Shireen Rao

In vitro Activation of Protein Kinase C by β-N-oxalyl-l-α, β-diaminopropionic acid, the Lathyrus sativus Neurotoxin

Abstractβ-N-oxalyl-l-α,β-diaminopropionic acid (l-ODAP) toxicity has been associated with lathyrism; a spastic paraparesis caused by excessive dietary intake of the pulse Lathyrus sativus. We investigated the effect of Lathyrus neurotoxin l-ODAP on protein kinase C (PKC) activity under in vitro conditions. l-ODAP activated phosphorylation activity of purified chick brain PKC. Both lysine-rich (histone III-S) and arginine-rich (protamine sulfate) substrate phosphorylation was enhanced in the presence of l-ODAP. The activation is concentration dependent, and maximal activation is observed at 100 μM concentration. Protamine sulfate phosphorylation was enhanced by 47%, whereas histone III-S phosphorylation was enhanced by 50% over PS/PDBu/Ca2+ dependent activity. The nontoxic d-isomer (d-ODAP) did not affect both histone III-S and protamine sulfate phosphorylation activity. These results indicate that l-ODAP taken up by neuronal cells could also contribute to PKC activation and so be associated with toxicity.

Pharmacokinetics of a Fixed-Dose Combination of Atorvastatin and Metformin Extended Release versus Concurrent Administration of Individual Formulations

AbstractBackground: Type 2 diabetes mellitus is associated with a 2- to 4-fold increased risk of coronary heart disease (CHD). Combined therapy with an antihyperglycaemic agent and an HMG-CoA reductase inhibitor (statin) is indicated for the treatment of diabetic patients at risk of CHD. Patients with type 2 diabetes are generally considered to be at equivalent cardiovascular disease risk to patients with established CHD, and should have low-density lipoprotein (LDL) cholesterol levels reduced to <100mg/dL or by 30–40%. Atorvastatin is the drug of choice for lowering LDL cholesterol levels. Metformin is the first therapeutic option in type 2 diabetes patients who are overweight or obese because it may also prevent vascular complications and mortality. Hence, a fixed-dose combination (FDC) of atorvastatin 10 mg and metformin 500 mg extended release (ER) was developed for patients with type 2 diabetes with or without hyperlipidaemia. Objectives: This study set out to establish bioequivalence between treatment 1 (test) — atorvastatin/metformin ER 10 mg/500 mg FDC, and treatment 2 (reference) — atorvastatin 10 mg (Lipitor®) and metformin 500 mg (Glucophage® XR) administered concurrently as individual tablets. Methods: The study was a randomized, open-label, two-treatment, two-period, two-sequence, single-dose, crossover, bioequivalence study in 40 male subjects of Asian origin aged 18–45 years. The order of receiving the test and reference treatments for each subject during both the periods of the study was determined according to an SAS®-generated randomization schedule. The two treatments were separated by a washout period of 11 days. Blood samples were collected pre-dose and up to 72 hours post-dose in each period for determination of plasma atorvastatin/metformin concentrations and calculation of the respective pharmacokinetic parameters. ANOVA was performed on the lognormal-transformed pharmacokinetic parameters. A 90% confidence interval (CI) for the ratios of the test and reference product averages (least squares means) was calculated for atorvastatin and metformin to establish bioequivalence. Results: The 90% CIs for atorvastatin and metformin were within the bioequivalence acceptance criteria of 80–125%. The 90% CIs obtained for atorvastatin for maximum plasma concentration (Cmax) area under the plasma concentration-time curve (AUC) from time zero to time of last measurable concentration (AUClast) and AUC from time zero to infinity (AUC∞) were (88.11, 106.93), (91.18, 107.94) and (89.25, 106.60), respectively. The 90% CIs observed for metformin for Cmax, AUClast, AUC∞ and AUC from time zero to 24 hours (AUC24) were (113.3, 124.0), (102.65, 117.97), (101.87, 116.82) and (102.44, 117.53), respectively. The two treatments were well tolerated by the study subjects. Conclusion: Atorvastatin/metformin ER 10 mg/500 mg FDC has similar bioavailability to the co-administration of separate atorvastatin 10 mg and metformin 500 mg tablets. The FDC tablets show similar safety and tolerability profiles to their individual components. Therefore, atorvastatin/metformin ER 10 mg/500 mg FDC tablets can be used safely in clinical settings to decrease the pill burden and increase patient compliance with therapy.

