Shriram M. Pathak

Enhanced oral absorption of saquinavir with Methyl-Beta-Cyclodextrin-Preparation and in vitro and in vivo evaluation.

Saquinavir (SQV) is a weak base compound, whose solubility is strongly influenced by pH variations. Thus, in the present work, we thought it worthy of interest to investigate in-depth the combined effect of pH control and cyclodextrin (CyD) complexation on SQV solubilization. Phase-solubility studies were performed by adding excess drug to buffered (pH from 1.1 to 7.4) aqueous solutions containing increasing concentrations of Methyl-Beta-CyD (M-β-CyD) in order to evaluate the role of the unionized species of SQV in improving solubility by CyD complexation and to be able to select the most suitable conditions for optimizing drug solubilization. Our study reveals that the integrated approach of pH adjustment and CyD complexation can be successfully used for improving the CyD solubilizing power towards an ionizable drug such as SQV, thus allowing a smaller quantity of CyD to solubilize a given amount of drug, offering clear economic and technologic advantages as well. When biopharmaceutics of the optimized cyclodextrin-based formulation of SQV was studied in Wistar rats after intravenous and oral administrations, we found that inclusion of SQV into M-β-CyD could dramatically improve its oral bioavailability and decrease the variation of its oral pharmacokinetics. Compared to the control, the presence of M-β-CyD significantly increased the area under the plasma concentration-time curve (439.7±161.35 to 2312.03±159.53, p<0.01) and the peak plasma concentration (117.24±35.77 to 1347.88±276.76, p<0.01) of orally administered SQV. The modulating effect of M-β-CyD on the bidirectional transport of SQV was also investigated using a modified Ussing chamber system. The results demonstrated that the enhancing effect of M-β-CyD on the oral bioavailability of SQV is due not only to its solubilizing effect on SQV but also, at least in part, to the inhibitory effect of M-β-CyD on the P-glycoprotein (P-gp) mediated efflux of SQV in the gastrointestinal tract. The present results suggest that M-β-CyD is particularly useful in designing oral preparations of SQV with an enhanced bioavailability and a reduced variability in absorption.

A simple and rapid high performance liquid chromatographic method with fluorescence detection for the estimation of fexofenadine in rat plasma-Application to preclinical pharmacokinetics

A sensitive high performance liquid chromatographic (HPLC) method involving fluorescence detection was developed for the determination of fexofenadine (FEX), known to have low oral bioavailability, in rat plasma. In order to understand the effect of various chromatographic factors on the separation of analytes and to simultaneously optimize the resolution and analysis run time, a response surface method was used. The chromatographic separation was achieved using a Supelco C(18)-DB (250 mm x 4.6mm I.D./5 microm particle size) column with mobile phase comprising of ammonium acetate buffer and acetonitrile (63:37, v/v), delivered isocratically at a flow rate of 1.0 mL min(-1). Diphenhydramine was used as an internal standard (I.S.). The statistical evaluation of the method was examined and the method was found to be precise and accurate with a linearity range of 1-500 ng mL(-1) (r>0.9980). The intra- and inter-day precision studies showed good reproducibility with coefficients of variation (C.V.) less than 12.26%. The advantages of our method are small sample volume (100 microL), short time of analysis (13 min) and a simple sample extraction and clean-up as compared to the previously published methods. The established method provides a reliable bioanalytical methodology to carry out FEX pharmacokinetics in rat plasma.

Evaluation of Chemical Interactions of Maleic acid with Sodium Hypochlorite and Chlorhexidine gluconate

INTRODUCTION The elimination of microorganisms from the root canal system necessitates the use of combination of irrigating solutions to enhance their antimicrobial property. The combination of irrigants and their interaction sometimes could be detrimental to the outcome of the root canal therapy. The purposes of this study were (1) to evaluate the interaction between 7% maleic acid (MA) and 2% chlorhexidine gluconate solution (CHX) and to find out the availability of individual irrigant and (2) to determine the free available chlorine content when 7% MA was mixed with 2.5% sodium hypochlorite (NaOCl) solution. METHODS Interaction between MA and CHX was assessed by high-performance liquid chromatography. Available chlorine content in NaOCl was evaluated by the standard iodine/thiosulfate titration method. RESULTS It was observed that more than 90% free MA and CHX were available when MA was combined with CHX. It was also observed that there was no precipitate formation when 7% MA was mixed with 2% CHX. Available chlorine content decreased significantly in the MA/NaOCl mixture. CONCLUSIONS There were no adverse interactions or precipitate formation observed when MA was combined with CHX, but the available chlorine content was reduced when NaOCl was mixed with MA.

Pre-clinical evidence of enhanced oral bioavailability of the P-glycoprotein substrate talinolol in combination with morin.

Most known interactions between herbal extracts and drugs involve the inhibition of drug‐metabolizing enzymes, but little is yet known about the possible role of transporters in these interactions. In order to evaluate the effect of one of such prominent flavonoids, morin, on P‐glycoprotein related efflux carriers, measurements of transport characteristics through Ussing chambers, in situ perfusion and in vivo drug absorption studies were performed with the transported, yet not metabolized model compound talinolol.

