Pharmacokinetics of ibuprofen enantiomers in rats after intravenous and oral administration of ibuprofen arginate 布洛芬精氨酸注射及口服给药在大鼠体内的立体选择性药代动力学

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1 88 药学学报 Acta Pharmaceutica Sinica 2012, 47 (1): Pharmacokinetics of ibuprofen enantiomers in rats after intravenous and oral administration of ibuprofen arginate WANG Xiao-lin, HAN Jing, ZHANG Dan, LIU Hui-chen * (Department of Clinical Pharmacology, Aerospace Center Hospital, Beijing , China) Abstract: The pharmacokinetics of ibuprofen enantiomers were studied in rats after intravenous and oral administration of ibuprofen arginate by means of a chiral HPLC method. The pharmacokinetics of ibuprofen was stereoselective after intravenous and oral administration of ibuprofen arginate. The pharmacokinetic stereoselectivity was higher after oral administration than that after intravenous administration. The systematic (R)-( )-to-(s)-(+) inversion might be more important than the presystematic one in the stereoselective pharmacokinetics after oral administration. Oral administration of ibuprofen arginate resulted in a very rapid absorption of (S)-(+)-ibuprofen (eutomer), and the absolute bioavailabilities of (S)-(+)-ibuprofen and (R)-( )-ibuprofen were about 100% and 80%, respectively. Based on the systemic exposure of (S)-(+)-ibuprofen, it could be concluded that the pharmacological actions might be similar when ibuprofen arginate was given orally and intravenously, except some differences in the onset of action. Key words: ibuprofen arginate; enantiomer; chiral HPLC; pharmacokinetics CLC number: R969 Document code: A Article ID: (2012) 布洛芬精氨酸注射及口服给药在大鼠体内的立体选择性药代动力学 * 王晓琳, 韩静, 张丹, 刘会臣 ( 航天中心医院临床药理室, 北京 ) 摘要 : 利用手性高效液相色谱法研究大鼠注射及口服布洛芬精氨酸之后布洛芬对映体的药代动力学 布洛芬精氨酸注射及口服给药后, 布洛芬对映体的药代动力学呈现立体选择性, 且口服给药后立体选择性程度更高 与系统前转化相比, R- 布洛芬至 S- 布洛芬的系统转化在口服给药后立体选择性药代动力学中起更重要的作用 布洛芬精氨酸口服给药后, 优势对映体 S- 布洛芬迅速吸收, S- 布洛芬与 R- 布洛芬的绝对生物利用度分别为 100% 和 80% 基于研究发现的 S- 布洛芬体内系统性暴露, 可以推断布洛芬精氨酸注射及口服给药后的药理作用相似, 仅在作用的起始阶段存在差异 关键词 : 布洛芬精氨酸 ; 对映体 ; 手性高效液相色谱 ; 药代动力学 Ibuprofen is one of the most widely used 2- arylpropionic acid (2-APA) nonsteroidal anti-inflammatory drugs (NSAID) due to its antipyretic, analgesic and anti-inflammatory effects with few adverse events [1]. Ibuprofen contains a chiral carbon atom within the propionic acid side chain moiety and is usually Received , Accepted *Corresponding author Tel: , Fax: , liu-huichen@163.com marketed commercially as the racemic mixture of (S)-(+)- and (R)-( )-enantiomers. Actually, it is well recognized that the antiinflammatory activity is almost exclusively produced by (S)-(+)-ibuprofen (eutomer), which was also found less gastric toxicity than the racemate in analgesic and anti-inflammatory tests [2]. (R)-( )-ibuprofen (distomer) is bioinverted unidirectionally to its antipode [3 5]. The (R)-( )-to-(s)-(+) inversion may occur presystematically

2 WANG Xiao-lin, et al: Pharmacokinetics of ibuprofen enantiomers in rats after intravenous and oral administration of ibuprofen arginate 89 and systematically, and the systematic inversion contributes predominantly to the total inversion when ibuprofen is rapidly absorbed [6 8]. The extent of (R)- ( )-to-(s)-(+) inversion is absorption-rate-dependent, and modifications on dosage form may affect the extent of inversion and bioavailability of (S)-(+)-ibuprofen [9]. Ibuprofen arginate is a salt formulation of ibuprofen designed to reach target concentrations rapidly. The administration of the soluble granular formulation of ibuprofen arginate resulted in very rapid absorption of the (S)-(+)-ibuprofen. When racemic ibuprofen was administered, t max of the (S)-(+)-isomer given in soluble granular formulation was much shorter than that of the isomer given in conventional oral solid formulations, such as capsules or tablets [10]. Furthermore, the antiinflammatory effects of ibuprofen can be enhanced by L-arginine, and the gastrointestinal side effects of ibuprofen can be counteracted by L-arginine, because L-arginine is the substrate of the nitric oxide (NO) synthesizing enzymes, They promote the generating of NO, which is a short-lived messenger owning much biological functions [11]. The injection of ibuprofen arginate provides a therapeutic option for patients who have difficulty in swallowing oral agents. This study was conducted to compare the pharmacokinetic stereoselectivity of ibuprofen after intravenous and oral administration of ibuprofen arginate and provide primary references for the interpretation of pharmacological phenomena. Materials and methods Chemicals Ibuprofen arginate (purity > 99.0%) and its injection (ph 7.4) were prepared in our laboratory. 