Improved HPLC method for determination of four PPis, omeprazole, pantoprazole, lansoprazole and rabeprazole in human plasma

PURPOSE: To develop a simple and rapid HPLC method for measuring of four proton-pump inhibitors (PPIs), omeprazole (OPZ), pantoprazole (PPZ), lansoprazole (LPZ) and rabeprazole (RPZ) concentrations in human plasma. METHODS: Following a single step liquid-liquid extraction analytes along with an internal standard (IS) were separated using an isocratic mobile phase of phosphate buffer (10 mM)/acetonitrile (53/47, v/v adjusted pH to 7.3 with triethylamine) at flow rate of 1 mL/min on reverse phase TRACER EXCEL 120 ODS-A column at room temperature. RESULTS: Total analytical run time for selected PPIs was 10 min. The assays exhibited good linearity (r2>0.99) over the studied range of 20 to 2500 ng/mL for OPZ, 20 to 4000 ng/mL for PPZ, 20 to 3000 ng/mL for LPZ and 20 to 1500 ng/mL for RPZ. The recovery of method was equal or greater than 80 and lower limit of quantification (LLOQ) was 20 ng/mL for four PPIs. Coefficient of variation and error at all of the intra-day and inter-day assessment were less than 9.2 for all compounds. CONCLUSIONS: The results indicated that this method is a simple, rapid, precise and accurate assay for determination of four PPIs concentrations in human plasma. This validated method is sensitive and reproducible enough to be used in pharmacokinetic studies and also is time- and cost-benefit when selected PPIs are desired to be analyzed

AN IN VITRO INVESTIGATION INTO THE MECHANISM OF THE CLINICALLY RELEVANT DRUG-DRUG INTERACTION BETWEEN OMEPRAZOLE OR ESOMEPRAZOLE AND CLOPIDOGREL