IMI – Oral biopharmaceutics tools project – Evaluation of bottom-up PBPK prediction success part 3: Identifying gaps in system parameters by analysing In Silico performance across different compound classes

&NA; Three Physiologically Based Pharmacokinetic software packages (GI‐Sim, Simcyp® Simulator, and GastroPlus™) were evaluated as part of the Innovative Medicine Initiative Oral Biopharmaceutics Tools project (OrBiTo) during a blinded “bottom‐up” anticipation of human pharmacokinetics. After data analysis of the predicted vs. measured pharmacokinetics parameters, it was found that oral bioavailability (Foral) was underpredicted for compounds with low permeability, suggesting improper estimates of intestinal surface area, colonic absorption and/or lack of intestinal transporter information. Foral was also underpredicted for acidic compounds, suggesting overestimation of impact of ionisation on permeation, lack of information on intestinal transporters, or underestimation of solubilisation of weak acids due to less than optimal intestinal model pH settings or underestimation of bile micelle contribution. Foral was overpredicted for weak bases, suggesting inadequate models for precipitation or lack of in vitro precipitation information to build informed models. Relative bioavailability was underpredicted for both high logP compounds as well as poorly water‐soluble compounds, suggesting inadequate models for solubility/dissolution, underperforming bile enhancement models and/or lack of biorelevant solubility measurements. These results indicate areas for improvement in model software, modelling approaches, and generation of applicable input data. However, caution is required when interpreting the impact of drug‐specific properties in this exercise, as the availability of input parameters was heterogeneous and highly variable, and the modellers generally used the data “as is” in this blinded bottom‐up prediction approach. Graphical Abstract Figure. No caption available.

IMI - Oral biopharmaceutics tools project - Evaluation of bottom-up PBPK prediction success part 2 : An introduction to the simulation exercise and overview of results

&NA; Orally administered drugs are subject to a number of barriers impacting bioavailability (Foral), causing challenges during drug and formulation development. Physiologically‐based pharmacokinetic (PBPK) modelling can help during drug and formulation development by providing quantitative predictions through a systems approach. The performance of three available PBPK software packages (GI‐Sim, Simcyp®, and GastroPlus™) were evaluated by comparing simulated and observed pharmacokinetic (PK) parameters. Since the availability of input parameters was heterogeneous and highly variable, caution is required when interpreting the results of this exercise. Additionally, this prospective simulation exercise may not be representative of prospective modelling in industry, as API information was limited to sparse details. 43 active pharmaceutical ingredients (APIs) from the OrBiTo database were selected for the exercise. Over 4000 simulation output files were generated, representing over 2550 study arm‐institution‐software combinations and approximately 600 human clinical study arms simulated with overlap. 84% of the simulated study arms represented administration of immediate release formulations, 11% prolonged or delayed release, and 5% intravenous (i.v.). Higher percentages of i.v. predicted area under the curve (AUC) were within two‐fold of observed (52.9%) compared to per oral (p.o.) (37.2%), however, Foral and relative AUC (Frel) between p.o. formulations and solutions were generally well predicted (64.7% and 75.0%). Predictive performance declined progressing from i.v. to solution and immediate release tablet, indicating the compounding error with each layer of complexity. Overall performance was comparable to previous large‐scale evaluations. A general overprediction of AUC was observed with average fold error (AFE) of 1.56 over all simulations. AFE ranged from 0.0361 to 64.0 across the 43 APIs, with 25 showing overpredictions. Discrepancies between software packages were observed for a few APIs, the largest being 606, 171, and 81.7‐fold differences in AFE between SimCYP and GI‐Sim, however average performance was relatively consistent across the three software platforms. Graphical abstract Figure. No caption available.

IMI – oral biopharmaceutics tools project – evaluation of bottom-up PBPK prediction success part 1: Characterisation of the OrBiTo database of compounds

&NA; Predicting oral bioavailability (Foral) is of importance for estimating systemic exposure of orally administered drugs. Physiologically‐based pharmacokinetic (PBPK) modelling and simulation have been applied extensively in biopharmaceutics recently. The Oral Biopharmaceutical Tools (OrBiTo) project (Innovative Medicines Initiative) aims to develop and improve upon biopharmaceutical tools, including PBPK absorption models. A large‐scale evaluation of PBPK models may be considered the first step. Here we characterise the OrBiTo active pharmaceutical ingredient (API) database for use in a large‐scale simulation study. The OrBiTo database comprised 83 APIs and 1475 study arms. The database displayed a median logP of 3.60 (2.40–4.58), human blood‐to‐plasma ratio of 0.62 (0.57–0.71), and fraction unbound in plasma of 0.05 (0.01–0.17). The database mainly consisted of basic compounds (48.19%) and Biopharmaceutics Classification System class II compounds (55.81%). Median human intravenous clearance was 16.9 L/h (interquartile range: 11.6–43.6 L/h; n = 23), volume of distribution was 80.8 L (54.5–239 L; n = 23). The majority of oral formulations were immediate release (IR: 87.6%). Human Foral displayed a median of 0.415 (0.203–0.724; n = 22) for IR formulations. The OrBiTo database was found to be largely representative of previously published datasets. 43 of the APIs were found to satisfy the minimum inclusion criteria for the simulation exercise, and many of these have significant gaps of other key parameters, which could potentially impact the interpretability of the simulation outcome. However, the OrBiTo simulation exercise represents a unique opportunity to perform a large‐scale evaluation of the PBPK approach to predicting oral biopharmaceutics. Graphical abstract Figure. No caption available.