100 mg ml 1 racemic ibuprofen (mixture of 50% (R)-( )- and 50% (S)-(+)-ibuprofen) were dissolved in saline for injection. (S)-(+)-ibuprofen (purity > 99.8%) was purchased from the Suzhou No.4 Pharmaceutical Factory (Suzhou, China). Naproxen (internal standard, IS, purity > 99.5%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 2-Propanol and n-hexane (Fisher, USA) were of HPLC grade. Ultrapure water (18.3 MΩ cm, 25 ) was prepared by deionized water passing through the A-K Lab Pure Water System. Other chemicals such as sodium dihydrogen phosphate, anhydrous sodium acetate, sodium hydroxide and glacial acetic acid were of analytical grade. Animals and drug administration Male Sprague-Dawley (SD) rats, weighing g, were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). Permission number of experimental animal production was SCXK-(military) The animals were fasted for 12 h with free access to water before administration of ibuprofen arginate. Sixteen rats were randomly divided into two groups based on their body weight and administered by oral or by intravenous injection into the caudal vein at 20 mg kg 1 of ibuprofen. Blood samples (about 0.3 ml) were collected from orbital canthus vein before drug administration and post-dose at 2 (intravenously dosed rats only), 5, 15, 30, 45 (orally dosed rats only), 60, 90, 120, 240, 360, 480 and 600 min. Plasma was separated by centrifugation (4, r min 1, 10 min) and stored at 70 until analysis. Sample preparations Liquid-liquid extraction was applied to extract the analytes from the plasma samples. Frozen plasma samples were thawed at room temperature and vortex-mixed briefly. An aliquot (100 µl) of plasma was transferred to a glass tube followed by IS working solution (20 μg ml 1, 10 µl), then 100 µl of 0.1 mol L 1 sodium acetate (ph 4.0, adjusted with glacial acetic acid) was added, and then the mixture was vortexed for 30 s. This mixture was added by 3 ml of n-hexane/2-propanol (95 5, v/v), and vortexed for 2 min, then centrifuged at r min 1 for 5 min. The organic phase was transferred to another glass tube and evaporated to dryness at 40 under a gentle stream of nitrogen. The residue was reconstituted in 100 µl of NaH 2 PO 4 (0.1 mol L 1, ph 7.0) and 20 µl was injected into the HPLC system. HPLC analysis Shimadzu Prominence 20A (Shimadzu Corporation, Tokyo, Japan) HPLC system includes two LC-20 AT solvent delivery modules, a DGU-20A 3 on-line degasser, an SIL-20A autosampler, a CTO-20A column oven, an SPD-M20A photodiode array detector (PDA), and a CBM-20A system controller. Separation of ibuprofen enantiomers was achieved using a Chiral-AGP column (100 mm 4 mm, 5 µm, Daicel, Tokyo, Japan) fitted with a Chiral-AGP guard column (10 mm 3 mm, 5 µm, Daicel, Tokyo, Japan) at a flow rate of 0.5 ml min 1. The mobile phase was 0.1 mol L 1 NaH 2 PO 4 (ph 7.0) containing 0.25% 2-propanol (v/v). The column temperature was maintained at 30, and the PDA detector was adjusted over the range of nm (219 nm for ibuprofen and naproxen). Method validation Specificity was assessed by

