- Email: [email protected]
Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of pioglitazone enantiomers in rats
Journal of Chromatography B, 951–952 (2014) 143–148
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of pioglitazone enantiomers in rats Bin Du a , Li Pang a , Yanhua Yang a , Guopeng Shen b,∗ , Zhenzhong Zhang a,∗ a b
School of Pharmaceutical Sciences, Zhengzhou University, 100 Science Road, Zhengzhou 450001, PR China School of Chemical Engineering and Energy, Zhengzhou University, 100 Science Road, Zhengzhou 450001, PR China
a r t i c l e
i n f o
Article history: Received 3 November 2013 Accepted 10 January 2014 Available online 16 January 2014 Keywords: Pioglitazone Enantiomer separation Pharmacokinetics HPLC
a b s t r a c t A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the pioglitazone enantiomers in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation of pioglitazone enantiomers and internal standard (I.S., dexamethasone acetate) was achieved on a cellulose tris (3,5-dichlorophenylcarbamate) column known as Chiralpak IC with a mobile phase of hexane–isopropanol (70:30, v/v) at a flow rate of 1.0 mL/min. The ultraviolet (UV) detection wavelength was set at 225 nm. Baseline separation of pioglitazone enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Linear calibration curves were obtained over the range of 0.25–50 g/mL in plasma for both enantiomers (R2 > 0.9990) with quantitation limit of 0.25 g/mL. The mean extraction recoveries were 82.37–91.38% for pioglitazone enantiomers and 95.76% for I.S. from rat plasma. The mean relative error (R.E. %) of accuracy and the mean relative standard deviation (R.S.D. %) of intra-day and inter-day precision for both enantiomers were <10%. The method was validated with accuracy, precision, recovery and stability and used to determine the pharmacokinetics of pioglitazone enantiomers, after a single oral administration of racemic pioglitazone (30 mg/kg). The differences between the pharmacokinetic parameters Cmax , AUC0–24 , AUC0–∞ , CL/F of (+)-pioglitazone and (–)-pioglitazone were significant, suggesting that the disposition of pioglitazone in rats may be enantioselective. Moreover, the plasma levels of (+)- and (–)-pioglitazone in female rats were apparently higher than that in male rats, respectively. © 2014 Published by Elsevier B.V.
1. Introduction Pioglitazone (PIO) is an oral anti-hyperglycemic agent that acts primarily by activating peroxisome proliferators–activator receptor gamma (PPAR-␥) [1]. It is used as monotherapy or in combination with sulfonylurea or insulin in the management of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus, NIDDM) [2–4]. The chemical structure of PIO was shown in Fig. 1, with one chiral centre in the thiazolidinedione ring which was attached to a carbonyl group. It is a relatively new member of the thiazolidinedione class and commercially available as the hydrochloride salt. Investigations have shown that PIO was used as the racemic mixture clinically [5]. Examination of literature reveals that the primary method for PIO enantioseparation in vitro is capillary electrochromatography (CEC) [5,6]. In addition, several nonstereoselective analytical methods have been described for quantitative determination of racemic PIO in biological fluids, including HPLC-UV with the run time of
∗ Corresponding authors. Tel.: +86 371 67781902; fax: +86 371 67781908. E-mail addresses: [email protected] (G. Shen), [email protected] (Z. Zhang). 1570-0232/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2014.01.013
less than 20 min [7–9] and LC-MS/MS with a lower limit of quantification (LLOQ) [10,11]. However, the literature on stereoselective pharmacokinetic study of PIO enantiomers has not been reported at present. It is necessary to realize that the safety and efficacy data for a drug evaluated as a mixture of enantiomers are still valid. In this paper, we were interested to develop a simple, specific and accurate chiral HPLC method for quantitative determination of (+)-, (−)-PIO in plasma and its application to stereoselective pharmacokinetics study in rats. According to the report in the literature [12], there are sex differences in the plasma levels of PIO racemate in rats. To our knowledge, no comparison of the pharmacokinetics of PIO enantiomers in male and female rats was reported up to now. Therefore we studied and analyzed the sex differences in pharmacokinetics of pioglitazone enantiomers. 2. Materials and methods 2.1. Chemicals and reagents The racemic pioglitazone reference substance (purity 99.5%) and dexamethasone acetate (IS, purity 99.0%, see Fig. 1) were supplied by Henan Provincial Institute of Food and Drug Control
144
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
Fig. 1. The chemical structures of (A) PIO and (B) dexamethasone acetate.
(Zhengzhou, China). HPLC grade n-hexane, isopropanol, ethanol and ethyl acetate of analytical reagent grade were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). All other reagents purchased from Tianjin Tjshield Chemicals Co., Ltd (Tianjin, China) were of analytical reagent grade. 0.45 m pore size filers (Millipore, MA, USA) were used to filter the mobile phase and solutions. 2.2. Animals Age-matched male (240–270 g) and female (220–250 g) Sprague-Dawley (SD) rats were obtained from the Zhengzhou University Medical Laboratory Animal Center (Zhengzhou, China). Animals were housed under good laboratory conditions (temperature 25 ± 2◦ C, 50 ± 20% relative humidity) and allowed to acclimatize for at least 1 week before initiation of studies. Prior to the drug administration, rats were fasted overnight (12 h) and had free access to water throughout the experimental period. The handling and treatments of all animals used in this study were in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). The animal use and care protocol was reviewed and approved by the ethics committee of the Zhengzhou University. 2.3. Chromatographic conditions Chromatographic studies were performed on Agilent 1100 HPLC system (Agilent, Palo Alto, CA, USA) equipped with autosampler, thermostated-column device and a variable-wavelength UV Detector. The chromatographic separations were carried out on a Chiralpak IC column (250 mm × 4.6 mm i.d., 5 m, from Daicel Chemical Industries Ltd., Tokyo, Japan). The mobile phase was hexane-isopropanol (70:30, v/v). All separations were performed isocratically at a flow rate of 1.0 mL/min with UV detection at 225 nm. Column temperature was maintained at 35 ± 1 ◦ C. The injection volume was 20 L. Agilent ChemStation® software was used for data acquisition and integration.
centrifuge tubes. Ethanol (20 L) and the I.S. solution (30 L) of 50 g/mL were added and vortex-mixed for 1 min. The mixture was extracted with ethyl acetate (1 mL) using a vortex mixer for 3 min and then centrifuged at 3000 × g for 10 min. The organic layer (900 L) was transferred into clean test tubes and evaporated to dryness at 35 ◦ C using a gentle stream of nitrogen. The residue was reconstituted with 100 L ethanol and mixed by vortexing for 5 min, and then centrifugated at 12000 × g for 15 min at 4 ◦ C. Aliquot (20 L) was injected into HPLC system for analysis.
2.6. Bioanalytical method validation 2.6.1. Calibration curve Blank plasma samples were spiked with 20 L of racemic pioglitazone working standard solutions and 30 L of I.S. working solution in order to generate concentrations of the enantiomers ranging from 0.25 to 50 g/mL. The calibration samples were prepared as described above. The concentration of each isomer was looked upon as half of the racemic concentration since the two enantiomers were almost the same in the peak area. The calibration curves were obtained by plotting the peak area ratios of each enantiomer and I.S. versus the concentration of enantiomer spiked in the samples by least-squares linear regression analysis. Leastsquares linear regression analysis was performed using Microsoft® Excel. The limit of quantitation (LOQ) for each enantiomer was considered as the concentration that produced a signal-to-noise (S/N) ratio of 10.
