http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–8 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.988772
RESEARCH ARTICLE
Gender differences in corydaline pharmacokinetics in rats Ji Won Jung1, Mi Ran Choi1, Yong Sam Kwon2, Jin Seok Jeong2, Miwon Son2, and Hee Eun Kang1 1
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College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon, Republic of Korea and 2Research Center, Dong-A Pharmaceutical Co., Yongin, Republic of Korea Abstract
Keywords
1. Corydaline, an isoquinoline alkaloid, is one of the major active constituents in a new prokinetic botanical agent, DA-9701. It has been recommended that preclinical pharmacokinetic studies of natural medicines include both genders. Therefore, in this study, the pharmacokinetics of corydaline in male and female rats was evaluated following intravenous and oral administration of pure corydaline or DA-9701. 2. After intravenous administration of corydaline, the area under the plasma concentration– time curve (AUC) was significantly greater (by 46.4%) in female rats compared to male rats due to a 29.3% reduction in non-renal clearance in female rats. The gender difference in corydaline hepatic metabolic clearance was supported by a significantly slower metabolism of corydaline in hepatic microsomes of female rats mediated via male-specific (CYP2C11 and CYP3A2) or male-dominant (CYP3A1) CYP isozymes. 3. Following oral administration of pure corydaline or DA-9701, the AUC and Cmax values of corydaline in female rats were significantly greater (by 793% and 466% increase for corydaline administration or by 501% and 143% increase for DA-9701 administration) than in male rats. Greater F values of corydaline in female rats could be due to smaller hepatic first-pass extraction as a result of slower hepatic metabolism of corydaline. 4. However, we observed a comparable disappearance of corydaline in male and female human liver microsomes, consistent with little gender difference in CYP2C9 and CYP3A activities in humans compared to that in rats. Thus, gender differences in corydaline metabolism are not expected to occur in humans.
Corydaline, CYP2C, CYP3A, DA-9701, gender, pharmacokinetics, rats
Introduction The corydalis tuber has anti-inflammatory, anti-allergic, and analgesic effects (Kubo et al., 1994; Matsuda et al., 1995; Yuan et al., 2004) that have led to its use as an analgesic or anti-spasmodic agent in Asian countries (Lee & Suh, 2012). Corydalis tuber from the roots of Corydalis yanhusuo W. T. Wang is the primary constituent of the herbal medicine DA9701, a new botanical drug marketed as oral tablets (MotilitoneÕ ) for the treatment of functional dyspepsia in Republic of Korea since May 2011. DA-9701 is formulated with dried 50% ethanolic extracts of Pharbitidis semen from the seed of Pharbitis nil Choisy and Corydalis tuber from the roots of Corydalis yanhusuo W. T. Wang (1:5, w/w). An investigational new drug (IND) application of DA-9701 for a phase II clinical trial will be submitted to the United States Federal Drug Administration (FDA) this year. In addition, a bridging study for marketing DA-9701 in China is in preparation.
Address for correspondence: Prof. Hee Eun Kang, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon 420-743, Republic of Korea. E-mail: [email protected]
History Received 14 October 2014 Revised 13 November 2014 Accepted 13 November 2014 Published online 28 November 2014
The effects of DA-9701 as a prokinetic agent have been reported in various animal models (Jung et al., 2013; Kim et al., 2012; Lee et al., 2008). These effects are likely mediated by the induction of pacemaker currents in the interstitial cells of Cajal of the small intestine, which are required for gastrointestinal motility (Choi et al., 2009), along with antagonistic effects on the dopamine D2 receptor and agonistic effects on serotonin 5-HT4, 5-HT1A, and 5-HT1B receptors (Kim et al., 2012). DA-9701 produces strong gastroprokinetic effects at an effective dose of 0.3–3 mg/kg in rats and has a superior safety profile compared to conventional prokinetics, such as cisapride, mosapride, itopride and domperidone (Lee et al., 2008). Corydaline [(13S,13aR)-2,3,9,10-tetramethoxy-13-methyl6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline; Figure 1], an isoquinoline alkaloid, is an active ingredient in Corydalis tuber and DA-9701. High-performance liquid chromatography (HPLC) performed for batch-to-batch quality control revealed that corydaline is one of the major constituents in DA-9701 (Kwon & Son, 2013). Corydaline promotes gastric emptying and small intestinal transit, and facilitates gastric accommodation (Lee et al., 2010). Various metabolites of corydaline have been found and O-demethylation and
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Figure 1. Chemical structure of corydaline.
hydroxylation are the major metabolic pathways in human liver microsomes and hepatocytes (Ji et al., 2012). Cytochrome P450 (CYP) 2C9 and 3A4 were reported as major enzymes involved in the formation of the major metabolite, O-desmethylcorydaline (M1) from corydaline (Ji et al., 2011a). Corydaline inhibits activities of metabolic enzymes in human liver microsomes with potent inhibition of CYP 2C19 and 2C9, and moderate inhibition of UDP-glucuronosyl transferase 1A1 and 1A9 (Ji et al., 2011b). Recently, we reported the pharmacokinetics of corydaline in rats following various doses of pure corydaline and DA-9701 (Jung et al., 2014). Corydaline showed dose-dependent pharmacokinetics after intravenous and oral administration (1.1–4.5 mg/kg) due to saturation of corydaline metabolism at higher doses. As a result, the extent of absolute oral bioavailability (F) of corydaline following its oral administration varied considerably (9.10–21.1%) according to the dose administered. The Technical Requirements for the Study of New Natural Medicines issued recently by the State Food and Drug Administration in China recommended that a preclinical pharmacokinetic study of a new natural medicine in both genders be performed and gender differences confirmed. Knowledge on gender differences in pharmacokinetics can help understanding of possible gender-different preclinical data of the drug. Therefore, in this study, the pharmacokinetics of corydaline in male and female rats were investigated following intravenous and oral administration of pure corydaline or oral administration of DA-9701.