3 90 药学学报 Acta Pharmaceutica Sinica 2012, 47 (1): assay of blank plasma from six rats without given any drug. Linear calibration curves were obtained by plotting the peak area ratios of analytes to IS against the concentration over the range of μg ml 1 of analytes in plasma. The accuracy (relative error, RE) and precision (relative standard deviation, RSD) were determined by analyzing the two enantiomers in plasma samples with three concentrations (1, 8 and 80 μg ml 1 ), and the lower limit of quantitation (LLOQ) was defined as the concentration below which RE was > ±15% or the inter-day RSD > 15%. The extraction recovery of analytes was determined by comparing the peak areas of analytes in extracted standard samples with mean peak areas of analytes obtained by direct injection of standard solutions of analytes and IS at corresponding concentrations. Ibuprofen was stable in human plasma after three cycles of freezing and thawing, long-term storage at low temperature and storage for 16 h at ambient temperature, and it was also stable in reconstitution solutions after liquid-liquid extraction for 24 h [12, 13], so the stability of ibuprofen was not tested in this experiment. Pharmacokinetic analysis Non-compartmental analysis was performed on the calculation of the pharmacokinetic parameters of (S)-(+)- and (R)-( )- ibuprofen for each rat. The maximum plasma concentration (C max ) and the time required to reach C max (t max ) were directly obtained from the raw data. The area under the plasma concentration-time curve (AUC 0 t ) was obtained by the linear trapezoidal rule up to the last sampling point with detectable levels. The AUC 0 t was extrapolated to infinity (AUC 0 ) by adding the quotient of last measurable plasma concentration (C t ) and the terminal elimination rate constant (K e ). K e was determined by linear least squares regression of the terminal portion of the plasma log-transformed concentration-time curve and the corresponding elimination half-life (t 1/2 ) was calculated as 0.693/K e. The clearance (CL) for intravenous administration and CL/F for oral administration were derived from the ratio dose/auc 0. The distribution volume (V) for intravenous administration and V/F for oral administration were estimated by the ratio CL/K e and CL/F/K e, respectively. The bioavailability was calculated as the following equation: AUC 0 ( ) = po F 100% (1) AUC 0 (iv ) Statistical analysis All pharmacokinetic results were expressed as mean ± standard deviation. Statistical differences between the two enantiomers after oral or intravenous administration were determined using paired t-test, and grouped t-test was used to compare difference in each enantiomer between two administration routes. Statistical analyses were performed using SAS V8 (SAS Institute Inc., USA). The value of P < 0.05 was considered statistically significant. Results and discussion 1 Method validation Representative chiral chromatograms of blank rat plasma, rat plasma spiked with 16 μg ml 1 of ibuprofen and IS, rat plasma samples obtained after a single intravenous and oral dose of ibuprofen arginate were shown in Figure 1. No detectable interference was found in any samples analyzed. The enantiomeric elution order was decided by the assay of the rat plasma spiked with (S)-(+)-ibuprofen. The retention times for (R)-( )-ibuprofen, (S)-(+)-ibuprofen and IS were about 6.7, 8.4 and 11.4 min, respectively. The typical regression equations for (S)-(+)-ibuprofen and (R)-( )-ibuprofen were y = x (r= ) and y = x (r= ). The lower limit of quantitation (LLOQ) was 0.5 μg ml 1 for the two enantiomers with RE within ±15% and RSD lower than 15%. The precision, accuracy and extraction recovery results of analytes were presented in Table 1. Intra- and inter-day precisions as RSD were <15% and accuracies as RE were within ±15% for ibuprofen enantiomers. The mean extraction recoveries of (S)-(+) and (R)-( )-ibuprofen were (83.7 ± 2.2) % and (83.3 ± 1.9) %, respectively. The extraction recovery of IS was (81.8 ± 2.8) %. Table 1 Precision, accuracy (n = 6, 3 days) and extraction recovery (mean ± SD, n = 6) for the determination of ibuprofen enantiomers in rat plasma Concentration /µg ml 1 Enantiomer Intra-day precision RSD/% Inter-day precision Accuracy RE/% Extraction recovery /% 1.0 (low) (S)-(+) ± 0.6 (R)-(-) ± (middle) (S)-(+) ± 1.4 (R)-(-) ± (high) (S)-(+) ± 2.1 (R)-(-) ± Stereoselective pharmacokinetic results The plasma concentration-time curves of (S)-(+)- and (R)-( )-ibuprofen in SD rats after a single intravenous

