Chromatographic separation was performed on a C18 column using a mobile phase of methanol: 10mM phosphate buffer (80:20 v/v) adjusted to pH 3.0 with orthophosphoric acid, at a flow rate of 1.0 ml/min and UV detection at 230 nm. The retention time of glimepiride and gliclazide was found to be 5.5 and 4.0 min and separation was complete in less than 10 min. The method was validated for linearity, accuracy and precision were found to be acceptable over the range of 0.5 - 500 μg/mL for glimepiride. The method was found suitable to analyse rat serum samples for application in pharmacokinetic, pharmacodynamic, bioavailability/bioequivalence studies.

Interaction study between Ocimum sanctum and Glimepiride(sulfonylurea derivative) in diabetic rats

The present study was intended to discover the interaction between Ocimum sanctum(O. sanctum) and Glimepiride, a sulfonylurea derivative used in the treatment of type-2 diabetes, in diabetic rats to suggest an alteration in the dose of Glimepiride in case of hypoglycaemia. LD50 studies for the aqueous extract of O. sanctum leaf were carried out in albino mice up to the dose level of 2000 mg/kg. O. sanctum at low, medium and high doses(100, 200 and 400 mg/kg, per oral), respectively and Glimepiride ?(half) therapeutic dose, therapeutic dose and 2 time therapeutic dose(0.036, 0.072 and 0.144 mg/200 g, per oral) were selected. After the treatment with O. sanctum and Glimepiride as single doses and in various combinations of these two drugs in streptozotocin-induced diabetic rats serum glucose levels in all the groups were analysed by the glucose oxidase-peroxidase method. When administered alone as single doses both aqueous extract of O. sanctum leaf and Glimepiride produced a significant anti-diabetic effect in diabetic rats. The anti-diabetic effect observed with combination of aqueous extract of O. sanctum leaf and Glimepiride was significant and more when compared to either of the drugs given alone. There was a definite interaction that necessitates dose readjustment of Glimepiride and further glucose levels are to be frequently monitored to avoid complication of hypoglycemic condition with aqueous extract of O. sanctum leaf and Glimepiride combination.

Effect of Lifestyle Modification and Oral Anti-Diabetic Drugs on Metabolic Parameters in Recently Diagnosed Patientswith Uncomplicated Type 2 Diabetes Mellitus in Eastern India

BACKGROUND: Oral anti-diabetic drugs (OADs) are often advised for initial treatment for patients with Type 2 Diabetes Mellitus (T2DM). Their effects on glycemic control, lipid profile, insulin resistance and beta cell function has not been systematically studied in India. The objective of this study was to evaluate the effect of lifestyle modification and OADs on metabolic parameters in recently diagnosed uncomplicated T2DM patients. MATERIAL METHODS: A total of consecutive sixty four (64) cases of recently diagnosed uncomplicated T2DM in the age group of 30 - 60 years were studied. They were evaluated for weight, body mass index (BMI), fasting plasma glucose (FPG), 2hr post glucose plasma glucose (2hrPGPG), HbA1c, lipid profile, serum fasting insulin, c-peptide, HOMA-IR and HOMA-β. They were divided into four groups according to increasing order of HbA1c values (6.5% - 6.9%, 7% - 7.5%, 7.6% - 8.5%, 8.6% - 8.9%). These four groups were subjected to lifestyle modification (LSM), monotherapy with metformin (1 g) and LSM, dual drug therapy i.e. metformin (1 g), glimepiride (1 mg) and LSM, triple drug therapy i.e. metformin (1 g), glimepiride 1 mg, sitagliptin 100 mg) and LSM respectively. These patients were followed up after three months of therapy. They were evaluated for the same metabolic parameters and compared with their baseline value. Fourteen (14) patients were lost to follow up. RESULTS: We found 91%, 92.8%, 53.3% and 60% of our patients from above four different groups achieved target glycemic control (HbA1c ≤ 6.5%). In all the four groups, significant improvement in glycemic status, lipid profile, HOMA-IR and HOMA-β were observed (p value CONCLUSION: In our study, early Initiation of LSM along with OADs either as monotherapy or in combination therapy according to HbA1c value showed significant improvement in glycemic control, insulin resistance and beta cell function.

临床试验CHICAGO:糖尿病药物对动脉粥样硬化的影响

CHICAGO是carotid intima-media THIC kiness in atherosclerosis using pioglitazone study的缩称。它是一项Ⅲb期的多中心、双盲、随机化、2组的研究，比较pioglitazone与glimepiride的作用。前者属thiazolidinediones类药物，它增高肌肉及脂肪组织对胰岛素的敏感度；glimepiride属sulfonylurca类药物，它刺激胰岛素分泌。

Release of exosomes and microvesicles harbouring specific RNAs and glycosylphosphatidylinositol-anchored proteins from rat and human adipocytes is controlled by histone methylation

The transfer of proteins and nucleic acids from donor to acceptor cells via small membrane vesicles has been implicated with (patho)physiological consequences. Previously the upregulation of esterification and downregulation of lipolysis in small rat adipocytes upon incubation with exosomes and microvesicles (EMVs) released from large adipocytes and harbouring the glycosylphosphatidylinositol (GPI)-anchored proteins, Gce1 and CD73, transcripts specific for FSP27 and GPAT3, and microRNAs, miR-16 and miR-222 was demonstrated. Here the release of EMVs from large (but not small) primary and differentiated and human rat adipocytes in response to palmitate, H2O2 and the anti-diabetic sulfonylurea, glimepiride, is shown to be significantly reduced upon inhibition of histone H3 lysine9 methyltransferase G9a by trans-2-phenylcyclopropylamine (tPCPA) and histone H3 lysine4 demethylase LSD1 by BIX01294. Inhibition of EMV release by tPCPA and BIX01294 was not caused by apoptosis but accompanied by upregulation of the H2O2-induced stimulation of lipid synthesis and downregulation of lipolysis in large (but not small) primary and differentiated rat and human adipocytes. In contrast, the simultaneous presence of tPCPA and BIX-01294 had almost no effect on the induced release of EMVs and lipid metabolism. These findings argue for regulation of the release of EMVs harbouring specific GPI-anchored proteins, transcripts and microRNAs from rat and human adipocytes by histone H3 methylation at lysines 4 and 9 in interdependent fashion. Thus the EMV-mediated transfer of lipogenic and anti-lipolytic information between large and small adipocytes in response to certain physiological and pharmacological stimuli seems to be controlled by epigenetic mechanisms.