Materials and methods Materials Corydaline and DA-9701 (Lot No. CP2410, 1.38% of corydaline) were supplied by Dong-A Pharmaceutical Co. (Yongin, Republic of Korea). Corydaline was a product of Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and DA-9701 was a product of Dong-A Pharmaceutical Co. Verapamil, the reduced form of b-nicotinamide adenine dinucleotide phosphate (NADPH; as tetrasodium salt), polysorbate (Tween)-80, dimethylsulfoxide (DMSO) and ammonium acetate were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Human liver microsomes pooled from either males or females were products of BD Biosciences (Woburn, MA). Other chemicals were of reagent or HPLC grade. Animals Animal study protocols were approved by the Department of Laboratory Animals, Institutional Animal Care and Use
Committee on the Sungsim Campus of The Catholic University of Korea (Approval No. 2011-015; Bucheon, Republic of Korea). Age-matched (8–9 weeks old) male (280–330 g) and female (190–230 g) Sprague–Dawley (SD) rats, were purchased from Charles River Company Korea (Seoul, Republic of Korea). The procedures for housing and handling the rats were in accord with a reported method (Kim et al., 2013). Disappearance of corydaline in hepatic microsomal fractions Hepatic microsomal fraction from male and female rats (n ¼ 4 each) were prepared according to a reported method (Lee & Lee, 2008). Protein contents in the microsomal fraction were measured using a reported method (Bradford, 1976). The hepatic microsomal fractions (equivalent to 0.5 mg protein) were mixed with 5 mL of methanol containing corydaline to final concentrations of 2 and 20 mM, and 25 mL of 0.1 M phosphate buffer (pH 7.4) containing 1 mM NADPH. The volume was adjusted to 0.25 mL by adding 0.1 M phosphate buffer (pH 7.4). The components were incubated in a thermomixer [Thermomixer 5436; Eppendorf, Hamburg, Germany; 37 C, 500 revolutions per min (rpm)]. At 0, 10, 20, 30, 45, and 60 min of incubation, 20 mL were collected and added to an Eppendorf tube containing 50 mL of acetonitrile with 10 ng/mL verapamil (internal standard) and vortex-mixed to terminate the reaction. The disappearance of corydaline in male and female pools of human liver microsomes (equivalent to 1-mg protein) was also measured in triplicate according to the method described above. Intravenous administration of corydaline For intravenous administration, the carotid artery and jugular vein were cannulated using procedures reported previously (Kim et al., 2013). Corydaline [dissolved in 1:2:7 (v/v/v) DMSO: Tween 80: distilled water] at a dose of 1.1 mg (2 mL)/ kg was infused for 1 min via the jugular vein of male and female rats (n ¼ 7, each). A blood sample of 50 mL was collected via the carotid artery at time 0 (prior to dosing), 1 (at the end of the infusion), 5, 15, 30, 60, 90, 120, 180, 240, 360 and 480 min after the start of infusion. Blood samples were immediately centrifuged and 20 mL of each plasma sample was stored at 70 C until used for the liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of corydaline. The preparation and handling of the 24-h urine samples (Ae0–24 h) and gastrointestinal tract (GI) samples (including its contents and feces) at 24 h (GI24 h) were performed according to a reported method (Kim et al., 2013). Oral administration of corydaline and DA-9701 For the oral administration study, rats were fasted overnight with free access to water, and the carotid artery was cannulated using procedures reported previously (Kim et al., 2013). Corydaline at a dose of 4.5 mg (3 mL)/kg or the equivalent dose of DA-9701 (328 mg/kg) [dissolved in 1:2:12 (v/v/v) DMSO: Tween 80: distilled water] was administered orally using a gastric gavage tube to male and female rats (n ¼ 7 and 8, respectively, for corydaline; n ¼ 8 and 9, respectively, for
Gender-different PKs of corydaline in rats
DOI: 10.3109/00498254.2014.988772
DA-9701). A blood sample of 50 mL was collected via the carotid artery at 0, 5, 15, 30, 45, 60, 90, 120, 180, 240, 360 and 480 min after oral administration of the drug. Other procedures for the oral study were same as those for the intravenous study.
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Measurement of protein binding of corydaline in plasma from male and female rats using equilibrium dialysis Protein binding of corydaline to fresh plasma from male and female rats (n ¼ 5 and 4, respectively) was assessed by equilibrium dialysis (Jung et al., 2014). Fresh plasma (0.2 mL) was dialyzed against 0.2 mL of isotonic Sørensen phosphate buffer (pH 7.4) containing 3% (w/v) dextran (‘the buffer’) to minimize volume shift (Boudinot & Jusko, 1984) using a 0.25 mL micro-equilibrium dialyzer (Harvard Apparatus, Holliston, MA) and a regenerated cellulose membrane (molecular weight cut-off 10 kDa, Harvard Apparatus). To reduce the equilibrium time of corydaline between the buffer and plasma compartments, the drug was spiked into the plasma side (Øie & Guentert, 1982) to a final concentration of 1 mg/mL. The spiked dialysis cell was incubated in a waterbath shaker kept at 37 C and at a rate of 50 oscillations per min (opm). After 24 h, two 20 mL aliquots were removed from each compartment and stored at 70 C until LC-MS/MS analysis.