Clopidogrel is a thienopyridine antiplatelet prodrug that was approved by the US FDA in 1997 and quickly supplanted ticlopidine as the primary drug therapy for reducing atherothrombotic events. It is converted to its pharmacologically active metabolite H4, which irreversibly inactivates the P2Y12 receptor on platelets, through two sequential reactions that are catalyzed mainly by CYP2C19. Common clinical practice involved the coadministration of a proton pump inhibitor (PPI, including omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole) with clopidogrel to decrease the risk of upper gastrointestinal bleeding. This practice was formalized for high risk patients by the American Heart Association (and others) in 2008. By 2009, numerous publications described an unexpected decrease in clopidogrel efficacy when coadministered with PPIs, prompting both the US Food & Drug Administration (FDA) and European Medicines Agency (EMA) to issue recommendations discouraging the concomitant use of PPIs and clopidogrel. Proton pump inhibitors are also metabolized by CYP2C19. It seemed reasonable to conclude that, despite their relatively short plasma half-lives, PPIs might competitively inhibit CYP2C19, thereby reducing the efficacy of clopidogrel. In 2010, as numerous publications emerged, both regulatory agencies restricted subsequent warnings to only omeprazole and esomeprazole. The interaction between clopidogrel and PPIs, and the potential mechanisms responsible for it, continues to be a subject of much debate in 2015. This dissertation describes research that contributes to the progress made in understanding the basis for the interaction between clopidogrel and PPIs since the time of the initial regulatory statements, and in particular, why only omeprazole and esomeprazole are implicated in this drug interaction. The initial studies in this dissertation identified omeprazole (a racemic mixture of R- and S-enantiomers) and esomeprazole (the S-enantiomer) as not only competitive inhibitors, but more importantly, metabolism-dependent inhibitors (MDIs) of CYP2C19 in human liver microsomes (HLM), human hepatocytes and recombinant CYP2C19. In contrast, lansoprazole and pantoprazole did not cause metabolism-dependent inhibition (MDI) of CYP2C19. In addition to its clinical relevance, these observations are important because they underscore the importance of using a low concentration of enzyme and a short incubation time with the CYP marker substrate in order to detect MDI of CYP enzymes in vitro. In many previous studies of CYP2C19 inhibition by omeprazole or esomeprazole, the concentration of HLM was too high and/or the substrate incubation time was too long to detect MDI. The kinetic parameters for CYP2C19 inactivation by omeprazole, namely kinact and KI, were determined and used in a physiologically based pharmacokinetic (PBPK) model to predict the degree of CYP2C19 inactivation under clinical conditions. Omeprazole and esomeprazole were subsequently shown to be irreversible MDIs of CYP2C19, which explained why the decrease in clopidogrel efficacy could not be prevented in clinical studies by simply separating the doses of clopidogrel from omeprazole or esomeprazole. Subsequent studies demonstrated that, like the parent drug, two of the three major metabolites of omeprazole are also irreversible MDIs of CYP2C19. The kinetic parameters for CYP2C19 inactivation by these metabolites were determined and, along with those for omeprazole and esomeprazole, used in a mechanistic static model to predict the reduction of H4 formation from clopidogrel under clinical conditions. The model slightly overpredicted (by a factor of 2) the ability of omeprazole to block the conversion of clopidogrel to H4, its pharmacologically active metabolites, but otherwise established that inactivation of CYP2C19 is the likely mechanism for the clinical interaction between omeprazole/esomeprazole and clopidogrel. Esomeprazole and its two inhibitory metabolites, namely omeprazole sulfone and 5 O desmethylomeprazole, were subsequently determined to meet several criteria for mechanism-based inhibition (a special case of irreversible MDI). In addition, studies were initiated to test the hypothesis that the mechanism of CYP2C19 inactivation by esomeprazole and its metabolites involves the formation of a benzylic radical (on the 5,,S-methyl group) that binds covalently to the heme moiety. This hypothesis was based on the observation that the 5,,S methyl group is present on the pyridine ring of those compounds that irreversibly inactivate CYP2C19, namely omeprazole, esomeprazole, omeprazole sulfone and 5 O desmethylomeprazole, but absent from those compounds that did not inactivate CYP2C19, namely lansoprazole, pantoprazole and 5,,S-hydroxyomeprazole. Based on this hypothesis, the investigational PPI, tenatoprazole, which contains a 5,,S-methyl group, was correctly predicted to cause MDI of CYP2C19 whereas ilaprazole and rabeprazole, which lack a 5,,S-methyl group, did not cause MDI of CYP2C19. These results suggest that the investigational PPI, tenatoprazole, but not the clinically used PPIs ilaprazole or rabeprazole, may compromise the therapeutic effectiveness of clopidogrel. Finally, studies were performed in an attempt to provide direct evidence for the proposed mechanism of inactivation of CYP2C19 by esomeprazole, namely the formation of a heme adduct. The potential for the formation of a heme adduct in incubations of esomeprazole in HLM was evaluated by UHPLC analysis with UV/VIS detection and high resolution mass spectrometry (HRMS) with post-acquisition mass-defect filtering to identify heme and heme-containing adducts. Incubation of esomeprazole with NADPH-fortified HLM resulted in a substantial decrease in the amount of heme detectable by UHPLC with either UV absorbance or HRMS and appeared to show the formation of a heme adduct based on mass-defect filtering and isotopic distribution. However, the putative heme adduct was subsequently identified as a dimer of esomeprazole sulfone (a metabolite of esomeprazole formed by CYP3A4/5). Although an adduct between heme and a metabolite of esomeprazole was not ultimately identified, the potential for an unusual analytical artifact was revealed; namely, that sulfur-containing drugs can be converted to metabolites that closely resemble a heme adduct based on mass-defect filtering and isotopic distribution. In summary, this dissertation supports the hypothesis that irreversible inactivation of CYP2C19 is the mechanism by which omeprazole and esomeprazole reduce the efficacy of clopidogrel. This property is not shared by lansoprazole, pantoprazole, rabeprazole or ilaprazole. These findings support regulatory agencies¡¦ recommendations that, in order to reduce the risk of gastrointestinal bleeding, clopidogrel should not be coadministered with omeprazole or esomeprazole but should be coadministered with other PPIs that do not inactivate CYP2C19