β-N-oxalyl-L-α,β-diaminopropionic acid regulates mitogen-activated protein kinase signaling by down-regulation of phosphatidylethanolamine-binding protein 1.

J. Neurochem. (2011) 118, 176–186.

Pharmacokinetics of a fixed-dose combination of atorvastatin and metformin extended release versus concurrent administration of individual formulations: a randomized, open-label, two-treatment, two-period, two-sequence, single-dose, crossover, bioequivalence study.

BACKGROUND Type 2 diabetes mellitus is associated with a 2- to 4-fold increased risk of coronary heart disease (CHD). Combined therapy with an antihyperglycaemic agent and an HMG-CoA reductase inhibitor (statin) is indicated for the treatment of diabetic patients at risk of CHD. Patients with type 2 diabetes are generally considered to be at equivalent cardiovascular disease risk to patients with established CHD, and should have low-density lipoprotein (LDL) cholesterol levels reduced to <100 mg/dL or by 30-40%. Atorvastatin is the drug of choice for lowering LDL cholesterol levels. Metformin is the first therapeutic option in type 2 diabetes patients who are overweight or obese because it may also prevent vascular complications and mortality. Hence, a fixed-dose combination (FDC) of atorvastatin 10 mg and metformin 500 mg extended release (ER) was developed for patients with type 2 diabetes with or without hyperlipidaemia. OBJECTIVES This study set out to establish bioequivalence between treatment 1 (test) - atorvastatin/metformin ER 10 mg/500 mg FDC, and treatment 2 (reference) - atorvastatin 10 mg (Lipitor®) and metformin 500 mg (Glucophage® XR) administered concurrently as individual tablets. METHODS The study was a randomized, open-label, two-treatment, two-period, two-sequence, single-dose, crossover, bioequivalence study in 40 male subjects of Asian origin aged 18-45 years. The order of receiving the test and reference treatments for each subject during both the periods of the study was determined according to an SAS®-generated randomization schedule. The two treatments were separated by a washout period of 11 days. Blood samples were collected pre-dose and up to 72 hours post-dose in each period for determination of plasma atorvastatin/metformin concentrations and calculation of the respective pharmacokinetic parameters. ANOVA was performed on the lognormal-transformed pharmacokinetic parameters. A 90% confidence interval (CI) for the ratios of the test and reference product averages (least squares means) was calculated for atorvastatin and metformin to establish bioequivalence. RESULTS The 90% CIs for atorvastatin and metformin were within the bioequivalence acceptance criteria of 80-125%. The 90% CIs obtained for atorvastatin for maximum plasma concentration (C(max))(,) area under the plasma concentration-time curve (AUC) from time zero to time of last measurable concentration (AUC(last)) and AUC from time zero to infinity (AUC(∞)) were (88.11, 106.93), (91.18, 107.94) and (89.25, 106.60), respectively. The 90% CIs observed for metformin for C(max,) AUC(last,) AUC(∞) and AUC from time zero to 24 hours (AUC(24)) were (113.3, 124.0), (102.65, 117.97), (101.87, 116.82) and (102.44, 117.53), respectively. The two treatments were well tolerated by the study subjects. CONCLUSION Atorvastatin/metformin ER 10 mg/500 mg FDC has similar bioavailability to the co-administration of separate atorvastatin 10 mg and metformin 500 mg tablets. The FDC tablets show similar safety and tolerability profiles to their individual components. Therefore, atorvastatin/metformin ER 10 mg/500 mg FDC tablets can be used safely in clinical settings to decrease the pill burden and increase patient compliance with therapy.