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the rat plasma samples were 0.5–5000 ng/mL, and those in the urine and GI24 h samples were 2.5–5000 ng/mL. The mean intra- and inter-day coefficients of variation (CV) of the analysis on 3 consecutive days were below 5.05%, and the assay accuracies ranged from 86.5 to 112%. Pharmacokinetic analysis The total area under the plasma concentration–time curve from time zero to last measured time, t (AUC0–t) and that from time zero to infinity (AUC) were calculated using the trapezoidal rule-extrapolation method (Chiou, 1978). Standard methods (Gibaldi & Perrier, 1982) were used to calculate the following pharmacokinetic parameters using a non-compartmental analysis (WinNonlinÕ ; Pharsight Corporation, Mountain View, CA): the time-averaged total body, renal, and non-renal clearances (CL, CLR, and CLNR, respectively), terminal halflife, mean residence time (MRT), and apparent volume of distribution at steady state (Vss). The maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) were read directly from the experimental data. For comparison, the extent of absolute oral bioavailability (F) was calculated by dividing the dose-normalized AUC values after oral administration by the dose-normalized AUC values after intravenous administration. Statistical analysis
LC-MS/MS analysis of corydaline The LC-MS/MS system comprised an Agilent 6460 triple quadrupole mass spectrometer with an Agilent 1260 LC system (Agilent, Waldbronn, Germany). Instrument control and data acquisition were performed using the Agilent MassHunter Workstation software (Version B. 04. 01). Concentrations of corydaline in the samples were determined using the LC-MS/MS method developed in our laboratory (Jung et al., 2014). In brief, 50 mL of acetonitrile containing 10 ng/mL of verapamil as the internal standard (IS) was added to 20 mL of each biological sample. After mixing and centrifugation (16,000 g, 5 min), the supernatant was collected and 3 mL injected directly onto a reversed-phase HPLC column (KinetexÕ C18; 4.6 mm i.d. 50 mm l.; particle size, 2.6 mm; Phenomenex, Torrance, CA). The mobile phase, 2 mM ammonium acetate buffer: acetonitrile [40:60 (v/v)], was run through the column at a flow rate of 0.4 mL/min. The temperature of the column was maintained at 35 C and the autosampler at 4 C. The eluent was monitored using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source with positive ion (ESI+; 2–10 min) modes, with multiple reaction monitoring. The instrument parameters were set as follows: nitrogen gas temperature of 210 C, sheath gas temperature of 400 C, gas flow of 5 L/min, sheath gas flow of 12 L/min, nebulizer of 55 psi, and electrospray voltage of 3.5 kV. The fragmentor voltage was set at 165 V for corydaline, and 115 V for the IS. The collision energy for corydaline and the IS was 28 and 20 eV, respectively. The precursor to product ion transitions for corydaline and IS were m/z 370.2 ([M + H]+) ! 192.1 and m/z 455.3 ([M + H]+) ! 165, respectively. The retention times of corydaline and the IS were approximately 5.2 and 8.3 min, respectively. The calibration ranges of corydaline in
A p value 50.05 was deemed to indicate statistical significance using an unpaired two-tailed t-test between the two means. All data were expressed as means ± SD, except medians (range) for Tmax.
Results Disappearance of corydaline in hepatic microsomal fractions from male and female rats Body weight-normalized total hepatic microsomal protein contents were not significantly different between male and female rats. The percentages of the spiked amounts of corydaline (2 and 20 mM) remaining after 10-, 20-, 30-, 45-, and 60-min incubations with hepatic microsomal fractions from male and female rats are shown in Figure 2. In female rats, the remaining values at each incubation time were significantly greater than those in male rats. Following 60min incubation, 80% of spiked amounts were recovered in female rats, while 13–30% of spiked amounts were recovered in male rats. These data suggested that hepatic metabolism of corydaline was slower in female rats than in male rats. Pharmacokinetics of corydaline after its intravenous administration to male and female rats The mean arterial plasma concentration–time profiles of corydaline after its intravenous administration at a dose of 1.1 mg/kg to male and female rats are shown in Figure 3. The relevant pharmacokinetic parameters are listed in Table 1. Note that the AUC0–8 h and AUC of corydaline in female rats were 32.4% and 46.4% greater, respectively, than that in male rats, as a result of 28.9% slower CL and 29.3% slower CLNR in female rats. We also observed 167% faster CLR, 30.9%
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Figure 3. Mean arterial plasma concentration–time profiles of corydaline following its 1-min intravenous administration at a dose of 1.1 mg/kg to male and female rats (n ¼ 7 each). Vertical bars represent SD.
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Table 1. Pharmacokinetic parameters of corydaline after its intravenous administration (1.1 mg/kg) to male and female rats. Parameters
Male rats (n ¼ 7)
Female rats (n ¼ 7)
Body weight (g) AUC0–8 h (mg min/mL) AUC (mg min/mL) Terminal half-life (min) CL (mL/min/kg) CLR (mL/min/kg) CLNR (mL/min/kg) Vss (mL/kg) MRT (min) Ae0–24 h (% of dose) GI24 h (% of dose)
310 ± 11.6 105 ± 10.7 112 ± 11.0 188 ± 35.1 9.90 ± 1.02 0.0214 ± 0.00681 9.88 ± 1.02 1150 ± 289 116 ± 25.1 0.221 ± 0.0831 0.275 ± 0.109
227 ± 4.88*** 139 ± 29.4* 164 ± 41.3** 246 ± 36.6* 7.04 ± 1.48** 0.0571 ± 0.0242** 6.99 ± 1.48** 1250 ± 291 183 ± 50.6** 0.874 ± 0.465** 0.588 ± 0.335*
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Figure 2. Mean percentages of the spiked amounts of corydaline, 2 mM (A) and 20 mM (B), remaining in hepatic microsomes from male and female rats (n ¼ 4 each) at 10, 20, 30, 45, and 60 min after the start of incubation. Vertical bars represent SD. Significant difference from male rats, ***p50.001.
longer terminal half-life, 57.8% longer MRT, 295% greater Ae0–24 h, and 114% greater GI24 h values in females than in male rats. Pharmacokinetics of corydaline after its oral administration to male and female rats The mean arterial plasma concentration–time profiles of corydaline after its oral administration at a dose of 4.5 mg/kg to male and female rats are shown in Figure 4. The relevant pharmacokinetic parameters are listed in Table 2. Note that the AUC0–8 h, AUC, and Cmax values of corydaline in female rats were 695%, 793%, and 466% greater than in male rats, respectively. Moreover, the F value of corydaline in female rats (55.7%) was considerably greater than that in male rats
Data are expressed as mean ± SD. The value was significantly different from male rats (*p50.05, **p50.01, ***p50.001).