ALTERATION OF PHARMACOKINETIC PARAMETERS OF PROTON PUMP INHIBITORS USING TRANSDERMAL DRUG DELIVERY SYSTEM

Objective: The present study was aimed to find out the effect of transdermal patches of proton pump inhibitors pantoprazole and esomeprazole on the alteration of pharmacokinetic parameters of these drugs. Methods: The transdermal patches were formulated by the solvent evaporation technique using polymers HPMC E5 with PVP K 30 and HPMC E5 with Eudragit L100 in different ratios. The best formulation from each of the drug pantoprazole and esomeprazole was selected and administered to rabbits and the plasma drug concentration was compared with the marketed formulation. The pharmacokinetic parameters such as maximum plasma concentration (Cmax), time to reach Cmax (tmax), area under the curve (AUC), area under first moment curve (AUMC), elimination rate constant (λz), biological half-life (t1/2), and mean residence time (MRT) were determined. Results: The plasma drug concentration vs time curve shows the extended-release of the drugs pantoprazole and esomeprazole when compared with the marketed formulation. The results show that there is no change in the peak plasma concentration, but a significant difference was observed in all the pharmacokinetic parameters. The AUC showed 6 fold increase for pantoprazole from 8.91 to 55.20 μg*h/ml and 3.5 fold increase for the drug esomeprazole from 7.86 to 28.53 μg*h/ml, and the mean residence time also showed 2 fold increase for the transdermal patches when compared with the marketed formulations. Conclusion: The increase in tmax, AUC, and MRT values of the formulated transdermal patches with the values of the marketed formulation of both the drugs, revealed that the transdermal patches can be used to deliver the drug for an extended period and also can alter the pharmacokinetics of pantoprazole and esomeprazole

A New Solid-Phase Extraction Method for Determination of Pantoprazole in Human Plasma Using High-Performance Liquid Chromatography

BACKGROUND: A new simple, selective and accurate high-performance liquid chromatographic (HPLC) method utilising solid-phase extraction for the determination of pantoprazole in human plasma samples has been developed. AIM: The purpose of this paper was developing a new HPLC method suitable for the determination of pantoprazole in plasma samples, which enables simple and rapid isolation and concentration of the analysed drug.METHODS: The chromatographic separation was accomplished on a LiChroCart LiChrospher 60 RP select B column using a mobile phase composed of 0.2 % (V/V) water solution of triethylamine (pH 7) and acetonitrile (58:42, V/V) followed by UV detection was at 280 nm. The solid-phase extraction method using LiChrolut RP-18 (200 mg, 3 ml) was applied to the obtained good separation of investigated drug from endogenous plasma components. Best results were achieved when plasma samples were buffered with 0.1 mol/L KH2PO4 (pH 9) before extraction, eluted and reconstituted with acetonitrile and 0.001 mol/L NaOH after extraction, respectively. RESULTS: The standard calibration curves showed good linearity within the range of 25.0-4000.0 ng/mL with a correlation coefficient greater than 0.996. Retention times of pantoprazole and internal standard, lansoprazole was 4.1 and 6.0 min respectively. The limit of quantification was 25.0 ng/mL. For intra- and inter-day precision relative standard deviations ranged from 4.2 to 9.3%. The relative errors for stability investigations were ranged from 0.12 to -10.5%. CONCLUSION: This method has good precision and accuracy and was successfully applied to the pharmacokinetic and bioequivalence study of 40 mg pantoprazole in healthy volunteers

Design, development and evaluation of controlled release multiple unit pellets for potential delivery of antiulcerants