QbD-Driven Development and Validation of a Bioanalytical LC–MS Method for Quantification of Fluoxetine in Human Plasma

The current studies describe the Quality by Design (QbD)-based development and validation of a LC-MS-MS method for quantification of fluoxetine in human plasma using fluoxetine-D5 as an internal standard (IS). Solid-phase extraction was employed for sample preparation, and linearity was observed for drug concentrations ranging between 2 and 30 ng/mL. Systematic optimization of the method was carried out by employing Box-Behnken design with mobile phase flow rate (X1), pH (X2) and mobile phase composition (X3) as the method variables, followed by evaluating retention time (Rt) (Y1) and peak area (Y2) as the responses. The optimization studies revealed reduction in the variability associated with the method variables for improving the method robustness. Validation studies of the developed method revealed good linearity, accuracy, precision, selectivity and sensitivity of fluoxetine in human plasma. Stability studies performed in human plasma through freeze-thaw, bench-top, short-term and long-term cycles, and autosampler stability revealed lack of any change in the percent recovery of the drug. In a nutshell, the developed method demonstrated satisfactory results for analysis of fluoxetine in human plasma with plausible utility in pharmacokinetic and bioequivalence studies.

Development and validation of an improved LC-MS/MS method for the quantification of desloratadine and its metabolite in human plasma using deutrated desloratadine as internal standard.

Purpose: For the determination of desloratadine (DES) and 3-OH desloratadine (3-OHD) in human plasma using deutrated desloratadine (DESD5) as internal standard (IS), a novel stability indicating liquid chromatography-tandem mass spectrometric method was developed and validated to support the clinical advancement. Materials and Methods: The solid-phase extraction method used for sample preparation and calibration range was 100-11,000 pg/ml, for which a quadratic regression (1/x2) was best fitted. The blank plasma was screened and observed free from any endogenous interference. Results: The accuracy (% nominal) at low limit of quantification LLOQ level for DES and 3-OHD was 100.4% and 99.9% whereas precision (%CV) was 4.6 and 5.1%. They (DES and 3-OHD) were stable in human plasma after five freeze-thaw cycles, at room temperature for 23.8 hour, bench top stability for 6.4 hour. Conclusion: This method fulfills all the regulatory requirements for selectivity, sensitivity, precision, accuracy, stability, goodness of fit, and ruggedness of the method for the determination of DES and 3-OHD in human plasma.

The pharmacodynamic equivalence of Orlistat 60 mg capsule. An open label, balanced, randomized, multiple-dose, cross-over pharmacodynamic end-point bioequivalence study in healthy, adult, human Asian Indian subjects under fed conditions

