emin-vitroem-drugdrug-interaction-studies-of-apixaban-with-atorvastati

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Poly-pharmacy of cardiovascular drugs is a common cause of a few unexpected drug-drug interactions (DDI) which may lead to the occurrence of the adverse effects of the drugs co-administered. The High Performance Thin Layer Chromatography method was adopted for the analysis of apixaban and atorvastatin. The method validation for the individual drugs was carried out according to the ICH guidelines. The validated method was applied for the in vitro drug-drug interaction study is at the biological pH of stomach at 4.0, of blood at 7.4 and intestinal condition at 9.0. The aim of the study is to evaluate the effect of atorvastatin under the simulated conditions of the human body. The in vitro drug-drug interaction studies suggest that the drug apixaban does not have any profound change at pH 4 and 9 when present with atorvastatin. When apixaban and atorvastatin are present at a pH 7.4 the significant variation in the detector response is indicative of the decrease in the level of apixaban

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In vitro Drug-Drug Interaction Studies of Apixaban with Atorvastatin by HPTLC Method Sangeetha RK 1 and Ravi TK 2 1 Department of Pharmaceutical Analysis College of Pharmacy Sri Ramakrishna Institute of Paramedical Sciences Coimbatore-641044 India 2 Principal and HOD Department of pharmaceutical analysis College of pharmacy Sri Ramakrishna Institute of paramedical Sciences Coimbatore-641044 India Corresponding author: Sangeetha RK Assistant Professor Department of Pharmaceutical Analysis College of Pharmacy Sri Ramakrishna Institute of Paramedical Sciences Coimbatore - 641044 India Tel: +91 9566545310 Email: geetkarangmail.com Received date: September 11 2018 Accepted date: October 3 2018 Published date: October 13 2018 Copyright: © 2018 Sangeetha RK. This is an open-access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Abstract Poly-pharmacy of cardiovascular drugs is a common cause of a few unexpected drug-drug interactions DDI which may lead to the occurrence of the adverse effects of the drugs co-administered. The High Performance Thin Layer Chromatography method was adopted for the analysis of apixaban and atorvastatin. The method validation for the individual drugs was carried out according to the ICH guidelines. The validated method was applied for the in vitro drug-drug interaction study is at the biological pH of stomach at 4.0 of blood at 7.4 and intestinal condition at 9.0. The aim of the study is to evaluate the effect of atorvastatin under the simulated conditions of the human body. The in vitro drug-drug interaction studies suggest that the drug apixaban does not have any profound change at pH 4 and 9 when present with atorvastatin. When apixaban and atorvastatin are present at a pH 7.4 the significant variation in the detector response is indicative of the decrease in the level of apixaban. Keywords: Drug interaction Apixaban Atorvastatin HPTLC method. Introduction Apixaban 1-4-methoxyphenyl-7-oxo-6-4-2-oxopiperidin-1- ylphenyl-4567-tetrahydro-1H-pyrazolo34-cpyridine-3- carboxamide 1 is indicated to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fbrillation. Its molecular formula is C 25 H 25 N 5 O 4 which corresponds to a molecular weight of 459.5. Apixaban appears as a white-to-pale yellow non-hygroscopic crystalline powder with an aqueous solubility of 0.028 mg/mL at 24°C. Te reported methods of estimation of apixaban by spectroscopic 23 and liquid chromatographic methods 45 were found in the literatures. Te other methods reported were surveyed for the selection of research work 6-8. Till date there are no in vitro interaction studies reported for apixaban by HTPLC. Further apixaban is not ofcial in any pharmacopeia. Atorvastatin calcium is a synthetic lipid-lowering agent. Atorvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A dHMG-CoA reductase. Tis enzyme catalyzes the conversion of HMG-CoA to mevalonate an early and rate-limiting step in cholesterol biosynthesis. Atorvastatin calcium is R-R R -2-4-fuorophenyl-ß δ- dihydroxy-5-1-methylethyl-3-phenyl-4- phenylaminocarbonyl-1H-pyrrole-1-heptanoic acid calcium salt 