2.6.2. Extraction recovery The recovery of each enantiomer was determined by comparing the peak area ratios of the analytes to I.S. in the regularly pre-treated QC samples at three concentration levels (five samples each) with those of spiked post-extraction samples. Similarly, the recovery of I.S. was determined at 50 g/mL [13]. The absolute recoveries of the analytes and I.S. need not be 100%, but the extent of recovery should be consistent, precise and reproducible.
2.4. Preparation of stock and standard solutions The stock solutions of racemic PIO and I.S. were prepared in ethanol (1.0 mg/mL). A series of standard working solutions of racemic PIO were prepared by further dilution of the stock solution with ethanol to achieve concentrations ranging from 5 to 500 g/mL for racemic PIO. The I.S. working solution (50 g/mL) was prepared by the dilution of the stock dexamethasone acetate solution with ethanol. Quality control (QC) working solutions of 1.5, 8.0 and 80.0 g/mL were prepared in the same way as that of the standard working solutions. All the solutions were stored at 4 ◦ C and brought to room temperature before use. 2.5. Preparation of samples Sample preparation was carried out by liquid–liquid extraction. Aliquots (150 L) of plasma sample were pipetted into 1.5 mL
2.6.3. The accuracy and precision Intra- and inter-day precision and accuracy of the method were evaluated, based on the data from QC plasma samples at three different concentrations (1.5, 8.0 and 80 g/mL) in five replicates. The precision and accuracy were assessed by analyzing five QC samples at each concentration within a day and mean values of five replicates with the same concentrations on five consecutive days, respectively. Accuracy was expressed as percent relative error (R.E. %) [(measured concentration − spiked concentration)/spiked concentration] × 100% [14], while precision was quantitated by calculating intra- and inter-relative standard deviation values (R.S.D. %). The criteria for acceptability of the data included an accuracy within ±15% relative error from the nominal values and a precision of within 15% R.S.D except for LOQ, where it should not exceed ±20% [15].
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
2.6.4. Stability The stability of PIO enantiomers in rat plasma was tested using QC samples at three different concentrations (1.5, 8 and 80 g/mL), all three copies. Stability of each enantiomer in the biomatrix during 6 h (bench-top) was determined at ambient temperature (25 ± 2 ◦ C). Freezer stability was assessed by analyzing the QC samples stored at −20 ◦ C for at least 30 days. The freeze-thaw stability was determined over three cycles within 3 days. In each freeze-thaw cycle, the spiked plasma samples were frozen for 24 h at −20 ◦ C and thawed at room temperature. The samples were processed using the same procedure as described in the sample preparation section. Residue of racemate PIO was kept for 15 days at −20 ◦ C to evaluate residue stability, before sample injection analysis. For the acceptance criterion of stability, the R.S.D. % compared to the freshly prepared standard should be within 15%. 2.7. Preliminary pharmacokinetic study in rats The validated method was applied to a pharmacokinetic study of PIO in healthy male and female SD rats (5 animals per group). Two commercially available tablets labeled by containing 15 mg pioglitazone hydrochloride were grinded and the contents were suspended in 0.5% CMC-Na solution to produce concentration of 3 mg/mL. Each rat was given a single oral dose of 30 mg/kg by oral gavage after overnight fasting. Immediately before each administration the suspension was vortexed for few seconds. Water was available at all times during the experiment. Whole blood (400 L) was collected via the orbital venous plexus from rats into heparinized centrifuge tubes at 0.3, 0.5, 1, 2, 4, 6, 8, 10, 12, 18 and 24 h following administration. The blood was immediately centrifuged at 3000 × g for 5 min and plasma (150 L) was transferred into tagged centrifuge tubes. The plasma samples were analyzed immediately or stored at −20 ◦ C. 2.8. Data analysis Pharmacokinetic parameters were calculated by noncompartmental approach using DAS 3.0 (Bozhiyin T&S Co., Ltd, Beijing, China). The maximum plasma concentration (Cmax ) and the time to reach maximal plasma concentration (Tmax ) were obtained directly from raw data. Other noncompartmental model pharmacokinetic parameters were obtained from the DAS output. All statistical analyses were done with IBM SPSS Statistics 19 (Inc., Chicago, IL, USA). Data are expressed as the mean values ± standard deviation (S.D.). Statistical significance of the differences in pharmacokinetic parameters between two enantiomers was analyzed by paired student’s t test. P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Effects of assay conditions on the separation In this study, the baseline resolution of PIO enantiomers and the I.S. in rat plasma were achieved in normal phase liquid chromatography using the Chiralpak IC column, which is a cellulose tris (3,5-dichlorophenylcarbamate) - based stationary phase on silica. The effect of mobile phase composition on the separation, together with the effects of column temperature and flow rate, was investigated to choose the optimum separation conditions. Different combinations of hexane, ethanol and isopropanol were tested to select the mobile phase that would give an optimum separation and selectivity for PIO enantiomers. As organic modifier, the percentage of isopropanol in the mobile phase had a stronger effect on the separation than ethanol. With decreasing the percentages of the isopropanol in the mobile phase from 50% to 20%, the chromatographic run time and resolution increased from 7.36 to
145
42.47 min and from 2.51 to 4.20, respectively. When the percentage of isopropanol was more than 30%, retention time was shortened, but the impurity peak interfered the separation of PIO enantiomers and the I.S. When the percentage of isopropanol was 20%, retention time exceeded 40 min. Therefore, the mobile phase was decided to be n-hexane and isopropanol (70:30, v/v). The effect of column temperature ranging from 20 to 35◦ C was investigated on the PIO enantiomers separation. Across the tested temperature range, a raise in temperature shortened the elution time from 34.74 to 22.47 min and reduced the column pressure, however the resolution was decreased from 4.20 to 3.43. In order to maintain high resolution and avoid long analysis time and high column pressure, the column temperature was set at 35◦ C. Under the optimum mobile phase and column temperature conditions, the influence of flow rate was examined by changing from 0.6 to 1.2 mL/min. It was found that the high flow rate resulted in relatively low resolution and short analysis time. As the assay was being developed and optimized for the rapid quantitative determination of PIO enantiomers, cutting the run time becomes very important. Therefore, with regards to the right balance of resolution and elution time, the flow rate was set at 1.0 mL/min. 3.2. Specificity and chromatography Under the optimal conditions mentioned above, the peaks of I.S. and (−)-PIO, (+)-PIO were well resolved in rat plasma, with retention times of 11.87, 18.67 and 22.47 min, respectively. The order of elution was determined by using a Polar 3001 Automatic Polarimeter (Optical Activity Ltd, Cambridgeshire, UK). Representative chromatograms of rat blank plasma, rat blank plasma spiked with standard solution of PIO racemate and the I.S., and plasma sample obtained at 2 h and 24 h after oral administration were shown in Fig. 2A–D, respectively. Comparison of blank plasma (Fig. 2A) with blank plasma spiked with PIO racemate and I.S. (Fig. 2B) showed that no endogenous peak from plasma was found to interfere with the elution of PIO enantiomers or the I.S. Similarly, no interfering peaks were observed in the chromatograms of plasma sample obtained at 2 h and 24 h after a 30 mg/kg oral dose of PIO racemate (Fig. 2C and D). This has indicated that appropriate selectivity of the elaborated procedure and a stereoselective HPLC method with good specificity was developed. 