AIM AND OBJECTIVES: The main aim of the present study was to formulate and evaluate enteric coated, controlled release multiple unit pellets of Esomprazole magnesium and Rabeprazole sodium using natural and synthetic polymers. OBJECTIVES: 1. To formulate Esomeprazole magnesium and Rabeprazole Sodium Controlled release Multiple Unit Pellets using various concentrations of Hydroxy Propyl Methyl Cellulose K15, Hydroxy Propyl Methyl Cellulose K100, Ethyl Cellulose, and Xanthan gum by Extrusion Spheronization process. 2. To evaluate the pre - compression parameters like drug excipient interaction study, bulk density, tapped density, compressibility index and angle of repose. 3. To evaluate various post compression evaluation parameters like friability, drug content and in vitro dissolution studies and Scanning Electron Microscopy (SEM) study. 4. To formulate gastric protection for the prepared multiple unit pellets using Hydroxy Propyl Methyl Cellulose Phthalate as enteric polymer. 6. To determine the mechanism and kinetics of drug release. 7. To conduct stability studies on optimized formulation as per ICH guidelines. SUMMARY: In the modern era of Pharmaceutical research much attention has been focussed on patient’s health in terms of therapeutic efficacy and safety. Modified Dosage Form (MRDF) has always been more effective therapeutic alternative to conventional or immediate release. The term modified release drug product is used to describe product that alter the timing and /or the rate of release of the drug substances. There are two types of MRDF .i) Delayed release ii) Extended release. The terms Controlled release (CR), Sustained release (SR) Prolong release (PR) has been used synonymously with extended release dosage forms. Controlled release drug delivery is one which delivers the drug at a predetermined rate for locally (or) systemically for a specified period of time. Controlled release drug delivery system aim to maintain plasma concentration of drugs within the therapeutic window for a longer period of time. Delayed release products are formulated with acid resistance (or) enteric coating protects acid labile drug substance from the gastric environment (or) to prevent adverse events such as irritation. A peptic ulcer is an open sore on the lining of the stomach or duodenum. Gastric and duodenal ulcer is produced by an imbalance between mucosal defences particularly gastric acid and pepsin. In addition, H.pylori infection is a major factor in the pathogenic of peptic ulcer. Proton pump inhibitors (PPI) rank among the top 10 prescribed classes of drugs and are commonly used to treat acid reflux, indigestion and peptic ulcers. PPIs are among the most widely sold drugs in the world and the first one in antiulcer medicine is omeprazole(WHO model list of essential medicines). Among seven available PPI drugs Esomeprazole magnesium and Rabeprazole sodium are classical examples of proton pump inhibitors and are approved by FDA for the treatment of GERD, Peptic ulcer and maintains of erosive esophagites. These drugs will degrading in acidic environment of stomach and will lead to therapeutic in efficacy so it is necessary to bypass the acidic pH of the stomach which can be achieved by formulating delayed release dosage forms by using different enteric polymers. The goal of any drug delivery system is to provide a therapeutic amount of drug to the proper site in the body to achieve promptly and then maintain the desired drug concentration. Many marketed Esomeprazole and Rabeprazole tablets/pellets are available as enteric coated formulation only. The main aim of the present study was to develop and evaluate enteric coated, controlled release tablets of Esomeprazole magnesium and Rabeprazole sodium using natural and synthetic polymers gives better and more uniform drug absorption and greater bioavailability. To design the multiple unit pellets, we have developed two different spheroid unit of uniform drug content with varying in polymer concentration to achieve rate controlled drug release as per our specification. The first group spheroid unit which contain only drug with spheroidizing polymers was prepared to achieve the minimum effective concentration. The second group of spheroid unit was prepared by using the controlled release polymers HPMC K100, HPMC K15, Ethyl cellulose and Xanthan gum. Among the four polymers HPMC K100 the drug Polymer ratio 1:1.5 shows good controlled release characters in Esomeprazole magnesium and HPMC K 15 the drug and polymer ratio 1:2 shows good controlled release profile in Rabeprazole sodium. The MUPS were prepared by Extrusion -Spheronization a promising pelletization technique. In this process the pellets were prepared by mixing the drug with excipient along with binder solution the resultant mass was extruded through extruder followed by spheronizer and finally dried. The possible interactions between drugs and distinct polymers were investigated via FT-IR Studies. Results proved that Rabeprazole sodium and Esomeprazole magnesium was found to be compatible with excipient as no disappearance of the peaks or shift of the peaks indicating that the drugs are compatible with ingredients. The micromeritics evaluation like Bulk density, Tapped density, Angle of repose, Carr’s index and Hauser’s ratio of the prepared pellets shows good flow property. The post formulation parameters like friability, drug content were carried out and found to be within acceptable limit. SEM study shows the surface morphology of the optimized formulations E2 and R6 the pellets was compact, continuous and was porous in nature, demonstrated the spherical nature of the pellets. Based on in vitro dissolution profile the enteric coated, controlled release multiple unit pellets of Esomeprazole magnesium and Rabeprazole sodium was developed using HPMCK100 in the ratio 1:1.5 ( drug :polymer) and HPMC K15 in the ratio 1:1.5 ( drug :polymer) respectively as controlled release polymer and Hypromellose phthalate HP55 as enteric coated polymer. The optimized formulations E2 (Esomeprazole) and R6 (Rabeprazole) had better resistant to 0.1N HCl and better cumulative percent drug release as compared to other formulation. After 12 hours E2 shows 97.88% and R6 shows 97.59 % cumulative percent drug release as compared to other formulation. So E2 (Esomeprazole) R6 (Rabeprazole) was selected as optimized formulation from the trail batches. The in vivo pharmacokinetic plasma concentration and time curve parameters shows that less plasma concentration fluctuation, lower Cmax, prolonged tmax and MRT of formulated MUPS than that of marketed enteric coated formulations. Stability study revealed there was no significant change in in vitro release profile. All the parameters were within limit after 90 days. CONCLUSION: From the above research finding it can be concluded that controlled release of Esomeprazole Magnesium and Rabeprazole Sodium Multiple Unit Pellets could be developed by using HPMC K100 the drug Polymer ratio 1:1.5 and HPMC K 15 the drug and polymer ratio 1:2 prepared by Extrusion -Spheronization to achieve better bioavailability and extended drug release. Further, the first group spheroid unit could maintain the minimum effective concentration and the second group spheroid unit could release the medicament in control release manner. Hence the prepared Multiple Unit Pellets could achieve both enteric coating and controlled release approach for the potential delivery of Antiulcerants