4 WANG Xiao-lin, et al: Pharmacokinetics of ibuprofen enantiomers in rats after intravenous and oral administration of ibuprofen arginate 91 Figure 2 Plasma concentration-time profiles of ibuprofen enantiomers in rats after a single (A) intravenous and (B) oral dose of ibuprofen (20 mg kg 1 ). Data were shown as means ± SD (n = 8) Figure 1 Representative chromatograms of (A) standard solution containing racemic ibuprofen (2 μg ml 1 ) and IS, (B) extract from blank rat plasma, (C) extract from blank rat plasma spiked with ibuprofen (16 μg ml 1 ) and IS, (D) extract from a plasma sample collected from a rat 30 min after a single intravenous dose of ibuprofen (20 mg kg 1 ), and (E) extract from a plasma sample collected from a rat 30 min after a single oral dose of ibuprofen (20 mg kg 1 ). Peak I: (R)-( )-ibuprofen; Peak II: (S)-(+)-ibuprofen; Peak III: IS and oral dose of ibuprofen arginate (equivalent to 20 mg kg 1 ibuprofen) were shown in Figure 2. The pharmacokinetic parameters of ibuprofen enantiomers were listed in Table 2. 3 The comparison of (S)-(+)- and (R)-( )-ibuprofen The pharmacokinetics of ibuprofen was stereoselective after intravenous and oral administration of ibuprofen arginate, and the AUC values were significantly greater for (S)-(+)-ibuprofen than for (R)-( )- ibuprofen. In view of the preferred metabolism of (S)-(+)-ibuprofen [8], the greater AUC values and lower CL/F values of (S)-(+)-ibuprofen were mainly due to the unidirectional inversion of (R)-( )-ibuprofen to its antipode [14]. The t max of (R)-( )-ibuprofen was significantly shorter than that of (S)-(+)-ibuprofen, which is probably due to the higher affinity of (R)-( )- ibuprofen with plasma protein [15] and the inversion of (R)-( )-ibuprofen. 4 The comparison of intravenous and oral administration The predominant differences in pharmacokinetic parameters (except t max and C max ) between the two administration routes existed in the AUC, t 1/2 and V (or V/F) of (R)-( )-ibuprofen, but the P values were close to 0.05 (P = 0.04 for all parameters). The active (S)-(+)-ibuprofen presented similar values for two routes. The (S)-(+)/(R)-( ) ratio of the AUC values of ibuprofen enantiomers differed significantly between

5 92 药学学报 Acta Pharmaceutica Sinica 2012, 47 (1): Table 2 The pharmacokinetic parameters of ibuprofen enantiomers in rats after a single intravenous and oral dose of 20 mg kg 1 ibuprofen (mean ± SD, n = 8). * P < 0.05, ** P < 0.01, *** P < vs (S)-(+)-ibuprofen; # P < 0.05 vs intravenous administration Parameter Intravenous administration Oral administration (S)-(+)-ibuprofen (R)-( )-ibuprofen (S)-(+)-ibuprofen (R)-( )-ibuprofen t max /min ± ± 3.54 * C max /mg L ± ± *** AUC 0 t /mg min L ± ± *** ± ± ***# AUC 0 /mg min L ± ± *** ± ± ***# t 1/2 /min ± ± 3.59 ** ± ± ***# CL or CL/F /ml min kg ± ± 3.11 *** 3.37 ± ± 4.00 *** V or V/F /ml kg ± ± ± ± # F /% ± ± 8.98 the two administration routes (P = 0.03; 4.32 ± 0.69 for intravenous administration, 5.32 ± 1.01 for oral administration), and it indicated that the pharmacokinetic stereoselectivity was higher after oral administration, which might be due to that there was only systematic (R)-( )-to-(s)-(+) inversion occurred after intravenous administration. It was also shown that the presystematic and systematic inversion both existed and the latter might play a more important role in the pharmacokinetic stereoselectivity after oral administration of ibuprofen arginate. 5 Chiral inversion Following oral administration of ibuprofen arginate, the absolute bioavailability of (R)-( )-ibuprofen was about 80%. There is less than 20% of (R)-( )-ibuprofen inverted to its antipode during the absorption course, which agreed with the previously reported value for the conversion of (R)-( )-ibuprofen in human [16], since (R)- ( )-ibuprofen would also undergo other presystematic metabolism via glucuronidation and oxidation [17]. 