3.3. Assay validation 3.3.1. Calibration curve and recovery Peak area ratios of each enantiomer of PIO to the I.S. were measured and acted as a surrogate for quantitation. The results were fitted to linear regression analysis without the use of a weighting factor as well as weighting factor. The weighting options did not show any improvement in the performance of standard curve and quality control samples. Therefore, we have opted to the linear regression without weighting factor for validation of the analytical data. The mean linear regression equations of (−)-PIO and (+)PIO, obtained by a least-squares method, were y = 0.1434x + 0.0082, y = 0.1447x + 0.0883, respectively (y is the peak area ratio of the enantiomer to I.S. and x is the concentration of each enantiomer in plasma) over a concentration range of 0.5–100 g/mL racemic PIO added to rat plasma which is equivalent to 0.25–50 g/mL of each enantiomer. The correlation coefficient (R2 ) of the calibration curve generated during the validation was 0.9998 for both enantiomers, demonstrating linearity over the entire standard range tested. The LOQ of this assay was 0.25 g/mL for both isomers at a signal-to-noise ratio of about 10. The absolute recovery for individual enantiomer was estimated at three different concentrations (1.5, 8 and 80 g/mL of PIO racemate in plasma). The mean percentage recoveries for two
146
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
Fig. 2. Typical chromatograms of rat blank plasma (A), rat blank plasma spiked with standard solution (80 g/mL) of PIO racemate and the I.S. (50 g/mL) (B), plasma sample (8.35, 9.58 g/mL for (−)-, (+)-PIO, respectively) obtained at 2 h after a single dose of 30 mg/kg of racemic (C) and plasma sample (0.34, 0.81 g/mL for (−)-, (+)-PIO, respectively) obtained at 24 h after a single dose of 30 mg/kg of racemic (D). 1: (−)-PIO; 2: (+)-PIO; IS: internal standard substance.
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
147
Table 1 Precision and accuracy for the determination of PIO enantiomers in plasma (n = 5). Analyte
Added (g/mL)
Found (g/mL)
Relative error (%)
Intra-day R.S.D (%)
Inter-day R.S.D (%)
(−)-PIO
0.75 4.00 40.00 0.75 4.00 40.00
0.68 ± 0.08 4.25 ± 0.15 42.37 ± 5.37 0.68 ± 0.11 4.16 ± 0.09 41.44 ± 5.85
−9.33 +6.25 +5.92 –9.33 +4.00 +3.60
6.24 5.43 3.40 4.73 4.48 2.45
3.22 3.08 1.68 4.86 4.73 1.68
(+)-PIO
enantiomers ranged from 82.37 ± 5.76% to 91.38 ± 2.50%. A mean percentage recovery of 95.76 ± 2.69% was obtained for the I.S. at 50 g/mL. 3.3.2. Accuracy and precision The results for accuracy and precision throughout the standard curve for (−)-PIO and (+)-PIO are presented in Table 1. The R.S.D. values of intra-day and inter-day precision for two enantiomers were less than 6.24%. The R.E. values of accuracy for two enantiomers varied between –9.33% and 6.25%, and between –9.33% and 4.00%, respectively. All the values of accuracy and precision including LOQ fell within the limits which were considered as acceptable. 3.3.3. Stability In this study, short-term, long-term, freeze-thaws and residue stabilities of two enantiomers in plasma were investigated using low-, middle- and high-quality control samples. The mean R.S.D.% values of PIO enantiomers in all stability tests were within 10%, demonstrating that plasma samples spiked with PIO enantiomers were stable on the bench-top for at least 6 h, at −20 ◦ C for at least 30 days, from −20 ◦ C to ambient temperature for at least three freeze/thaw cycles and PIO residue was stable at −20 ◦ C for at least 15 days. The concentrations of PIO enantiomers remained almost unchanged during stability experiments. These studies further suggested that rat plasma samples containing PIO enantiomers can be handled under normal laboratory conditions without significant conversion or racemization.
Table 3 Pharmacokinetic parameters of pioglitazone enantiomers following oral administration of racemic pioglitazone in five male rats (30 mg/kg). Paramters
(−)-PIO
Cmax (g/mL) Tmax (h) t1/2 (h) AUC0–24 (g h/mL) AUC0–∞ (g h/mL) CL/F (L/h kg)
5.59 2.60 6.97 43.52 47.60 0.64
± ± ± ± ± ±
(+)-PIO 1.81** 1.34 1.73 5.64** 5.35** 0.08**
10.17 2.80 6.75 86.94 95.35 0.32
± ± ± ± ± ±
1.86 1.10 3.07 5.29 3.22 0.01
Statistically significant difference versus (+)-PIO. ** P < 0.01 (mean ± S.D., n = 5).
plasma concentration of (+)-PIO was higher than that of (−)-PIO at each time point in female rats, as shown in Fig. 3A. The Cmax and AUC0–24 , AUC0–∞ of (+)-PIO were about 2.3 and 2.6, 3.4 times as high as those of (−)-PIO, respectively, indicating greater adsorption and distribution of (+)-PIO [16]. Compared with female rats, PIO enantiomers were relatively fast absorbed from tablets in male rats. The plasma concentrations of (−)-PIO and (+)-PIO reached Cmax at approximately 2.6 h and 2.8 h (Tmax ) after dosing and then decreased with a terminal phase t1/2 of about 6.97 h and 6.75 h, respectively. The mean plasma levels of (+)-PIO were found to be
3.4. Pharmacokinetics in rats After a single oral administration of 30 mg/kg racemic PIO to male and female SD rats, the plasma concentrations of two enantiomers were determined by the described method. The mean plasma concentration versus time profiles of both enantiomers of PIO in the female and male rats were illustrated individually in Fig. 3A and B respectively. Pharmacokinetic parameters of two enantiomers calculated by the non-compartmental approach in female and male rats were listed in Tables 2 and 3, respectively. The (−)-PIO and (+)-PIO were very slow absorbed from tablets in female rats resulting in the same Tmax value of 9.2 h. However, the (−)-PIO showed a faster elimination and a shorter t1/2 . The Table 2 Pharmacokinetic parameters of PIO enantiomers following oral administration of racemic PIO in five female rats (30 mg/kg). Parameters Cmax (g/mL) Tmax (h) t1/2 (h) AUC0–24 (g h/mL) AUC0–∞ (g h/mL) CL/F (L/h kg)
(−)−PIO 9.44 9.20 2.63 77.14 77.81 0.39
± ± ± ± ± ±
(+)−PIO **
2.77 1.78 0.71 5.09** 4.65* 0.02**
Statistically significant difference versus (+)−PIO. * P < 0.05. ** P < 0.01 (mean ± S.D., n = 5).