(9.14%). The GI24 h value was 815% greater in female rats compared to male rats, but the values in male and female rats were both negligible at 51% of the oral dose. Pharmacokinetics of corydaline after oral administration of DA-9701 to male and female rats The mean arterial plasma concentration–time profiles of corydaline after oral administration of DA-9701 at a dose equivalent to 4.5 mg/kg corydaline to male and female rats are shown in Figure 5. The relevant pharmacokinetic parameters are listed in Table 3. Note that the AUC0–8 h, AUC, and Cmax values of corydaline in female rats were 398%, 501%, and 143% greater than in male rats, respectively. As a result, the F value of corydaline in female rats (29.7%) was also considerably greater than that in male rats (7.22%). The Ae0– 24 h value was 475% greater in female rats compared to that in male rats, but the values of both male and female rats were negligible at 50.1% of the oral dose.
Gender-different PKs of corydaline in rats
DOI: 10.3109/00498254.2014.988772
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Figure 4. Mean arterial plasma concentration–time profiles of corydaline following its oral administration at a dose of 4.5 mg/kg to male (n ¼ 7) and female (n ¼ 8) rats. Vertical bars represent SD.
Figure 5. Mean arterial plasma concentration–time profiles of corydaline following oral administration of 328 mg/kg DA-9701 (equivalent to 4.5 mg/kg corydaline) to male (n ¼ 8) and female (n ¼ 9) rats. Vertical bars represent SD.
Table 2. Pharmacokinetic parameters of corydaline after its oral administration (4.5 mg/kg) to male and female rats.
Table 3. Pharmacokinetic parameters of corydaline after oral administration of DA-9701 (328 mg/kg, equivalent to 4.5 mg/kg corydaline) to male and female rats.
Parameters
Male rats (n ¼ 7)
Female rats (n ¼ 8)
Body weight (g) AUC0–8 h (mg min/mL) AUC (mg min/mL) Terminal half-life (min) CLR (mL/min/kg) Cmax (ng/mL) Tmax (min)a Ae0–24 h (% of dose) GI24 h (% of dose) F (%)b
291 ± 12.2 34.7 ± 13.7 41.9 ± 12.2 280 ± 206 0.0141 ± 0.00955 419 ± 233 30 (15–90) 0.0128 ± 0.00875 0.0615 ± 0.0370 9.14
200 ± 9.26*** 276 ± 142*** 374 ± 175*** 324 ± 137 0.0253 ± 0.0511 2370 ± 986*** 30 (15–45) 0.375 ± 0.912 0.563 ± 0.607* 55.7
Data are expressed as mean ± SD. The value was significantly different from male rats (*p50.05, ***p50.001). a Tmax is expressed as median (range). b The extent of absolute oral bioavailability (F) value was calculated by the equation; (AUCoral Doseiv,)/(AUCiv Doseoral).
Protein binding of corydaline to fresh rat plasma from male and female rats The plasma protein binding values of corydaline in male and female rats were 93.7 ± 1.36% (free fraction of 6.26%) and 95.7 ± 0.260% (free fraction of 4.34%), respectively. The values were significantly different between the two groups (p50.05, t-test). Almost complete recovery of corydaline (101 ± 4.70%) from both plasma and buffer sides indicated that non-selective adsorption of corydaline to equilibrium dialysis apparatus was negligible. Disappearance of corydaline in human hepatic microsomal fractions from male pool and female pool The percentages of the spiked amounts of corydaline (2 and 20 mM) remaining after 10-, 20-, 30-, 45-, and 60-min incubations with human liver microsomes from male and
Parameters
Male rats (n ¼ 8)
Female rats (n ¼ 9)
Body weight (g) AUC0–8 h (mg min/mL) AUC (mg min/mL) Terminal half-life (min) CLR (mL/min/kg) Cmax (ng/mL) Tmax (min)a Ae0–24 h (% of dose) GI24 h (% of dose) F (%)b
318 ± 8.84 26.5 ± 10.5 33.1 ± 12.3 252 ± 85.6 0.0166 ± 0.00732 269 ± 159 30 (15–90) 0.0114 ± 0.00440 0.952 ± 1.56 7.22
209 ± 10.2*** 132 ± 31.4*** 199 ± 68.3*** 312 ± 100 0.0136 ± 0.00897 655 ± 227** 60 (15–180) 0.0655 ± 0.0582* 0.639 ± 1.32 29.7
Data are expressed as mean ± SD. The value was significantly different from male rats (*p50.05, **p50.01, ***p50.001). a Tmax is expressed as median (ranges). b The extent of absolute oral bioavailability (F) value was calculated by the equation; (AUCoral Doseiv,)/(AUCiv Dose as corydalineoral).
female pools are shown in Figure 6. Significantly greater remaining values of corydaline in male pool microsomes were observed compared to those in female pool microsomes at 10, 20, and 30 min after the start of incubation with 20 mM corydaline. However, there was no statistically significant difference in recovery of corydaline after 45- and 60-min incubation.
Discussion The doses of corydaline used in this study, 1.1 mg/kg for the intravenous experiments and 4.5 mg/kg for the oral experiments, were determined based on the previous pharmacokinetic study on corydaline in rats (Jung et al., 2014). After intravenous administration of corydaline to female rats, we observed significantly greater AUC compared to male rats (Table 1) due to significantly slower CL and CLNR values.
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Figure 6. Mean percentages of the spiked amounts of corydaline, 2 mM (A) and 20 mM (B), remaining in human hepatic microsomes from male pool and female pool (in triplicate) at 10, 20, 30, 45, and 60 min after the start of incubation. Vertical bars represent SD. Significant difference from male, *p50.05, **p50.01, ***p50.001.
Based on negligible Ae0–24 h and GI24 h values (51% of the dose) after intravenous administration of corydaline, the contributions of CLR to the CL and gastrointestinal, including biliary, excretion of unchanged corydaline to its CLNR were negligible. This suggests that most of the administered corydaline was eliminated via non-renal metabolic clearance. Therefore, the significantly greater AUC and slower CLNR in female rats could be due to slower metabolism of corydaline. Corydaline is a drug with an intermediate hepatic extraction ratio, estimated to be 38.9–79.9% (Jung et al., 2014) with a reported equation (Lee & Chiou, 1983). Therefore, hepatic clearance of corydaline depends on the intrinsic clearance of the unbound drug (CLint), free (unbound) fraction in plasma (fu), and hepatic blood flow rate (QH) (Wilkinson & Shand, 1975). Gender differences in rat QH have not been published.