2:1 trihydrate. Te empirical formula of atorvastatin calcium is C 33 H 34 FN 2 O 5 2Ca•3H 2 O and its molecular weight is 1209.42.Atorvastatin calcium is a white to of-white crystalline powder that is insoluble in aqueous solutions of pH 4 and below. Atorvastatin calcium is very slightly soluble in distilled water pH 7.4 phosphate bufer and acetonitrile slightly soluble in ethanol and freely soluble in methanol 910. Te objective of the present work is to develop a method and validate as per the ICH parameters 11. Te developed method is applied for its determination as single drug for apixaban and atorvastatin and in combination. Te in vitro drug drug interaction studies between apixaban and atorvastatin indicates the potential interaction between the drugs at pH 7.4. Materials and Methods Te organic solvents like methanol toluene divchloromethane and tetrahydrofuran was of AR grade used for solution preparation and mobile phase system were procured from SD Fine Chemicals Ltd Mumbai. Te Active Pharmaceutical Ingredient Apixaban was procured from Sigma Aldrich Germany. Te tablet formulations of Apixaban and atorvastatin was procured from the local pharmacy in Coimbatore. Te drug atorvastatin selected for interaction was procured from Y arrow Chem Products Mumbai. Te HPTLC method was developed using precoated silica gel plate GF 254 Camag UV chamber for spot identifcation Camag linomat 5 applicator for sample application 20 x 20 cm and 10 x 10 cm chamber for plate development and Camag TLC Scanner for spot detection with W inCats Sofware. Te Digital pH meter MK VI was used for the pH measurement of the bufer and drug solutions. Chromatographic conditions Te solvent system consisting of Toluene: Dichloromethane: Tetrahydrofuran in the ratio of 2:2:6v/v/v with two drops of glacial acetic acid was found to separate the peaks of apixaban and atorvastatin. Te wavelength selected for the study was 279 nm with chamber saturation time of 20 minutes. Journal of Drug Metabolism Toxicology ISSN: 2157-7609 Journal of Drug Metabolism Toxicology Sangeetha et al. J Drug Metab Toxicol 2018 9:3 DOI: 10.4172/2157-7609.1000241 Research Article Open Access J Drug Metab Toxicol an open access journal ISSN: 2157-7609 Volume 9 • Issue 3 • 1000241

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Stock solution of apixaban and atorvastatin in the concentration of 1000 mcg/ml was prepared with methanol. Te working standard was prepared in the concentration of 100 mcg/ml with the same. Validation of HPTLC method for Apixaban and atorvastatin Linearity and range: Te method shows a linear correlation for apixaban between 200-700 ng/spot Figure 1 and 400-1400 ng/spot for atorvastatin Figure 2. Te peak area was subjected to regression analysis by the least square method. Te results are given in Table 1. Te correlation coefcient was more than 0.99 for both the drugs. Figure 1: 3D Densitogram of apixaban. Figure 2: 3D Densitogram of atorvastatin. Apixaban Atorvastatin Conc ng/ spot Rf value Peak Area Conc ng/ spot Rf value Peak Area 200 0.54 2574 400 0.68 3324 300 0.54 3294 600 0.69 4489 400 0.54 4014 800 0.69 5989 500 0.55 4567 1000 0.69 7262 600 0.55 5138 1200 0.7 7871 700 0.56 5587 1400 0.7 8850 Table 1: Linearity data. Intraday and interday precision: Te repeatability of the measurements were studied on the same day. Te interday precision was carried on three consecutive days. Te standard deviation and relative standard deviation was calculated. Te RSD was found to be below 1 indicating the method precise. Accuracy: Te recovery of the standard drug was evaluated at two diferent levels of 50 and 100 for the tablet formulation for both the drugs. Te recovery of the standard drug from the solution was calculated and found to be 93.80 and 97.40 for the selected brand xarelto 2.5 mg tablet. For the Atorva 5 mg tablet the recovery was 101.0 and 99.8 for the two levels carried out. Robustness and ruggedness: Te experimental parameters varied intentionally were the chamber saturation time by ± 2 minutes and mobile phase composition by ± 0.1 ml. Te peak area recorded were found to show no notable variation from the fxed parameters. Stability of the plate: Te plate was scanned at every two hours interval. Te peak area recorded was found