22.03 9.20 7.66 202.78 265.75 0.12
± ± ± ± ± ±
6.41 1.78 3.91 26.80 103.64 0.04
Fig. 3. The plasma concentrations (mean ± S.D.) time profile of (+)-PIO and (−)-PIO in female rats after receiving 30 mg/kg oral dose of racemic PIO (A). (N): (+)-PIO; (): (−)-PIO. The plasma concentrations (mean ± S.D.) time profile of (+)−PIO and (−)PIO in male rats after receiving 30 mg/kg oral dose of racemic PIO (B). (N): (+)-PIO; (): (−)-PIO.
148
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
higher than those of (−)-PIO in male rats, as shown in Fig. 3B. The Cmax , AUC0–24 , and AUC0–∞ of (+)-PIO were about 1.8, 2.0 and 2.0 times higher respectively than that of (−)-PIO, which indicates greater adsorption and distribution of (+)-PIO [16]. Though, for female and male rats, Tmax and t1/2 were not significantly different between (+)-PIO and (−)-PIO (P > 0.05), their Cmax , AUC0–24 , AUC0–∞ and CL/F were significantly different (P < 0.05 or P < 0.01), as shown in Tables 2 and 3. These results suggest that the disposition of PIO in rats is enantioselective. The enantiomer area ratios under the curve of plasma concentration versus time (AUCisomer (+) /AUCisomer (−) ) were more than 2.0, indicating that concentrations of the (+)PIO enantiomer were higher in plasma, which might be considered to have lower affinity for PPAR-␥ receptor [17]. Up to now, no report on differences of affinity at PPAR-␥ receptor of PIO enantiomers was available. However, there is the report on rosiglitazone, which also is one of the thiazolidinediones (TZDs) and acts by activating PPAR␥. The enantiomers of rosiglitazone showed differential activity at PPAR-␥ and (R) - (+) - rosiglitazone showed relatively weak affinity binding to PPAR-␥ [18]. So the reason caused stereoselectivity differences for PIO enantiomers may be different affinity at PPAR-␥ receptor. However, different metabolic rate or chiral inversion in rats could also lead to the stereoselectivity differences of PIO enantiomers in rats and further studies are required for elucidation of the detailed mechanisms. Compared with male rats, PIO enantiomers showed a slower adsorption and elimination in the female rats. Moreover, Cmax and AUC0–24 of PIO enantiomers were significantly higher in the female rats. Cmax of (−)-PIO and (+)-PIO in female rats were approximately 1.7 and 2.2 times than that in male rats. AUC0–24 of (−)-PIO and (+)PIO were 1.8 and 2.3 times higher in females than in males. These results suggested that there might be sex differences in pharmacokinetics of PIO enantiomers in rats, which were compatible with those described in the literature for PIO racemate [12]. The sex differences in some of cytochrome P450 (CYP) isoforms in rats have been reported, which may explain why the pharmacokinetics of PIO enantiomers are different in female and male rats [19–21]. 4. Conclusion A HPLC method was developed, validated and successfully applied to the simultaneous determination of PIO enantiomers in rat plasma and their stereoselective pharmacokinetics study in rats. Pharmacokinetic analysis showed that (+)-PIO had higher Cmax , AUC0–24 , AUC0–∞ than that of (−)-PIO after the oral administration of racemic PIO at 30 mg/kg, suggesting that the disposition of PIO in rats was enantioselective. These results presented here were a further contribution to the understanding of the kinetic behavior of PIO analogues. Nevertheless, further research needs to be carried out in order to ascertain the exact explanation for the observed pharmacokinetic stereoselectivity differences between PIO enantiomers in rats. By comparing and analyzing the pharmacokinetic
parameters of PIO enantiomers in male and female rats, we found that there were sex differences in the plasma levels of PIO enantiomers. Acknowledgements The author thanks Dr. J. Zhou for the helpful proposals and this work was financially supported by the National Natural Science Foundation of China (Grant No.81302717) and the Project of Zhengzhou Science and Technology Innovation Team (Grant No.2013sjxm020). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.01.013. References [1] K.S. Lakshmi, T. Rajesh, S. Sharma, IJPPS 1 (2009) 162–166. [2] J. Waugh, G.M. Keating, G.L. Plosker, S. Easthope, D.M. Robinson, Drugs 66 (2006) 85–109. [3] J. Chilcott, P. Tappenden, M. Lloyd, J.P. Wight, Clin. Ther. 23 (2001) 1792–1823. [4] B. Jamali, I. Bjørnsdottir, O. Nordfang, S.H. Hansen, J. Pharm. Biomed. 46 (2008) 82–87. [5] B. Jamali, G.C. Theill, L.L. Sørensen, J. Chromatogr. A 1049 (2004) 183–187. [6] B. Jamali, I. Bjoernsdottir, C. Cornett, S.H. Hansen, Electrophoresis 30 (2009) 2853–2861. [7] P. Venkatesh, T. Harisudhan, H. Choudhury, R. Mullangi, N.R. Srinivas, Biomed. Chromatogr. 20 (2006) 1043–1048. [8] M.S. Arayne, N. Sultana, A.Z. Mirza, J. Chromatogr. Sci. 49 (2011) 114–117. [9] P. Sripalakit, P. Neamhom, A. Saraphanchotiwitthaya, J. Chromatogr. B 843 (2006) 164–169. [10] Z.J. Lin, W. Ji, D. Desai-Krieger, L. Shum, Pharm. Biomed. Anal. 33 (2003) 101–108. [11] Y.H. Xue, K.C. Turner, J.B. Meeker, J. Pursley, M. Arnold, S. Unger, J. Chromatogr. B 795 (2003) 215–226. [12] Y. Fujita, Y. Yamada, M. Kusama, T. Yamauchi, J. Kamon, T. Kadowaki, T. Iga, Comp. Biochem. Physiol. C 136 (2003) 85–94. [13] Y.M. Lu, Z.M. Sun, Y.F. Zhang, X.Y. Chen, D.F. Zhong, J. Chromatogr. B 921 (2013) 27–34. [14] M.A. Radwan, H.H. Abdine, B.T. Al-Quadeb, H.Y. Aboul-Enein, K. Nakashima, J. Chromatogr. B 830 (2006) 114–119. [15] B. Du, L. Pang, H.Y. Li, S.J. Ma, Y. Li, X. Jia, Z.Z. Zhang, J. Chromatogr. B 932 (2013) 88–91. [16] S.S. Mustafa, R. Trivedi, N.V.S.R. Mamidi, R. Mullangi, N.R. Srinivas, J. Chromatogr. B 809 (2004) 23–30. [17] Z.Y. Hong, G.R. Fan, Y.F. Chai, X.P. Yin, J. Wen, Y.T. Wu, J. Chromatogr. B 826 (2005) 108–113. [18] V.L. Lanchote, A. Rocha, F.U.V. de Albuquerque, E.B. Coelho, P.S. Bonato, J. Chromatogr. B 765 (2001) 81–88. [19] D.J. Parks, N.C.O. Tomkinson, M.S. Villeneuve, S.G. Blanchard, T.M. Willson, Bioorg. Med. Chem. Lett. 8 (1998) 3657–3658. [20] Y. Imamura, M. Kaneko, H. Takada, M. Otagiri, H. Shimada, H. Akita, Comp. Biochem. Physiol. C 133 (2002) 587–592. [21] R. Kato, Y. Yamazoe, Toxicol. Lett. 64 (1992) 661–667.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of pioglitazone enantiomers in rats Bin Du a , Li Pang a , Yanhua Yang a , Guopeng Shen b,∗ , Zhenzhong Zhang a,∗ a b
School of Pharmaceutical Sciences, Zhengzhou University, 100 Science Road, Zhengzhou 450001, PR China School of Chemical Engineering and Energy, Zhengzhou University, 100 Science Road, Zhengzhou 450001, PR China
a r t i c l e
i n f o
Article history: Received 3 November 2013 Accepted 10 January 2014 Available online 16 January 2014 Keywords: Pioglitazone Enantiomer separation Pharmacokinetics HPLC
a b s t r a c t A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the pioglitazone enantiomers in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation of pioglitazone enantiomers and internal standard (I.S., dexamethasone acetate) was achieved on a cellulose tris (3,5-dichlorophenylcarbamate) column known as Chiralpak IC with a mobile phase of hexane–isopropanol (70:30, v/v) at a flow rate of 1.0 mL/min. The ultraviolet (UV) detection wavelength was set at 225 nm. Baseline separation of pioglitazone enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Linear calibration curves were obtained over the range of 0.25–50 g/mL in plasma for both enantiomers (R2 > 0.9990) with quantitation limit of 0.25 g/mL. The mean extraction recoveries were 82.37–91.38% for pioglitazone enantiomers and 95.76% for I.S. from rat plasma. The mean relative error (R.E. %) of accuracy and the mean relative standard deviation (R.S.D. %) of intra-day and inter-day precision for both enantiomers were <10%. The method was validated with accuracy, precision, recovery and stability and used to determine the pharmacokinetics of pioglitazone enantiomers, after a single oral administration of racemic pioglitazone (30 mg/kg). The differences between the pharmacokinetic parameters Cmax , AUC0–24 , AUC0–∞ , CL/F of (+)-pioglitazone and (–)-pioglitazone were significant, suggesting that the disposition of pioglitazone in rats may be enantioselective. Moreover, the plasma levels of (+)- and (–)-pioglitazone in female rats were apparently higher than that in male rats, respectively. © 2014 Published by Elsevier B.V.