Although the liver weights of female rats were significantly smaller than those of male rats, the values as a percentage of body weight were comparable between the two groups of rats. Therefore, little difference in QH values between male and female is expected, indicating that QH did not contribute to the slower metabolism of corydaline in female rats. We observed a 30.7% reduced free fraction of corydaline in plasma of female rats, as well as slower hepatic CLint compared to males, which could contribute to the slower hepatic clearance in female rats. The major human hepatic enzymes involved in the metabolism of corydaline are CYP2C9 and CYP3A4 (Ji et al., 2011a). They are 77% homologous to rat CYP2C11 and 73% homologous to rat CYP3A1 amino acid sequence (Soucek & Gut, 1992). Rat CYP3A2 also exhibits similar catalytic competence and functional analogies to humans CYP3A4 (Wojcikowski et al., 2012). These hepatic CYP enzymes in rats are expressed in a sex-specific manner; CYP2C11 and CYP3A2 are the male-specific forms expressed 10-fold higher than in females, and CYP3A1 is a male-dominant form (Lewis, 1996; Parkinson et al., 2004). Therefore, the slower metabolism of corydaline in female rats is likely due to the slower hepatic CLint compared to male rats. To confirm slower metabolism of corydaline in female rats, we evaluated in vitro hepatic intrinsic clearance of corydaline in microsomes from male and female rats. However, the metabolism of corydaline in female rats was so slow that the intrinsic clearance of corydaline could not be determined. Instead, we compared hepatic corydaline metabolic activity between male and female rats by measuring disappearance of corydaline in hepatic microsomal fractions within the incubation time. The concentrations of spiked corydaline were determined based on its in vivo hepatic concentrations such that the mean hepatic concentrations of corydaline were approximately 40 mM at 120 min and 13 mM at 360 min after oral administration of DA-9701 at a dose equivalent to 4.5 mg/kg corydaline (unpublished data). We observed significantly slower metabolism in hepatic microsomes of female rats compared to male rats of both low (2 mM) and high (20 mM) concentrations of corydaline (Figure 2), which confirmed the slower hepatic metabolism in female rats. After oral administration of corydaline, AUC and Cmax values in female rats were significantly greater than those in male rats (Table 2). The oral AUC of a drug that is eliminated primarily by the liver without reference to its hepatic extraction ratio is determined by the following equation (Benet & Hoener, 2002): AUCoral ¼ Fabs Fg Dose =ðf u CLint Þ in which Fabs is the fraction of administered drug that is absorbed into the gut wall, Fg is the fraction that passes through the gut wall unchanged, fu is free (unbound) fraction in plasma, and CLint is intrinsic hepatic (primary elimination organ) clearance of the unbound drug. Since absorption of corydaline from the GI tract is almost complete in both male and female rats based on a GI24 h 51% of the dose, there might be no difference in the Fabs between male and female rats. This is supported by the stability of corydaline in the human
Gender-different PKs of corydaline in rats
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DOI: 10.3109/00498254.2014.988772
intestinal flora and its high permeability through a Caco-2 cell monolayer via passive diffusion (Liu et al., 2013). Based on low F values and intermediate hepatic extraction ratio of corydaline, considerable GI first-pass extraction of corydaline was suspected following its oral administration. However, we could not reveal the possible differences in Fg between male and female rats due to negligible metabolism of corydaline in intestinal microsomal fractions from male and female rats (data not shown). It is obvious that the significantly smaller fu and slower CLint values in female compared to male rats contributed to the greater oral AUC of corydaline in female rats. The AUC and Cmax values of corydaline were also significantly greater in female rats than in male rats following oral administration of DA-9701 (Table 3). In female rats, smaller hepatic first-pass extraction of corydaline as a result of its slower hepatic metabolism than in male rats could result in greater F values following oral administration of corydaline or DA-9701. The considerably greater gender differences in the oral compared to the intravenous AUC could be due in part to the non-linear pharmacokinetic property of corydaline. In female rats, significant difference was observed in the oral exposure (AUC, p50.05; Cmax, p50.001) of corydaline when given equivalent dose as pure corydaline versus as DA-9701 (Tables 2 and 3). This contributed to the smaller gender differences in AUC and Cmax values of corydaline following DA-9701 administration than those of pure corydaline. One of the possible reasons for this phenomenon could be delayed absorption of corydaline in female rats based on the later Tmax following DA-9701 administration. When given as DA-9701, the slowed absorption rate of corydaline could increase its first-pass metabolism. Note that corydaline metabolism is not expected to show a gender difference in humans based on the comparable disappearance of corydaline in human liver microsomes from a male or female pool (Figure 6). The major human hepatic enzymes involved in the metabolism of corydaline, CYP2C9 and CYP3A4, show little gender difference in humans. Reported CYP2C9 activities of human liver microsomes from males and females showed no significant difference (Parkinson et al., 2004), although the CYP3A4 activity of human liver microsomes was slightly greater in females than in males, and a significant gender difference was reported in cryopreserved human hepatocytes (Parkinson et al., 2004). However, the gender difference in CYP3A activity in humans is small compared to the marked sex difference (>10-fold) in rats. In addition, CYP3A4 activity varies considerably within genders due to environmental factors that induce or suppress the enzyme. According to our preliminary study of corydaline metabolism in human liver microsomes, the pharmacokinetic gender differences in rats are likely not replicated in humans. Moreover, factors that affect CYP2C9 or CYP3A4, such as drug-drug interactions, are considered to be more clinically significant in terms of the pharmacokinetics of corydaline.
Conclusions This study revealed gender differences in corydaline pharmacokinetics in rats: AUC and F values were greater in female
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rats as a result of their slower corydaline metabolism. The gender difference in metabolic activity was due primarily to male-specific (CYP2C11 and CYP3A2) or male-dominant (CYP3A1) CYP isozymes involved in corydaline metabolism. However, the gender difference in corydaline pharmacokinetics identified in rats is not expected to occur in humans.