6 Rate of absorption Oral administration of ibuprofen arginate resulted in very rapid absorption of (S)-(+)-ibuprofen with t max less than 20 min and C max near to the concentration (about 68 μg ml 1 ) at 2 min after intravenous administration. This characteristic was also found in human subjects [6], which might be especially favorable when a very rapid analgesic effect is needed. Conclusions In the present study, the pharmacokinetics of ibuprofen enantiomers in rats after intravenous administration of ibuprofen arginate were firstly reported, and compared with those after oral administration. Based on the systematic exposure of (S)-(+)-ibuprofen (eutomer) found, it was concluded that the pharmacological actions might be similar when ibuprofen arginate was given orally and intravenously except some differences in the onset of action. References [1] Rainsford KD. Ibuprofen: pharmacology, efficacy and safety [J]. Inflammopharmacology, 2009, 17: [2] Bonabello A, Galmozzi MR, Canaparo R, et al. Dexibuprofen (S(+)-isomer ibuprofen) reduces gastric damage and improves analgesic and antiinflammatory effects in rodents [J]. Analg, 2003, 97: Anesth [3] Yang C, He W, Kong M, et al. Use of high-field nuclear magnetic resonance spectroscopy for the analysis of stereoselective metabolites of ibuprofen (R)-enantiomer in rat urine [J]. Pharm Sin ( 药学学报 ), 2000, 35: Acta [4] Kaiser DG, Vangiessen GJ, Reischer RJ, et al. Isomeric inversion of ibuprofen (R)-enantiomer in humans [J]. Sci, 1976, 65: J Pharm [5] Hao H, Wang G, Sun J, et al. Unidirectional inversion of ibuprofen in Caco-2 cells: developing a suitable model for presystemic chiral inversion study [J]. Biol Pharm Bull, 2005, 28: [6] Sádaba B, Campanero MA, Muñoz-Juarez MJ, et al. A comparative study of the pharmacokinetics of ibuprofen arginate versus dexibuprofen in healthy volunteers [J]. Clin Pharmacol, 2006, 62: Eur J [7] Ding G, Liu Y, Sun J, et al. Effect of absorption rate on pharmacokinetics of ibuprofen in relation to chiral inversion in humans [J]. J Pharm Pharmacol, 2007, 59: [8] Hao H, Wang G, Sun J. Enantioselective pharmacokinetics of ibuprofen and involved mechanisms [J]. 2005, 37: Drug Metab Rev,

6 WANG Xiao-lin, et al: Pharmacokinetics of ibuprofen enantiomers in rats after intravenous and oral administration of ibuprofen arginate 93 [9] Jamali F, Singh NN, Pasutto FM, et al. Pharmacokinetics of ibuprofen enantiomers in humans following oral administration of tablets with different absorption rates [J]. Pharm Res, 1988, 5: [10] Fornasini G, Monti N, Brogin G, et al. Preliminary pharmacokinetic study of ibuprofen enantiomers after administration of a new oral formulation (ibuprofen arginine) to healthy male volunteers [J]. Chirality, 1997, 9: [11] De Palma C, Di Paola R, Perrotta C, et al. Ibuprofen-arginine generates nitric oxide and has enhanced anti-inflammatory effects [J]. Pharmacol Res, 2009, 60: [12] Zhou H, Liang W, Hu P, et al. Method of quantitative determination of ibuprofen and pseudoephedrine in plasma by liquid chromatography-tandem mass spectrometry [J]. J Chin Mass Spectrom Soc ( 质谱学报 ), 2004, 25: [13] Li L, Duan X, Liu Q, et al. Rapid determination of ibuprofen in human plasma by liquid chromatography-tandem mass spectrometry method [J]. Chin Pharm J ( 中国药学杂志 ), 2008, 43: [14] Jamali F, Mehvar R, Russell AS, et al. Human pharmacokinetics of ibuprofen enantiomers following different doses and formulations: intestinal chiral inversion [J]. J Pharm Sci, 1992, 81: [15] Itoh T, Maruyama J, Tsuda Y, et al. Stereoselective pharmacokinetics of ibuprofen in rats: effect of enantiomerenantiomer interaction in plasma protein binding [J]. Chirality, 1997, 9: [16] Cheng H, Rogers JD, Demetriades JL, et al. Pharmacokinetics and bioinversion of ibuprofen enantiomers in humans [J]. Pharm Res, 1994, 11: [17] Tan SC, Patel BK, Jackson SH, et al. Stereoselectivity of ibuprofen metabolism and pharmacokinetics following the administration of the racemate to healthy volunteers [J]. Xenobiotica, 2002, 32: 化学生物学与药物研究 专刊出版预告 为了促进国内外化学生物学与药物研究的学术交流, 为相关研究人员提供展示最新研究成果的平台, 药学学报 将于 2012 年 3 月 ( 第 3 期 ) 出版一期 化学生物学与药物研究 专刊, 该专刊是由本刊编委会与编辑部共同策划和组织完成的, 计划刊发稿件 20 多篇 专刊中大多数稿件是约请本领域中具有一定学术影响力的专家撰写的综述和研究论文, 敬请读者关注 专刊还特别邀请中国医学科学院药物研究所王晓良教授和南京大学鞠熀先教授作特约编辑, 在此也对王教授和鞠教授的大力协助表示感谢 药学学报 编辑部