1. Introduction Pioglitazone (PIO) is an oral anti-hyperglycemic agent that acts primarily by activating peroxisome proliferators–activator receptor gamma (PPAR-␥) [1]. It is used as monotherapy or in combination with sulfonylurea or insulin in the management of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus, NIDDM) [2–4]. The chemical structure of PIO was shown in Fig. 1, with one chiral centre in the thiazolidinedione ring which was attached to a carbonyl group. It is a relatively new member of the thiazolidinedione class and commercially available as the hydrochloride salt. Investigations have shown that PIO was used as the racemic mixture clinically [5]. Examination of literature reveals that the primary method for PIO enantioseparation in vitro is capillary electrochromatography (CEC) [5,6]. In addition, several nonstereoselective analytical methods have been described for quantitative determination of racemic PIO in biological fluids, including HPLC-UV with the run time of
∗ Corresponding authors. Tel.: +86 371 67781902; fax: +86 371 67781908. E-mail addresses: [email protected] (G. Shen), [email protected] (Z. Zhang). 1570-0232/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2014.01.013
less than 20 min [7–9] and LC-MS/MS with a lower limit of quantification (LLOQ) [10,11]. However, the literature on stereoselective pharmacokinetic study of PIO enantiomers has not been reported at present. It is necessary to realize that the safety and efficacy data for a drug evaluated as a mixture of enantiomers are still valid. In this paper, we were interested to develop a simple, specific and accurate chiral HPLC method for quantitative determination of (+)-, (−)-PIO in plasma and its application to stereoselective pharmacokinetics study in rats. According to the report in the literature [12], there are sex differences in the plasma levels of PIO racemate in rats. To our knowledge, no comparison of the pharmacokinetics of PIO enantiomers in male and female rats was reported up to now. Therefore we studied and analyzed the sex differences in pharmacokinetics of pioglitazone enantiomers. 2. Materials and methods 2.1. Chemicals and reagents The racemic pioglitazone reference substance (purity 99.5%) and dexamethasone acetate (IS, purity 99.0%, see Fig. 1) were supplied by Henan Provincial Institute of Food and Drug Control
144
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
Fig. 1. The chemical structures of (A) PIO and (B) dexamethasone acetate.
(Zhengzhou, China). HPLC grade n-hexane, isopropanol, ethanol and ethyl acetate of analytical reagent grade were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). All other reagents purchased from Tianjin Tjshield Chemicals Co., Ltd (Tianjin, China) were of analytical reagent grade. 0.45 m pore size filers (Millipore, MA, USA) were used to filter the mobile phase and solutions. 2.2. Animals Age-matched male (240–270 g) and female (220–250 g) Sprague-Dawley (SD) rats were obtained from the Zhengzhou University Medical Laboratory Animal Center (Zhengzhou, China). Animals were housed under good laboratory conditions (temperature 25 ± 2◦ C, 50 ± 20% relative humidity) and allowed to acclimatize for at least 1 week before initiation of studies. Prior to the drug administration, rats were fasted overnight (12 h) and had free access to water throughout the experimental period. The handling and treatments of all animals used in this study were in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). The animal use and care protocol was reviewed and approved by the ethics committee of the Zhengzhou University. 2.3. Chromatographic conditions Chromatographic studies were performed on Agilent 1100 HPLC system (Agilent, Palo Alto, CA, USA) equipped with autosampler, thermostated-column device and a variable-wavelength UV Detector. The chromatographic separations were carried out on a Chiralpak IC column (250 mm × 4.6 mm i.d., 5 m, from Daicel Chemical Industries Ltd., Tokyo, Japan). The mobile phase was hexane-isopropanol (70:30, v/v). All separations were performed isocratically at a flow rate of 1.0 mL/min with UV detection at 225 nm. Column temperature was maintained at 35 ± 1 ◦ C. The injection volume was 20 L. Agilent ChemStation® software was used for data acquisition and integration.
centrifuge tubes. Ethanol (20 L) and the I.S. solution (30 L) of 50 g/mL were added and vortex-mixed for 1 min. The mixture was extracted with ethyl acetate (1 mL) using a vortex mixer for 3 min and then centrifuged at 3000 × g for 10 min. The organic layer (900 L) was transferred into clean test tubes and evaporated to dryness at 35 ◦ C using a gentle stream of nitrogen. The residue was reconstituted with 100 L ethanol and mixed by vortexing for 5 min, and then centrifugated at 12000 × g for 15 min at 4 ◦ C. Aliquot (20 L) was injected into HPLC system for analysis.
2.6. Bioanalytical method validation 2.6.1. Calibration curve Blank plasma samples were spiked with 20 L of racemic pioglitazone working standard solutions and 30 L of I.S. working solution in order to generate concentrations of the enantiomers ranging from 0.25 to 50 g/mL. The calibration samples were prepared as described above. The concentration of each isomer was looked upon as half of the racemic concentration since the two enantiomers were almost the same in the peak area. The calibration curves were obtained by plotting the peak area ratios of each enantiomer and I.S. versus the concentration of enantiomer spiked in the samples by least-squares linear regression analysis. Leastsquares linear regression analysis was performed using Microsoft® Excel. The limit of quantitation (LOQ) for each enantiomer was considered as the concentration that produced a signal-to-noise (S/N) ratio of 10.