Declaration of interest The authors have declared that there is no conflict of interest. This work was supported by the Global Leading Technology Program of the Office of Strategic R&D Planning (OSP) funded by the Ministry of Knowledge Economy, Republic of Korea (No. 10039321) and by the Research Fund, 2012 of The Catholic University of Korea (M-2012-B0002-00031).
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Lee MG, Chiou WL. (1983). Evaluation of potential causes for the incomplete bioavailability of furosemide: gastric first-pass metabolism. J Pharmacokinet Biopharm 11:623–40. Lee TH, Choi JJ, Kim DH, et al. (2008). Gastroprokinetic effects of DA9701, a new prokinetic agent formulated with Pharbitis Semen and Corydalis tuber. Phytomedicine 15:836–43. Lee TH, Son M, Kim SY. (2010). Effects of corydaline from Corydalis tuber on gastric motor function in an animal model. Biol Pharm Bull 33:958–62. Lewis DFV. (1996). Cytochromes P450 structure, function and mechanism. London: Taylor & Francis. Liu YZ, Yang XB, Yang XW, et al. (2013). Biotransformation by human intestinal flora and absorption-transportation characteristic in a model of Caco-2 cell monolayer of d-corydaline and tetrahydropalmatine. Zhongguo Zhong Yao Za Zhi 38:112–18. Matsuda H, Tokuoka K, Wu J, et al. (1995). Inhibitory effects of methanolic extract from corydalis tuber against types I–IV allergic models. Biol Pharm Bull 18:963–7.
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Øie S, Guentert TW. (1982). Comparison of equilibrium time in dialysis experiments using spiked plasma or spiked buffer. J Pharm Sci 71: 127–8. Parkinson A, Mudra DR, Johnson C, et al. (2004). The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes. Toxicol Appl Pharmacol 199:193–209. Soucek P, Gut I. (1992). Cytochromes P-450 in rats: structures, functions, properties and relevant human forms. Xenobiotica 22:83–103. Wilkinson GR, Shand DG. (1975). Commentary: a physiological approach to hepatic drug clearance. Clin Pharmacol Ther 18: 377–90. Wojcikowski J, Haduch A, Daniel WA. (2012). Effect of classic and atypical neuroleptics on cytochrome P450 3A (CYP3A) in rat liver. Pharmacol Rep 64:1411–8. Yuan CS, Mehendale SR, Wang CZ, et al. (2004). Effects of Corydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans: a controlled trial. J Clin Pharmacol 44:1323–7.
RESEARCH ARTICLE
Gender differences in corydaline pharmacokinetics in rats Ji Won Jung1, Mi Ran Choi1, Yong Sam Kwon2, Jin Seok Jeong2, Miwon Son2, and Hee Eun Kang1 1
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College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon, Republic of Korea and 2Research Center, Dong-A Pharmaceutical Co., Yongin, Republic of Korea Abstract
Keywords
1. Corydaline, an isoquinoline alkaloid, is one of the major active constituents in a new prokinetic botanical agent, DA-9701. It has been recommended that preclinical pharmacokinetic studies of natural medicines include both genders. Therefore, in this study, the pharmacokinetics of corydaline in male and female rats was evaluated following intravenous and oral administration of pure corydaline or DA-9701. 2. After intravenous administration of corydaline, the area under the plasma concentration– time curve (AUC) was significantly greater (by 46.4%) in female rats compared to male rats due to a 29.3% reduction in non-renal clearance in female rats. The gender difference in corydaline hepatic metabolic clearance was supported by a significantly slower metabolism of corydaline in hepatic microsomes of female rats mediated via male-specific (CYP2C11 and CYP3A2) or male-dominant (CYP3A1) CYP isozymes. 3. Following oral administration of pure corydaline or DA-9701, the AUC and Cmax values of corydaline in female rats were significantly greater (by 793% and 466% increase for corydaline administration or by 501% and 143% increase for DA-9701 administration) than in male rats. Greater F values of corydaline in female rats could be due to smaller hepatic first-pass extraction as a result of slower hepatic metabolism of corydaline. 4. However, we observed a comparable disappearance of corydaline in male and female human liver microsomes, consistent with little gender difference in CYP2C9 and CYP3A activities in humans compared to that in rats. Thus, gender differences in corydaline metabolism are not expected to occur in humans.
Corydaline, CYP2C, CYP3A, DA-9701, gender, pharmacokinetics, rats
Introduction The corydalis tuber has anti-inflammatory, anti-allergic, and analgesic effects (Kubo et al., 1994; Matsuda et al., 1995; Yuan et al., 2004) that have led to its use as an analgesic or anti-spasmodic agent in Asian countries (Lee & Suh, 2012). Corydalis tuber from the roots of Corydalis yanhusuo W. T. Wang is the primary constituent of the herbal medicine DA9701, a new botanical drug marketed as oral tablets (MotilitoneÕ ) for the treatment of functional dyspepsia in Republic of Korea since May 2011. DA-9701 is formulated with dried 50% ethanolic extracts of Pharbitidis semen from the seed of Pharbitis nil Choisy and Corydalis tuber from the roots of Corydalis yanhusuo W. T. Wang (1:5, w/w). An investigational new drug (IND) application of DA-9701 for a phase II clinical trial will be submitted to the United States Federal Drug Administration (FDA) this year. In addition, a bridging study for marketing DA-9701 in China is in preparation.