to be same till 10 hours afer the values varied. Te results are mean of six determinations. LOD and LOQ: Te limit of detection found by the detector was found to be 50 ng/spot for apixaban and 100 ng/spot for atorvastatin. Te limit of Quantitation was found to be 100 ng/spot and 200 ng/spot for apixaban and atorvastatin respectively. Analysis of formulations: Te Xarelto tablets 2.5 mg and the atorva 5 mg tablets were analysed for the percentage purity using the fxed chromatographic conditions. Te amount present in the tablet was found to be 103 for apixaban and 101.2 for atorvastatin. Te statistical estimation of six determinations were less than 1 for the RSD value for both drugs. In vitro drug-drug interaction studies Preparation of bufer solutions: Te pH 4.0 bufer was prepared in distilled water by weighing 3.725 g of potassium chloride in 1000 ml water. Te pH was adjusted upto 4.0 using 0.1 M HCl. Te bufer pH 7.4 was prepared by weighing accurately 0.6 g of potassium dihydrogen ortho-phosphate 6.4 g of disodium hydrogen orthophosphate and 5.85 g of sodium chloride in 1000 ml of HPLC grade water. Te pH 7.4 was adjusted using 0.1 M HCl. Te pH 9.0 bufer was prepared by weighing 4.98 g of ammonium chloride and dissolved in water afer which the volume was made up to 1000 ml with the same. Te pH was adjusted with ammonia solution up to 9.0. Preparation of solution: A 500+1000 mcg/ml of mixture stock solution for apixaban and atorvastatin was prepared in methanol. Te mixture of apixaban and atorvastatin working solution was prepared by diluting to a concentration of 50+100 mcg/ml with bufer solution. Te volume spotted for the analysis was 10 μl of the mixture solution. Method adopted for in vitro drug-drug interaction studies: Te mixture of drug apixaban and atorvastatin in the bufer solution was kept on a thermostat at 37°C. Te isolation and detection of apixaban and atorvastatin at the selective Rf value for the peak area and change in the response were tabulated. Te sampling was at an interval of one hour till 5 hours for the three bufer pH. Te blank bufer pH 4.0 7.4 and 9.0 were spotted to identify any interference with the drug peaks. Te spots of each mixture were studied for the peak area Rf value and the changes at regular intervals Figures 3-5. Te Table 2 illustrates the change in detector response from zero hours to fve hours. Citation: Sangeetha RK Ravi TK 2018 In vitro Drug-Drug Interaction Studies of Apixaban with Atorvastatin by HPTLC Method. J Drug Metab Toxicol 9: 241. doi:10.4172/2157-7609.1000241 Page 2 of 4 J Drug Metab Toxicol an open access journal ISSN: 2157-7609 Volume 9 • Issue 3 • 1000241

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Figure 3: HPTLC chromatograms of apixaban+atorvastatin mixture at 5 hours at pH 4.0. Figure 4: HPTLC chromatograms of apixaban+atorvastatin mixture at 5 hours at pH 7.4. Figure 5: HPTLC chromatograms of apixaban+atorvastatin mixture at 5 hours at pH 9.0. pH Time Hr Mixture Rf value Peak area Changes in peak area 4 0 0.58 6391 - 0.78 17718 - 5 0.63 6727 5.27 0.8 18592 4.92 7.4 0 0.96 1080 - 1.4 893 - 5 1.04 602 44.25 1.48 600 32.81 9 0 0.54 1141 - 0.73 1543 - 5 0.57 1110 2.82 0.76 2041 32.27 Table 2: In vitro drug-drug interaction studies of apixaban with atorvastatin. Results and Discussion Te HPTLC method was developed using diferent organic solvents based on the polarity and solubility of the drugs. Te proportional change in the ratio of Tetrahydrofuran had a signifcant efect in the Rf value of the spots. So the solvent system made up of Toluene: Dichloromethane: Tetrahydrofuran in the ratio of 2:2:6 has been selected. Te solvent composition carried the drug apixaban and atorvastatin to the same distance on the plate. So to have an acceptable separation between the two drugs selected two drops of glacial acetic acid was added which resulted in good resolution of both the drugs. Te experimental criterion such as the distance travelled by the solvent front chamber saturation time and the peak characteristics including the peak symmetry area and Rf value were fxed afer numerous trials. Te method developed was validated for the linearity range accuracy interday precision intraday precision ruggedness robustness limit of detection and limit of