2.6.2. Extraction recovery The recovery of each enantiomer was determined by comparing the peak area ratios of the analytes to I.S. in the regularly pre-treated QC samples at three concentration levels (five samples each) with those of spiked post-extraction samples. Similarly, the recovery of I.S. was determined at 50 g/mL [13]. The absolute recoveries of the analytes and I.S. need not be 100%, but the extent of recovery should be consistent, precise and reproducible.
2.4. Preparation of stock and standard solutions The stock solutions of racemic PIO and I.S. were prepared in ethanol (1.0 mg/mL). A series of standard working solutions of racemic PIO were prepared by further dilution of the stock solution with ethanol to achieve concentrations ranging from 5 to 500 g/mL for racemic PIO. The I.S. working solution (50 g/mL) was prepared by the dilution of the stock dexamethasone acetate solution with ethanol. Quality control (QC) working solutions of 1.5, 8.0 and 80.0 g/mL were prepared in the same way as that of the standard working solutions. All the solutions were stored at 4 ◦ C and brought to room temperature before use. 2.5. Preparation of samples Sample preparation was carried out by liquid–liquid extraction. Aliquots (150 L) of plasma sample were pipetted into 1.5 mL
2.6.3. The accuracy and precision Intra- and inter-day precision and accuracy of the method were evaluated, based on the data from QC plasma samples at three different concentrations (1.5, 8.0 and 80 g/mL) in five replicates. The precision and accuracy were assessed by analyzing five QC samples at each concentration within a day and mean values of five replicates with the same concentrations on five consecutive days, respectively. Accuracy was expressed as percent relative error (R.E. %) [(measured concentration − spiked concentration)/spiked concentration] × 100% [14], while precision was quantitated by calculating intra- and inter-relative standard deviation values (R.S.D. %). The criteria for acceptability of the data included an accuracy within ±15% relative error from the nominal values and a precision of within 15% R.S.D except for LOQ, where it should not exceed ±20% [15].
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
2.6.4. Stability The stability of PIO enantiomers in rat plasma was tested using QC samples at three different concentrations (1.5, 8 and 80 g/mL), all three copies. Stability of each enantiomer in the biomatrix during 6 h (bench-top) was determined at ambient temperature (25 ± 2 ◦ C). Freezer stability was assessed by analyzing the QC samples stored at −20 ◦ C for at least 30 days. The freeze-thaw stability was determined over three cycles within 3 days. In each freeze-thaw cycle, the spiked plasma samples were frozen for 24 h at −20 ◦ C and thawed at room temperature. The samples were processed using the same procedure as described in the sample preparation section. Residue of racemate PIO was kept for 15 days at −20 ◦ C to evaluate residue stability, before sample injection analysis. For the acceptance criterion of stability, the R.S.D. % compared to the freshly prepared standard should be within 15%. 2.7. Preliminary pharmacokinetic study in rats The validated method was applied to a pharmacokinetic study of PIO in healthy male and female SD rats (5 animals per group). Two commercially available tablets labeled by containing 15 mg pioglitazone hydrochloride were grinded and the contents were suspended in 0.5% CMC-Na solution to produce concentration of 3 mg/mL. Each rat was given a single oral dose of 30 mg/kg by oral gavage after overnight fasting. Immediately before each administration the suspension was vortexed for few seconds. Water was available at all times during the experiment. Whole blood (400 L) was collected via the orbital venous plexus from rats into heparinized centrifuge tubes at 0.3, 0.5, 1, 2, 4, 6, 8, 10, 12, 18 and 24 h following administration. The blood was immediately centrifuged at 3000 × g for 5 min and plasma (150 L) was transferred into tagged centrifuge tubes. The plasma samples were analyzed immediately or stored at −20 ◦ C. 2.8. Data analysis Pharmacokinetic parameters were calculated by noncompartmental approach using DAS 3.0 (Bozhiyin T&S Co., Ltd, Beijing, China). The maximum plasma concentration (Cmax ) and the time to reach maximal plasma concentration (Tmax ) were obtained directly from raw data. Other noncompartmental model pharmacokinetic parameters were obtained from the DAS output. All statistical analyses were done with IBM SPSS Statistics 19 (Inc., Chicago, IL, USA). Data are expressed as the mean values ± standard deviation (S.D.). Statistical significance of the differences in pharmacokinetic parameters between two enantiomers was analyzed by paired student’s t test. P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Effects of assay conditions on the separation In this study, the baseline resolution of PIO enantiomers and the I.S. in rat plasma were achieved in normal phase liquid chromatography using the Chiralpak IC column, which is a cellulose tris (3,5-dichlorophenylcarbamate) - based stationary phase on silica. The effect of mobile phase composition on the separation, together with the effects of column temperature and flow rate, was investigated to choose the optimum separation conditions. Different combinations of hexane, ethanol and isopropanol were tested to select the mobile phase that would give an optimum separation and selectivity for PIO enantiomers. As organic modifier, the percentage of isopropanol in the mobile phase had a stronger effect on the separation than ethanol. With decreasing the percentages of the isopropanol in the mobile phase from 50% to 20%, the chromatographic run time and resolution increased from 7.36 to
145
42.47 min and from 2.51 to 4.20, respectively. When the percentage of isopropanol was more than 30%, retention time was shortened, but the impurity peak interfered the separation of PIO enantiomers and the I.S. When the percentage of isopropanol was 20%, retention time exceeded 40 min. Therefore, the mobile phase was decided to be n-hexane and isopropanol (70:30, v/v). The effect of column temperature ranging from 20 to 35◦ C was investigated on the PIO enantiomers separation. Across the tested temperature range, a raise in temperature shortened the elution time from 34.74 to 22.47 min and reduced the column pressure, however the resolution was decreased from 4.20 to 3.43. In order to maintain high resolution and avoid long analysis time and high column pressure, the column temperature was set at 35◦ C. Under the optimum mobile phase and column temperature conditions, the influence of flow rate was examined by changing from 0.6 to 1.2 mL/min. It was found that the high flow rate resulted in relatively low resolution and short analysis time. As the assay was being developed and optimized for the rapid quantitative determination of PIO enantiomers, cutting the run time becomes very important. Therefore, with regards to the right balance of resolution and elution time, the flow rate was set at 1.0 mL/min. 3.2. Specificity and chromatography Under the optimal conditions mentioned above, the peaks of I.S. and (−)-PIO, (+)-PIO were well resolved in rat plasma, with retention times of 11.87, 18.67 and 22.47 min, respectively. The order of elution was determined by using a Polar 3001 Automatic Polarimeter (Optical Activity Ltd, Cambridgeshire, UK). Representative chromatograms of rat blank plasma, rat blank plasma spiked with standard solution of PIO racemate and the I.S., and plasma sample obtained at 2 h and 24 h after oral administration were shown in Fig. 2A–D, respectively. Comparison of blank plasma (Fig. 2A) with blank plasma spiked with PIO racemate and I.S. (Fig. 2B) showed that no endogenous peak from plasma was found to interfere with the elution of PIO enantiomers or the I.S. Similarly, no interfering peaks were observed in the chromatograms of plasma sample obtained at 2 h and 24 h after a 30 mg/kg oral dose of PIO racemate (Fig. 2C and D). This has indicated that appropriate selectivity of the elaborated procedure and a stereoselective HPLC method with good specificity was developed. 3.3. Assay validation 3.3.1. Calibration curve and recovery Peak area ratios of each enantiomer of PIO to the I.S. were measured and acted as a surrogate for quantitation. The results were fitted to linear regression analysis without the use of a weighting factor as well as weighting factor. The weighting options did not show any improvement in the performance of standard curve and quality control samples. Therefore, we have opted to the linear regression without weighting factor for validation of the analytical data. The mean linear regression equations of (−)-PIO and (+)PIO, obtained by a least-squares method, were y = 0.1434x + 0.0082, y = 0.1447x + 0.0883, respectively (y is the peak area ratio of the enantiomer to I.S. and x is the concentration of each enantiomer in plasma) over a concentration range of 0.5–100 g/mL racemic PIO added to rat plasma which is equivalent to 0.25–50 g/mL of each enantiomer. The correlation coefficient (R2 ) of the calibration curve generated during the validation was 0.9998 for both enantiomers, demonstrating linearity over the entire standard range tested. The LOQ of this assay was 0.25 g/mL for both isomers at a signal-to-noise ratio of about 10. The absolute recovery for individual enantiomer was estimated at three different concentrations (1.5, 8 and 80 g/mL of PIO racemate in plasma). The mean percentage recoveries for two
146
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
Fig. 2. Typical chromatograms of rat blank plasma (A), rat blank plasma spiked with standard solution (80 g/mL) of PIO racemate and the I.S. (50 g/mL) (B), plasma sample (8.35, 9.58 g/mL for (−)-, (+)-PIO, respectively) obtained at 2 h after a single dose of 30 mg/kg of racemic (C) and plasma sample (0.34, 0.81 g/mL for (−)-, (+)-PIO, respectively) obtained at 24 h after a single dose of 30 mg/kg of racemic (D). 1: (−)-PIO; 2: (+)-PIO; IS: internal standard substance.