Abstract Orlistat is a non-systemic treatment for obesity. The drug inhibits lipase in the gastrointestinal track mainly in the lumen of the stomach and small intestine by binding reversibly with the active site of gastric and pancreatic lipases, preventing the absorption of ∼30–35% of dietary fat. The undigested triglycerides are not absorbed, resulting in a caloric deficit and positive effect in weight control. The objective of this study was to assess the bioequivalence of orlistat administered as a generic and reference capsule formulations using a pharmacodynamic end-point. A total of 60 healthy volunteers followed a 5-day run-in diet period to be accustomed to a low fat diet; subjects were then randomized to receive under fed conditions oral doses of orlistat (60 mg) 3-times daily for 10 days as the generic (Ranbaxy Laboratories) or reference (Alli™, GlaxoSmithKline) capsule formulations. Subjects followed a standardized diet (2500 kcal/day, ∼30% as fat) for the entire study. Feces were collected over the last 2 days of the run-in period (baseline) and over the last 5 days of the two treatment periods. The amount of fat in meals and feces were assayed using the validated FTIR method with a limit of detection of 1.00 g%, respectively. Fecal fat excretion over 24 h (FFE24SS, calculated as the amount of fat excreted in feces adjusted by the amount of fat ingested) was used as a pharmacodynamic end-point to assess the bioequivalence between the two orlistat formulations. An analyses of variance (ANOVA) was performed on the log-transformed FFE24SS parameter to establish bioequivalence of the generic product with respect to the innovator formulation. Mean FFE24SS values at baseline and after repeated oral administrations of the generic and innovator formulations of orlistat were 0.88%, 40.55% and 40.54%, respectively. The ratio of least-squares means (LSM) of FFE24SS of the generic to the innovator formulation was 101.90%, with 90% confidence intervals of 97.94–106.01%. Orlistat was well tolerated by subjects in both periods of study and no serious adverse events were observed during the study. In conclusion, mean FFE24SS values were used as pharmacodynamic end-points to assess bioequivalence between two formulations of orlistat. Results from this study suggest that the test formulation of Orlistat capsule is bioequivalent to the reference marketed Alli™ when administered to healthy volunteers as a multiple dose under fed conditions.

A sensitive LC-ESI-MS/MS method for the quantification of low concentrations of buspirone in human plasma

A sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for the quantitation of low concentrations of buspirone in human plasma. The analyte and internal standard (buspirone D8) were extracted by solid phase extraction and the chromatographic separation was performed on a reverse phase column with a mobile phase consisting of acetonitrile–5 mM ammonium acetate–trifluoroacetic acid (90 : 10 : 0.001, v/v/v). The retention time of buspirone and internal standard were 0.95 and 0.94 min respectively. The protonated ion of the analyte and internal standard were quantified in the positive ion mode by multiple reaction monitoring (MRM). The mass transitions m/z 386.24 → 122.10 and 394.28 → 122.00 were used to measure the analyte and internal standard respectively. The assay was validated over the concentration range of 10.4–6690.4 pg mL−1, where the quadratic regression (1/x2) was best fitted. The lower limit of quantitation (LLOQ) was 10.4 pg mL−1 with a signal to noise ratio of more than 10 and the relative standard deviation was less than 15%. Buspirone and buspirone D8 were stable in human plasma when stored on a bench top for 6.43 h, in an autosampler (10 °C) for 70.05 h and after three freeze thaw cycles. Acceptable precision and accuracy were obtained for concentrations over the standard calibration curve range. The analytical run time of 3.0 min made this method suitable for the high throughput analysis of a large number of samples in short period of time. The validated method has been successfully applied in the analysis of buspirone levels in more than 500 human plasma samples in a pharmacokinetic study.

QbD-Driven Development and Validation of Liquid Chromatography Tandem Mass Spectrometric Method for the Quantitation of Sildenafil in Human Plasma

The present work was employing the Quality by Design approach for the development and validation of a LC-MS-MS method to support the clinical advancement in determination of sildenafil in human plasma using lorazepam as an internal standard. Sample preparation involved solid phase extraction and calibration range observed between 3 and 1,700 ng/mL. The method was systematically optimized by employing Box-Behnken design and used mobile phase flow rate, pH and composition of mobile phase as the critical factors, and assessing the design for retention time and peak area as the responses. A substantial decrease in the variability associated with the method variables was shown in optimization studies and confirmed enhanced method robustness. The present studies revealed that developed method achieves all the regulatory requirements for linearity, accuracy, precision, selectivity, sensitivity and stability for the determination of sildenafil in human plasma. There was not any significant change in the stability of the drug shown by stability studies, performed in human plasma through freeze-thaw cycles, bench-top stability, short-term stability, long-term stability and auto sampler stability. In short, this method shows satisfactory results for the analysis of sildenafil in human plasma and possesses high degree of utility in pharmacokinetic and bioequivalence studies.