Address for correspondence: Prof. Hee Eun Kang, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon 420-743, Republic of Korea. E-mail: [email protected]
History Received 14 October 2014 Revised 13 November 2014 Accepted 13 November 2014 Published online 28 November 2014
The effects of DA-9701 as a prokinetic agent have been reported in various animal models (Jung et al., 2013; Kim et al., 2012; Lee et al., 2008). These effects are likely mediated by the induction of pacemaker currents in the interstitial cells of Cajal of the small intestine, which are required for gastrointestinal motility (Choi et al., 2009), along with antagonistic effects on the dopamine D2 receptor and agonistic effects on serotonin 5-HT4, 5-HT1A, and 5-HT1B receptors (Kim et al., 2012). DA-9701 produces strong gastroprokinetic effects at an effective dose of 0.3–3 mg/kg in rats and has a superior safety profile compared to conventional prokinetics, such as cisapride, mosapride, itopride and domperidone (Lee et al., 2008). Corydaline [(13S,13aR)-2,3,9,10-tetramethoxy-13-methyl6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline; Figure 1], an isoquinoline alkaloid, is an active ingredient in Corydalis tuber and DA-9701. High-performance liquid chromatography (HPLC) performed for batch-to-batch quality control revealed that corydaline is one of the major constituents in DA-9701 (Kwon & Son, 2013). Corydaline promotes gastric emptying and small intestinal transit, and facilitates gastric accommodation (Lee et al., 2010). Various metabolites of corydaline have been found and O-demethylation and
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Figure 1. Chemical structure of corydaline.
hydroxylation are the major metabolic pathways in human liver microsomes and hepatocytes (Ji et al., 2012). Cytochrome P450 (CYP) 2C9 and 3A4 were reported as major enzymes involved in the formation of the major metabolite, O-desmethylcorydaline (M1) from corydaline (Ji et al., 2011a). Corydaline inhibits activities of metabolic enzymes in human liver microsomes with potent inhibition of CYP 2C19 and 2C9, and moderate inhibition of UDP-glucuronosyl transferase 1A1 and 1A9 (Ji et al., 2011b). Recently, we reported the pharmacokinetics of corydaline in rats following various doses of pure corydaline and DA-9701 (Jung et al., 2014). Corydaline showed dose-dependent pharmacokinetics after intravenous and oral administration (1.1–4.5 mg/kg) due to saturation of corydaline metabolism at higher doses. As a result, the extent of absolute oral bioavailability (F) of corydaline following its oral administration varied considerably (9.10–21.1%) according to the dose administered. The Technical Requirements for the Study of New Natural Medicines issued recently by the State Food and Drug Administration in China recommended that a preclinical pharmacokinetic study of a new natural medicine in both genders be performed and gender differences confirmed. Knowledge on gender differences in pharmacokinetics can help understanding of possible gender-different preclinical data of the drug. Therefore, in this study, the pharmacokinetics of corydaline in male and female rats were investigated following intravenous and oral administration of pure corydaline or oral administration of DA-9701.
Materials and methods Materials Corydaline and DA-9701 (Lot No. CP2410, 1.38% of corydaline) were supplied by Dong-A Pharmaceutical Co. (Yongin, Republic of Korea). Corydaline was a product of Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and DA-9701 was a product of Dong-A Pharmaceutical Co. Verapamil, the reduced form of b-nicotinamide adenine dinucleotide phosphate (NADPH; as tetrasodium salt), polysorbate (Tween)-80, dimethylsulfoxide (DMSO) and ammonium acetate were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Human liver microsomes pooled from either males or females were products of BD Biosciences (Woburn, MA). Other chemicals were of reagent or HPLC grade. Animals Animal study protocols were approved by the Department of Laboratory Animals, Institutional Animal Care and Use
Committee on the Sungsim Campus of The Catholic University of Korea (Approval No. 2011-015; Bucheon, Republic of Korea). Age-matched (8–9 weeks old) male (280–330 g) and female (190–230 g) Sprague–Dawley (SD) rats, were purchased from Charles River Company Korea (Seoul, Republic of Korea). The procedures for housing and handling the rats were in accord with a reported method (Kim et al., 2013). Disappearance of corydaline in hepatic microsomal fractions Hepatic microsomal fraction from male and female rats (n ¼ 4 each) were prepared according to a reported method (Lee & Lee, 2008). Protein contents in the microsomal fraction were measured using a reported method (Bradford, 1976). The hepatic microsomal fractions (equivalent to 0.5 mg protein) were mixed with 5 mL of methanol containing corydaline to final concentrations of 2 and 20 mM, and 25 mL of 0.1 M phosphate buffer (pH 7.4) containing 1 mM NADPH. The volume was adjusted to 0.25 mL by adding 0.1 M phosphate buffer (pH 7.4). The components were incubated in a thermomixer [Thermomixer 5436; Eppendorf, Hamburg, Germany; 37 C, 500 revolutions per min (rpm)]. At 0, 10, 20, 30, 45, and 60 min of incubation, 20 mL were collected and added to an Eppendorf tube containing 50 mL of acetonitrile with 10 ng/mL verapamil (internal standard) and vortex-mixed to terminate the reaction. The disappearance of corydaline in male and female pools of human liver microsomes (equivalent to 1-mg protein) was also measured in triplicate according to the method described above. Intravenous administration of corydaline For intravenous administration, the carotid artery and jugular vein were cannulated using procedures reported previously (Kim et al., 2013). Corydaline [dissolved in 1:2:7 (v/v/v) DMSO: Tween 80: distilled water] at a dose of 1.1 mg (2 mL)/ kg was infused for 1 min via the jugular vein of male and female rats (n ¼ 7, each). A blood sample of 50 mL was collected via the carotid artery at time 0 (prior to dosing), 1 (at the end of the infusion), 5, 15, 30, 60, 90, 120, 180, 240, 360 and 480 min after the start of infusion. Blood samples were immediately centrifuged and 20 mL of each plasma sample was stored at 70 C until used for the liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of corydaline. The preparation and handling of the 24-h urine samples (Ae0–24 h) and gastrointestinal tract (GI) samples (including its contents and feces) at 24 h (GI24 h) were performed according to a reported method (Kim et al., 2013). Oral administration of corydaline and DA-9701 For the oral administration study, rats were fasted overnight with free access to water, and the carotid artery was cannulated using procedures reported previously (Kim et al., 2013). Corydaline at a dose of 4.5 mg (3 mL)/kg or the equivalent dose of DA-9701 (328 mg/kg) [dissolved in 1:2:12 (v/v/v) DMSO: Tween 80: distilled water] was administered orally using a gastric gavage tube to male and female rats (n ¼ 7 and 8, respectively, for corydaline; n ¼ 8 and 9, respectively, for
Gender-different PKs of corydaline in rats
DOI: 10.3109/00498254.2014.988772
DA-9701). A blood sample of 50 mL was collected via the carotid artery at 0, 5, 15, 30, 45, 60, 90, 120, 180, 240, 360 and 480 min after oral administration of the drug. Other procedures for the oral study were same as those for the intravenous study.
