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Steroselective HPLC assay of donepezil enantiomers in tablets and plasma and its application to pharmacokinetics in Rats Bushra T. Mahasen A. *, Heba H. 2 Radwan 3 Abdine , Hassan Y. 4 Aboul-Enein and Kenichiro 5 Nakashima of Pharmaceutics, 2Department of Clinical Pharmacy, 3Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO Box 22452, Riyadh 11495; 4 Centre for Clinical Research (MBC-03-65), King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia; 5 Department of Clinical Pharmacy, Nagasaki University, 1-14, Bunkyo-machi, Nagassaki 852-8521, Japan. Introduction Results and Disscusion Experimental Chromatographic system and conditions: Donepezil (DH) is a reversible inhibitor of acetylcholinesterase. It is the second drug approved by the FDA for the treatment of mild to moderate dementia of the Alzheimer’s type. DH was demonstrated to be a potent and selective inhibitor of brain acetylcholinesterase with less adverse effects than physostigmine and tacrine [1-2]. DH has a chiral center adjacent to a carbonyl group. It is commercially available as the hydrochloride salt and is used as a racemic mixture in dosage forms. The donepzil enantiomers have differing extents of inhibition against acetylcholinestrerase in vivo and in vitro [3]; hence it was necessary to clarify the pharmacokinetics of each isomer during drug development course. The importance to develop new chiral discrimination separation systems to determine enantiomers in bulk substance, drug formulations and in biological fluids increases. This is because stereoisomers may have different pharmacological effects and also show differences in pharmacokinetic and pharmacodynamic properties [4]. Several pharmacokinetic studies [3, 5-7] including drug interactions with donepezil have been reported using high performance chromatographic (HPLC) method with UV detection [4], but methodology was not described. It should be mentioned that Matsui et al. [3] were the only group addressed the pharmacokinetics of DH enantiomers in human using liquid chromato-graphy–mass spectrometry, which was sensitive, but complicated. Aim of the work HPLC System: Waters' system Column: Chiralcel OD Mobile phase: n- hexane, isopropyl alcohol & triethylamine (87:12.9:0.1, v/v/v) Flow rate: 1.0 ml/min Detection: ultraviolet detection at 268 nm Injection volume: 75 µl 0.9 Determination of donepezil in pharmaceutical dosage forms: 0.9 0.6 Two commercially available formulations (Aricept® tablets) labelled to contain either 5 or 10 mg donepezil hydrochloride were analyzed. 0.6 0.3 0.3 0 0 0 Determination of DH in spiked rat plasma: B 1.2 10 20 I 0 -0.3 10 20 (-)R-DH (+)S-DH 0.1 0.01 Intra-day Nominal (ng/ml) Pharmacokinetics of DH in rats: 4 6 Time, h 8 Mean plasma concentrations (+ SD) time profile of (-)R and (+)S- donezepil enantiomers in rats after receiving 3mg/kg oral dose of racemic donepezil. A: Plasma samples free of drug (Blank). B: Plasma samples spiked with 200 ng/ml of donepezil HCl (B). I: (-)R-DH II: (+)S-DH Concentration 2 -0.3 Time, min Two hundred L of rat plasma samples were spiked with 20-50 L of DH stock solutions. Protein was precipitated with 600 L of acetonitrile. Vortexed for 1 min, & centrifuged at 20,000 rpm for 15 min. The supernatant was evaporated to dryness under a stream of N2. The residue was reconstituted in 100 L of the mobile phase. The absolute recoverry, Intra- and Interday accuracy and precision were evaluated by assaying quality controls with differrent concentrations of DH (0.05 - 2 g /ml). 12 Parameter (-)R-DH (+)S-DH Cmax*, ng/ml 143 ± 66 148 ± 110 Tmax*, h 5.2 ± 4 3.3 ± 2.7 AUC, mg h/L 3.94 2.91 Inter-day Recovery % Precision % 10 H. Sugimoto, Y. Yamanishi, Y. Iimura, Y. Kawakami, Curr. Med. Chem. 7 (2000) 303– 339. [2] E. Scarpini, P. Scheltens, H. Feldman, Lancet Neurology 2 (2003) 539–547. [3] K. Matsui, Y. Oda, H. Nakata, T.Yoshimura, J. Chromatography B, 729 (1999) 147–155. [4] J.P. Mason, A.J.Hutt Sterochemical Aspects of Drug Metabolism" Chapter 3 in "The impact of Sterochemistry in Drug Development and Use", H.Y. Aboul-Enein and I.W. Wainer (eds.) John Wiley & Sons Inc., New York, USA, 1997, pp. 45- 105. [5] J.W. Lee, S.L. Rogers, L.T. Friedhoff, M.R. Stiles, N.M. Cooper, Pharm. Res. 9 (1992) S350. [6] P.J. Tiseo, S.L. Rogers, L.T. Friedhoff, Clin. Pharmacol. Ther. 61(1997) 184. (PII75). [7] N. Yasui-Furukori, R. Furuya, T. Takahata, T. Tateishi, J. Chromatography B, 768 (2002) 261–265. Accuracy % Precision % Accuracy % (-)R-DH (+)S-DH (-)R-DH (+)S-DH (-)R-DH (+)S-DH (-)R-DH (+)S-DH (-)R-DH (+)S-DH Cl/F, L/h/kg 0.038 0.051 50 95.7 93.9 5.9 6.4 5.1 4.6 4.8 4.0 4.3 5.1 t½ , h 27.2 16.8 100 89 92 6.5 5.4 5.3 3.7 7.9 8.6 9.7 8 1000 90.7 88.9 7.1 4.4 3.9 2.9 5.7 8.1 9.3 10 Pharmacokinetic parameters of donepezil after 0.3 mg/kg PO dose in the rat (n=6). * Calculated from the actual data Single dose oral study has been performed. An equivalent to 3 mg drug/ Kg body weight of rats was calculated and as given as a suspension. 2000 97.3 95.5 4.3 3.1 1.2 2.8 6.5 7.1 2.7 4.5 Precision (RSD%), accuracy (relative error%) and recovery for determination of donepezil (-)R and (+)S-DH enantiomers in spiked rat plasma samples (n = 6). (+)S-DH Dosage Form Aricept® 5 mg Aricept® 10 mg II [1] 0 (-)R-DH The present study describes a stereospecific, isocratic, normal-phase HPLC method for the determination of the enantiomers of DH in tablets and plasma and its application to pharmacokinetic studies in rats. 1.5 A 1.2 Response Donepezil hydrochloride (DH), {2,3-dihydro-5,6dimethoxy-2-[[1-(phenyl -methyl)-4-piperidinyl] methyl]-1H-inden-1-one}hydrochloride 1.5 References 1 Enantiomer Concentrations,g/ml 1Department 1 Al-Quadeib , Content* (%) RSD (%) Content* (%) RSD (%) 95.6 96.9 8.3 3.2 96.2 97.3 9.6 3.4 Determination of the mean (-)R and (+)S- donezepil enantiomers content in pharmaceutical dosage forms (n = 6) Acknowledgment Special thanks to King Suad University, Research Center and to King Faisal Specialist Hospital and Research Center. Conclusion A new simple, stereospecific, reproducible, accurate and sensitive HPLC method has been successfully developed and applied to the determination of DH enantiomers in rats and in its commercial tablets. The results were used to generate profiles and to estimate the pharmacokinetic parameters of DH enantiomers up to 12 h after oral administration in rats. The disposition of the two enantiomers seems to be different.