High Performance Liquid Chromatographic Method for the Determination of Clobetasol in Rat Plasma and its Application to Skin Penetration

A simple a nd sensitive high per for ma nce liquid chromatography (HPLC) method was developed for qua ntification of clobeta sol (CLB) in r a t pla sma . Aripiprazole was used as an internal standard (IS). The present method uses protein precipitation with acetonitrile, followed by the liquid-liquid extraction with methyl tertiary butyl ether for extraction of the CLB from the matrix. Separation was carried out using Variance C 18 (250.0 × 4.6 mm, 5 μ m particle size) column and the effluent was monitored by an ultra violet (UV) detector at 240 nm. The mobile phase used was acetonitrile: phosphate buffer (pH 7.0; 25.0mM containing 0.2 %v/v triethylamine) (65: 35 % v/v) at a flow rate of 1.0 ml/min. The present method was va lida ted a s per the United States Food and Dr ug Administration (USFDA) guideline. This method was linear over the range of 25.0-1000.0 ng/ml with regression coefficient greater than 0.99. The mean recovery of CLB and IS were 70.8±1.9 and 83.32 ± 1.55 % respectively. The method was found to be precise, accurate and specific. The developed HPLC method was applied for the estimation of CLB in plasma after topical application of various zinc salts in rats to study the penetration of CLB through rat skin.

Novel liquid chromatographic method for simultaneous estimation of pioglitazone and glimepiride in rat plasma by solid phase extraction: application to preclinical pharmacokinetic studies.

The need for a reliable bioanalytical method is of primary importance during preclinical studies. The aim of the present study was simultaneous determination of pioglitazone (CAS 111025-46-8) (PIO) and glimepiride (CAS 93479-97-1) (GLM) in plasma of rats. A high-performance liquid chromatographic method has been developed and validated using C18 column and UV detector. A mobile phase composed of acetonitrile and ammonium acetate buffer pH 4.5 in the ratio of 55:45%. The plasma samples clean-up was carried out using solid phase cartridges. The method was in the linear range of 50-8000 ng/mL for PIO and 50-2000 ng/mL for GLM. The coefficient of regression was found to be > or = 0.99. Precision and accuracy were within the acceptable limits, as indicated by relative standard deviation varying from 1.5 to 6.1% for PIO and 3.1 to 7.0% for GLM whereas the accuracy ranged from 97.0 to 106.4% for PIO and 96.5 to 106.4% for GLM. The mean extraction recovery was found to be 90.2 +/- 4.5, 76.8 +/- 2.8 and 85.2 +/- 5.2% for PIO, GLM and internal standard, respectively. Moreover, PIO and GLM were stable in plasma, up to 30 days of storage at -70 degrees C and after being subjected to bench top, auto-sampler, and three freeze-thaw cycles. The developed method was applied for preclinical pharmacokinetic studies.

Validated HPLC method for quantitative determination of talinolol in rat plasma and application to a preclinical pharmacokinetic study

BACKGROUND A simple HPLC-UV method with a high reproducibility and sensitivity for the determination of talinolol in rat plasma was developed in this study. METHOD After liquid-liquid extraction, the compounds were separated on a Vydac(®) C18 monomeric column (250 × 4.6 mm inner diameter × 5-µm particle size) using a mobile phase composed of acetonitrile and potassium dihydrogen phosphate buffer (34:66 v/v), delivered isocratically at a flow rate of 1.0 ml min(-1). Escitalopram was used as an internal standard. The chromatographic peak-area ratio, based on UV absorbency at 245 nm, was used for quantitative analysis. RESULTS Calibration standards with concentrations over the range of 10-1000 ng ml(-1) were validated for routine sample analysis to support pharmacokinetic studies with talinolol in rats. The intra- and inter-day precision studies showed good reproducibility with coefficients of variation of less than 11.49%. The developed method is simpler and more sensitive than previously reported methods. DISCUSSION The analytical sensitivity and accuracy of this assay were adequate for characterization of talinolol in rat plasma and the assay has been applied successfully to the in vivo kinetic study of talinolol in rats. After talinolol (10 mg kg(-1)) was given orally, the maximum concentration and the AUC(0-∞) were 341.8 ± 99.4 ng ml(-1) and 976.26 ± 173.37 ng h ml(-1), respectively. The oral bioavailability was approximately 52.14 ± 9.26%. CONCLUSION The advantages of our method are a small sample volume (200 µl), short analysis time (13.5 min) and a simple sample extraction and clean-up compared with multiple extraction and washing steps and a longer analysis time in previously published methods.