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
147
Table 1 Precision and accuracy for the determination of PIO enantiomers in plasma (n = 5). Analyte
Added (g/mL)
Found (g/mL)
Relative error (%)
Intra-day R.S.D (%)
Inter-day R.S.D (%)
(−)-PIO
0.75 4.00 40.00 0.75 4.00 40.00
0.68 ± 0.08 4.25 ± 0.15 42.37 ± 5.37 0.68 ± 0.11 4.16 ± 0.09 41.44 ± 5.85
−9.33 +6.25 +5.92 –9.33 +4.00 +3.60
6.24 5.43 3.40 4.73 4.48 2.45
3.22 3.08 1.68 4.86 4.73 1.68
(+)-PIO
enantiomers ranged from 82.37 ± 5.76% to 91.38 ± 2.50%. A mean percentage recovery of 95.76 ± 2.69% was obtained for the I.S. at 50 g/mL. 3.3.2. Accuracy and precision The results for accuracy and precision throughout the standard curve for (−)-PIO and (+)-PIO are presented in Table 1. The R.S.D. values of intra-day and inter-day precision for two enantiomers were less than 6.24%. The R.E. values of accuracy for two enantiomers varied between –9.33% and 6.25%, and between –9.33% and 4.00%, respectively. All the values of accuracy and precision including LOQ fell within the limits which were considered as acceptable. 3.3.3. Stability In this study, short-term, long-term, freeze-thaws and residue stabilities of two enantiomers in plasma were investigated using low-, middle- and high-quality control samples. The mean R.S.D.% values of PIO enantiomers in all stability tests were within 10%, demonstrating that plasma samples spiked with PIO enantiomers were stable on the bench-top for at least 6 h, at −20 ◦ C for at least 30 days, from −20 ◦ C to ambient temperature for at least three freeze/thaw cycles and PIO residue was stable at −20 ◦ C for at least 15 days. The concentrations of PIO enantiomers remained almost unchanged during stability experiments. These studies further suggested that rat plasma samples containing PIO enantiomers can be handled under normal laboratory conditions without significant conversion or racemization.
Table 3 Pharmacokinetic parameters of pioglitazone enantiomers following oral administration of racemic pioglitazone in five male rats (30 mg/kg). Paramters
(−)-PIO
Cmax (g/mL) Tmax (h) t1/2 (h) AUC0–24 (g h/mL) AUC0–∞ (g h/mL) CL/F (L/h kg)
5.59 2.60 6.97 43.52 47.60 0.64
± ± ± ± ± ±
(+)-PIO 1.81** 1.34 1.73 5.64** 5.35** 0.08**
10.17 2.80 6.75 86.94 95.35 0.32
± ± ± ± ± ±
1.86 1.10 3.07 5.29 3.22 0.01
Statistically significant difference versus (+)-PIO. ** P < 0.01 (mean ± S.D., n = 5).
plasma concentration of (+)-PIO was higher than that of (−)-PIO at each time point in female rats, as shown in Fig. 3A. The Cmax and AUC0–24 , AUC0–∞ of (+)-PIO were about 2.3 and 2.6, 3.4 times as high as those of (−)-PIO, respectively, indicating greater adsorption and distribution of (+)-PIO [16]. Compared with female rats, PIO enantiomers were relatively fast absorbed from tablets in male rats. The plasma concentrations of (−)-PIO and (+)-PIO reached Cmax at approximately 2.6 h and 2.8 h (Tmax ) after dosing and then decreased with a terminal phase t1/2 of about 6.97 h and 6.75 h, respectively. The mean plasma levels of (+)-PIO were found to be
3.4. Pharmacokinetics in rats After a single oral administration of 30 mg/kg racemic PIO to male and female SD rats, the plasma concentrations of two enantiomers were determined by the described method. The mean plasma concentration versus time profiles of both enantiomers of PIO in the female and male rats were illustrated individually in Fig. 3A and B respectively. Pharmacokinetic parameters of two enantiomers calculated by the non-compartmental approach in female and male rats were listed in Tables 2 and 3, respectively. The (−)-PIO and (+)-PIO were very slow absorbed from tablets in female rats resulting in the same Tmax value of 9.2 h. However, the (−)-PIO showed a faster elimination and a shorter t1/2 . The Table 2 Pharmacokinetic parameters of PIO enantiomers following oral administration of racemic PIO in five female rats (30 mg/kg). Parameters Cmax (g/mL) Tmax (h) t1/2 (h) AUC0–24 (g h/mL) AUC0–∞ (g h/mL) CL/F (L/h kg)
(−)−PIO 9.44 9.20 2.63 77.14 77.81 0.39
± ± ± ± ± ±
(+)−PIO **
2.77 1.78 0.71 5.09** 4.65* 0.02**
Statistically significant difference versus (+)−PIO. * P < 0.05. ** P < 0.01 (mean ± S.D., n = 5).