Improvement and validation of a high-performance liquid chromatography in tandem mass spectrometry method for monitoring of omeprazole in Plasma

Omeprazole (OME) is a proton pump inhibitor (PPI) with 58% bioavailability after single oral dose, presenting large inter-individual variations and significant drug-drug interactions. A simple and rapid liquid chromatography in tandem with mass spectrometry (LC/MS-MS) with solid phase extraction (SPE) and isotope-labelled internal standard (IS) method was developed to monitor the plasma levels of OME for application in pharmacokinetics and drug-drug interactions studies. OME and its IS (OME-D3), were eluted with Zorbax extend C-18 rapid resolution (4.6 mm x 50 mm, 3.5 μm) at 25ºC, under isocratic conditions through a mobile phase consisting of 1 mM ammonium acetate, pH 8.5 (55%), and acetonitrile (ACN, 45%). The flow rate was 0.8 mL/min and the run time of chromatogram was 1.2 min. OME was detected and quantified by LC-MS/MS with positive electrospray ionization (ESI) that operates in multiple-reaction monitoring (MRM) mode. The method was linear in the range of 1.5- 2000 ng/mL for OME. The validation assays of accuracy and precision, matrix effect, extraction recovery and stability of the samples for OME did not deviate more than 20% for the lower limit of quantification (LLOQ) and no more than 15% for other quality controls (QCs), according to regulatory agencies.This work was also supported by FIS No. PI052124 and CA12/00122 to ARN and FPU12/02220 to AW

Bioanalytical Method Development and Validation of Esomeprazole in Human Plasma by Lc-Ms/Ms.

This studies to measure Bioavailability and/or establish Bioequivalence of a product are important elements in support of orally administered drug products in investigational new drug applications (INDs), new drug applications (NDAs) , abbreviated new drug applications (ANDAs), and their supplements. The systemic exposure profile determined during clinical trials in the IND period can serve as a benchmark for subsequent BE studies. Until recently, bioavailability (rate and extent of absorption of medicaments from drug delivery systems) of drugs was not emphasized. It was more or less assumed that if the physical and chemical integrities of a drug product were assured pharmacologic performance would be observed. It is now recognized that formulation factors can influence the biologic availability of a medicament from a dosage unit in mammalian systems. Consequently, it has become common practice to establish bioavailability by measurement of blood levels of drugs following administration of dosage forms. However, it should be noted that either bioavailability or bioequivalence data could be generated without analytical methodology to accurately measure drugs in biological fluids. Currently there is a need in the pharmaceutical environment to develop analytical methods for the determination of Esomeprazole in human plasma. The developed method could then be applied to clinical trials to obtain accurate pharmacokinetic parameters in human plasma. HPLC-UV and LC-MS/MS methods have been reported. Some of these methods use complicated extraction equipments, long and tedious extraction procedures, and large amounts of solvents or biological fluids for extraction while other methods have a long turn around time during analysis. Esomeprazole is a proton pump inhibitor which reduces gastric acid secretion through inhibition of H+/K+-ATPase in gastric parietal cells. By inhibiting the functioning of this enzyme, the drug prevents formation of gastric acid. The bioanalytical methodology described in this manuscript was specific, sensitive, accurate and precise. The method employed HPLC coupled with electrospray ionization mass spectrometric detection (LC-ESI-MS). The method involved a simple sample preparation by liquid- liquid extraction followed by isocratic chromatographic separations. A sensitive method that is precise and accurate over a linear assay range of 5.000 – 1000.000 ng/mL has been validated for the determination of Esomeprazole in Human plasma using LC-MS/MS Method. The LC-ESI-MS method was capable of estimating 5 ng/ml of Esomeprazole accurately in human plasma with high degree of reproducibility.The method can be useful for further BA/BE studies Pharmacokinetic studies