quantifcation 11. Te in vitro drug-drug interactions carried out highlights the eventual changes when both are present together in various biological fuids at the selected pH. Te conditions of study like the time of interaction was selected as the time to reach the peak plasma concentration is four hours for apixaban 1. Te Rf value of the drug apixaban in solution of the three pH 4.0 7.4 and 9.0 were run separately and the results were interpretated. At pH 4.0 and 9.0 the Rf value was found to be at .0.58 and 0.54 at zero hours and the changes in the responses were less than 10. At the pH 7.4 the peak area changes were 44 for apixaban and 33 for atorvastatin. Te peak area at zero hours was compared with the peak areas at the particular time of analysis. Te considerable changes in the peak area were calculated and tabulated in Table 2. Both the selected drugs are marketed in the potent dose of less than and equal to 10 mg. Te drug apixaban belongs to the class of anticoagulants and atorvastatin is a lipid lowering agent. Co- administration of both the class of drugs is a common regime. Te research work suggests no interactions between apixaban and atorvastatin at pH 4 and 9 whereas the responses at a pH 7.4 indicate decrease in the drug concentration of apixaban. Citation: Sangeetha RK Ravi TK 2018 In vitro Drug-Drug Interaction Studies of Apixaban with Atorvastatin by HPTLC Method. J Drug Metab Toxicol 9: 241. doi:10.4172/2157-7609.1000241 Page 3 of 4 J Drug Metab Toxicol an open access journal ISSN: 2157-7609 Volume 9 • Issue 3 • 1000241

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Conclusion Te method developed and validated can be applied for the routine analysis of Apixaban and atorvastatin. Te interaction method is an insight into the safe administration of apixaban and atorvastatin. Acknowledgements Te authors sincerely thank the SNR Sons Charitable Trust Coimbatore for providing the facilities needed for carrying out the research work. References 1. Pr Eliquis apixaban Product Monograph. Bristol-Myers Squibb Canada Co. Montreal Quebec H4S 0A4 and Pfzer Canada Inc. Kirkland Quebec H9J 2M5. 2. Dudhe PB Shelke PS Chavare PD 2017 Determination of apixaban from bulk and tablet dosage form by area under curve and frst order derivative spectrophotometric methods. Intl J Chem Tech Res 10: 703-711. 3. Ram Babu K Cherukuru N Ramsrinivas Rao G N 2015 Two novel validated RP-HPLC and UV spectrophotometric methods for the estimation of apixaban in bulk and pharmaceuticals dosage forms. Am J Pharm Tech Res 5: 450-460. 4. Shashikant Landge B 2015 Development and validation of stability indicating RP-HPLC method on core shell column for the determination of degradation and process related impurities of apixaban-an anticoagulant. Drug Am J Anal Chem 6: 539-550. 5. Hemant KJ V ishal KN 2017 Formulation development and stability indicating HPLC assay of tablets of apixaban. Int J Ph Pharma Sci 9: 24-32 6. Saeed Arayne M Najma Sultana Nawaz M 2014 Investigation of drug interaction studies of levocetirizne with HMG-CoA reductase inhibitors. Modern Chemistry Applications 2. 7. Delavenne X Mismetti P Basset T 2013 Rapid determination of apixaban concentration in human plasma by liquid chromatography/ tandem mass spectrometry: Application to pharmacokinetic study. J Pharm Biomed Anal 78–79. 8. Raghavan N Frost CE Yu Z He K Zhang H et al. 2009 Apixaban metabolism and pharmacokinetics afer oral administration to humans. Drug Metab Dispos 37: 74–81. 9. United States Pharmacopeia and National Formulary USP 34–NF 29 Te United States Pharmacopeial Convention Inc.: Rockville MD 2011. 10. Extractive spectrophotometric determination of Atorvastatin in bulk and pharmaceutical formulations 2003. Anal Lett 36: 2699-2711. 11. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals For Human Use ICH Harmonised Tripartite Guideline Validation of Analytical Procedures: Text And Methodology 2005. Citation: Sangeetha RK Ravi TK 2018 In vitro Drug-Drug Interaction Studies of Apixaban with Atorvastatin by HPTLC Method. J Drug Metab Toxicol 9: 241. doi:10.4172/2157-7609.1000241 Page 4 of 4 J Drug Metab Toxicol an open access journal ISSN: 2157-7609 Volume 9 • Issue 3 • 1000241

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