22.03 9.20 7.66 202.78 265.75 0.12
± ± ± ± ± ±
6.41 1.78 3.91 26.80 103.64 0.04
Fig. 3. The plasma concentrations (mean ± S.D.) time profile of (+)-PIO and (−)-PIO in female rats after receiving 30 mg/kg oral dose of racemic PIO (A). (N): (+)-PIO; (): (−)-PIO. The plasma concentrations (mean ± S.D.) time profile of (+)−PIO and (−)PIO in male rats after receiving 30 mg/kg oral dose of racemic PIO (B). (N): (+)-PIO; (): (−)-PIO.
148
B. Du et al. / J. Chromatogr. B 951–952 (2014) 143–148
higher than those of (−)-PIO in male rats, as shown in Fig. 3B. The Cmax , AUC0–24 , and AUC0–∞ of (+)-PIO were about 1.8, 2.0 and 2.0 times higher respectively than that of (−)-PIO, which indicates greater adsorption and distribution of (+)-PIO [16]. Though, for female and male rats, Tmax and t1/2 were not significantly different between (+)-PIO and (−)-PIO (P > 0.05), their Cmax , AUC0–24 , AUC0–∞ and CL/F were significantly different (P < 0.05 or P < 0.01), as shown in Tables 2 and 3. These results suggest that the disposition of PIO in rats is enantioselective. The enantiomer area ratios under the curve of plasma concentration versus time (AUCisomer (+) /AUCisomer (−) ) were more than 2.0, indicating that concentrations of the (+)PIO enantiomer were higher in plasma, which might be considered to have lower affinity for PPAR-␥ receptor [17]. Up to now, no report on differences of affinity at PPAR-␥ receptor of PIO enantiomers was available. However, there is the report on rosiglitazone, which also is one of the thiazolidinediones (TZDs) and acts by activating PPAR␥. The enantiomers of rosiglitazone showed differential activity at PPAR-␥ and (R) - (+) - rosiglitazone showed relatively weak affinity binding to PPAR-␥ [18]. So the reason caused stereoselectivity differences for PIO enantiomers may be different affinity at PPAR-␥ receptor. However, different metabolic rate or chiral inversion in rats could also lead to the stereoselectivity differences of PIO enantiomers in rats and further studies are required for elucidation of the detailed mechanisms. Compared with male rats, PIO enantiomers showed a slower adsorption and elimination in the female rats. Moreover, Cmax and AUC0–24 of PIO enantiomers were significantly higher in the female rats. Cmax of (−)-PIO and (+)-PIO in female rats were approximately 1.7 and 2.2 times than that in male rats. AUC0–24 of (−)-PIO and (+)PIO were 1.8 and 2.3 times higher in females than in males. These results suggested that there might be sex differences in pharmacokinetics of PIO enantiomers in rats, which were compatible with those described in the literature for PIO racemate [12]. The sex differences in some of cytochrome P450 (CYP) isoforms in rats have been reported, which may explain why the pharmacokinetics of PIO enantiomers are different in female and male rats [19–21]. 4. Conclusion A HPLC method was developed, validated and successfully applied to the simultaneous determination of PIO enantiomers in rat plasma and their stereoselective pharmacokinetics study in rats. Pharmacokinetic analysis showed that (+)-PIO had higher Cmax , AUC0–24 , AUC0–∞ than that of (−)-PIO after the oral administration of racemic PIO at 30 mg/kg, suggesting that the disposition of PIO in rats was enantioselective. These results presented here were a further contribution to the understanding of the kinetic behavior of PIO analogues. Nevertheless, further research needs to be carried out in order to ascertain the exact explanation for the observed pharmacokinetic stereoselectivity differences between PIO enantiomers in rats. By comparing and analyzing the pharmacokinetic
parameters of PIO enantiomers in male and female rats, we found that there were sex differences in the plasma levels of PIO enantiomers. Acknowledgements The author thanks Dr. J. Zhou for the helpful proposals and this work was financially supported by the National Natural Science Foundation of China (Grant No.81302717) and the Project of Zhengzhou Science and Technology Innovation Team (Grant No.2013sjxm020). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.01.013. References [1] K.S. Lakshmi, T. Rajesh, S. Sharma, IJPPS 1 (2009) 162–166. [2] J. Waugh, G.M. Keating, G.L. Plosker, S. Easthope, D.M. Robinson, Drugs 66 (2006) 85–109. [3] J. Chilcott, P. Tappenden, M. Lloyd, J.P. Wight, Clin. Ther. 23 (2001) 1792–1823. [4] B. Jamali, I. Bjørnsdottir, O. Nordfang, S.H. Hansen, J. Pharm. Biomed. 46 (2008) 82–87. [5] B. Jamali, G.C. Theill, L.L. Sørensen, J. Chromatogr. A 1049 (2004) 183–187. [6] B. Jamali, I. Bjoernsdottir, C. Cornett, S.H. Hansen, Electrophoresis 30 (2009) 2853–2861. [7] P. Venkatesh, T. Harisudhan, H. Choudhury, R. Mullangi, N.R. Srinivas, Biomed. Chromatogr. 20 (2006) 1043–1048. [8] M.S. Arayne, N. Sultana, A.Z. Mirza, J. Chromatogr. Sci. 49 (2011) 114–117. [9] P. Sripalakit, P. Neamhom, A. Saraphanchotiwitthaya, J. Chromatogr. B 843 (2006) 164–169. [10] Z.J. Lin, W. Ji, D. Desai-Krieger, L. Shum, Pharm. Biomed. Anal. 33 (2003) 101–108. [11] Y.H. Xue, K.C. Turner, J.B. Meeker, J. Pursley, M. Arnold, S. Unger, J. Chromatogr. B 795 (2003) 215–226. [12] Y. Fujita, Y. Yamada, M. Kusama, T. Yamauchi, J. Kamon, T. Kadowaki, T. Iga, Comp. Biochem. Physiol. C 136 (2003) 85–94. [13] Y.M. Lu, Z.M. Sun, Y.F. Zhang, X.Y. Chen, D.F. Zhong, J. Chromatogr. B 921 (2013) 27–34. [14] M.A. Radwan, H.H. Abdine, B.T. Al-Quadeb, H.Y. Aboul-Enein, K. Nakashima, J. Chromatogr. B 830 (2006) 114–119. [15] B. Du, L. Pang, H.Y. Li, S.J. Ma, Y. Li, X. Jia, Z.Z. Zhang, J. Chromatogr. B 932 (2013) 88–91. [16] S.S. Mustafa, R. Trivedi, N.V.S.R. Mamidi, R. Mullangi, N.R. Srinivas, J. Chromatogr. B 809 (2004) 23–30. [17] Z.Y. Hong, G.R. Fan, Y.F. Chai, X.P. Yin, J. Wen, Y.T. Wu, J. Chromatogr. B 826 (2005) 108–113. [18] V.L. Lanchote, A. Rocha, F.U.V. de Albuquerque, E.B. Coelho, P.S. Bonato, J. Chromatogr. B 765 (2001) 81–88. [19] D.J. Parks, N.C.O. Tomkinson, M.S. Villeneuve, S.G. Blanchard, T.M. Willson, Bioorg. Med. Chem. Lett. 8 (1998) 3657–3658. [20] Y. Imamura, M. Kaneko, H. Takada, M. Otagiri, H. Shimada, H. Akita, Comp. Biochem. Physiol. C 133 (2002) 587–592. [21] R. Kato, Y. Yamazoe, Toxicol. Lett. 64 (1992) 661–667.