Gradient high performance liquid chromatography method for simultaneous determination of ilaprazole and its related impurities in commercial tablets

AbstractA methodology (HPLC) proposed in this paper for simultaneously quantitative determination of ilaprazole and its related impurities in commercial tablets was developed and validated. The chromatographic separation was carried out by gradient elution using an Agilent C8 column (4.6 mm × 250 mm, 5 μm) which was maintained at 25 °C. The mobile phase composed of solvent A (methanol) and solvent B (solution consisting 0.02 mmol/l monopotassium phosphate and 0.025 mmol/l sodium hydroxide) was at a flow rate of 1.0 ml/min. The samples were detected and quantified at 237 nm using an ultraviolet absorbance detector. Calibration curves of all analytes from 0.5 to 3.5 μg/ml were good linearity (r ≥ 0.9990) and recovery was greater than 99.5% for each analyte. The lower limit of detection (LLOD) and quantification (LOQ) of this analytical method were 10 ng/ml and 25 ng/ml for all impurities, respectively. The stress studies indicated that the degradation products could not interfere with the detection of ilaprazole and its related impurities and the assay can thus be considered stability-indicating. The method precisions were in the range of 0.41–1.21 while the instrument precisions were in the range of 0.38–0.95 in terms of peak area RSD% for all impurities, respectively. This method is considered stability-indicating and is applicable for accurate and simultaneous measuring of the ilaprazole and its related impurities in commercial enteric-coated tablets

Validated Method Development for the Quantification of Cinitapride and Pantoprazole by Spectrophotometry and RP-HPLC in Bulk and Oral Dosge form.

The present study analysis of drugs plays a major role in the development, manufacture and therapeutic use of drug. Quantitative analysis of raw materials and the final product is necessary to ensure that they meet certain specifications and to ascertain the amount of each component in the final product. Standard analytical procedures for newly designed and synthesized drugs are not available in any of the pharmacopoeias. So it becomes necessary to develop newer, accurate, specific, simple, easy to perform, reliable and economic analytical techniques for the estimation of the new drugs. This thesis deals with pantoprazole (PNP) and cinitapride (CNP). Pantoprazole has been recently included in Indian Pharmacopoeia – 2010. Cinitapride is a very new drug, launched in 2007 and is not official in any of the pharmacopoeias. It has been very recently added in drug bank in 2012. The combination of the two drugs has been launched in 2011.Extensive literature survey reveals that only first derivative and HPTLC method for the determination of pantoprazole (PNP) and cinitapride (CNP) has been reported. There is no evidence for the estimation of PNP and CNP by UV-Visible spectrophotometry and RP-HPLC. CONCLUSION: The developed UV – Visible and RP-HPLC methods were found to be simple, precise, accurate and rapid methods for the analysis of Cinitapride and Pantoprazole in its pure form and in its pharmaceutical dosage formulation. Thus, all the above adopted methods can be effectively used for the routine analysis of Cinitapride and Pantoprazole in pharmaceutical dosage form. methods in bulk and in combined tablet dosage form. So, an attempt has been made is to develop a validated, simple, easy to perform, accurate, cost effective and rapid spectrophotometric methods for the estimation of CNP and PNP in bulk and combined oral dosage form