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Measurement of protein binding of corydaline in plasma from male and female rats using equilibrium dialysis Protein binding of corydaline to fresh plasma from male and female rats (n ¼ 5 and 4, respectively) was assessed by equilibrium dialysis (Jung et al., 2014). Fresh plasma (0.2 mL) was dialyzed against 0.2 mL of isotonic Sørensen phosphate buffer (pH 7.4) containing 3% (w/v) dextran (‘the buffer’) to minimize volume shift (Boudinot & Jusko, 1984) using a 0.25 mL micro-equilibrium dialyzer (Harvard Apparatus, Holliston, MA) and a regenerated cellulose membrane (molecular weight cut-off 10 kDa, Harvard Apparatus). To reduce the equilibrium time of corydaline between the buffer and plasma compartments, the drug was spiked into the plasma side (Øie & Guentert, 1982) to a final concentration of 1 mg/mL. The spiked dialysis cell was incubated in a waterbath shaker kept at 37 C and at a rate of 50 oscillations per min (opm). After 24 h, two 20 mL aliquots were removed from each compartment and stored at 70 C until LC-MS/MS analysis.
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the rat plasma samples were 0.5–5000 ng/mL, and those in the urine and GI24 h samples were 2.5–5000 ng/mL. The mean intra- and inter-day coefficients of variation (CV) of the analysis on 3 consecutive days were below 5.05%, and the assay accuracies ranged from 86.5 to 112%. Pharmacokinetic analysis The total area under the plasma concentration–time curve from time zero to last measured time, t (AUC0–t) and that from time zero to infinity (AUC) were calculated using the trapezoidal rule-extrapolation method (Chiou, 1978). Standard methods (Gibaldi & Perrier, 1982) were used to calculate the following pharmacokinetic parameters using a non-compartmental analysis (WinNonlinÕ ; Pharsight Corporation, Mountain View, CA): the time-averaged total body, renal, and non-renal clearances (CL, CLR, and CLNR, respectively), terminal halflife, mean residence time (MRT), and apparent volume of distribution at steady state (Vss). The maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) were read directly from the experimental data. For comparison, the extent of absolute oral bioavailability (F) was calculated by dividing the dose-normalized AUC values after oral administration by the dose-normalized AUC values after intravenous administration. Statistical analysis
LC-MS/MS analysis of corydaline The LC-MS/MS system comprised an Agilent 6460 triple quadrupole mass spectrometer with an Agilent 1260 LC system (Agilent, Waldbronn, Germany). Instrument control and data acquisition were performed using the Agilent MassHunter Workstation software (Version B. 04. 01). Concentrations of corydaline in the samples were determined using the LC-MS/MS method developed in our laboratory (Jung et al., 2014). In brief, 50 mL of acetonitrile containing 10 ng/mL of verapamil as the internal standard (IS) was added to 20 mL of each biological sample. After mixing and centrifugation (16,000 g, 5 min), the supernatant was collected and 3 mL injected directly onto a reversed-phase HPLC column (KinetexÕ C18; 4.6 mm i.d. 50 mm l.; particle size, 2.6 mm; Phenomenex, Torrance, CA). The mobile phase, 2 mM ammonium acetate buffer: acetonitrile [40:60 (v/v)], was run through the column at a flow rate of 0.4 mL/min. The temperature of the column was maintained at 35 C and the autosampler at 4 C. The eluent was monitored using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source with positive ion (ESI+; 2–10 min) modes, with multiple reaction monitoring. The instrument parameters were set as follows: nitrogen gas temperature of 210 C, sheath gas temperature of 400 C, gas flow of 5 L/min, sheath gas flow of 12 L/min, nebulizer of 55 psi, and electrospray voltage of 3.5 kV. The fragmentor voltage was set at 165 V for corydaline, and 115 V for the IS. The collision energy for corydaline and the IS was 28 and 20 eV, respectively. The precursor to product ion transitions for corydaline and IS were m/z 370.2 ([M + H]+) ! 192.1 and m/z 455.3 ([M + H]+) ! 165, respectively. The retention times of corydaline and the IS were approximately 5.2 and 8.3 min, respectively. The calibration ranges of corydaline in
A p value 50.05 was deemed to indicate statistical significance using an unpaired two-tailed t-test between the two means. All data were expressed as means ± SD, except medians (range) for Tmax.
Results Disappearance of corydaline in hepatic microsomal fractions from male and female rats Body weight-normalized total hepatic microsomal protein contents were not significantly different between male and female rats. The percentages of the spiked amounts of corydaline (2 and 20 mM) remaining after 10-, 20-, 30-, 45-, and 60-min incubations with hepatic microsomal fractions from male and female rats are shown in Figure 2. In female rats, the remaining values at each incubation time were significantly greater than those in male rats. Following 60min incubation, 80% of spiked amounts were recovered in female rats, while 13–30% of spiked amounts were recovered in male rats. These data suggested that hepatic metabolism of corydaline was slower in female rats than in male rats. Pharmacokinetics of corydaline after its intravenous administration to male and female rats The mean arterial plasma concentration–time profiles of corydaline after its intravenous administration at a dose of 1.1 mg/kg to male and female rats are shown in Figure 3. The relevant pharmacokinetic parameters are listed in Table 1. Note that the AUC0–8 h and AUC of corydaline in female rats were 32.4% and 46.4% greater, respectively, than that in male rats, as a result of 28.9% slower CL and 29.3% slower CLNR in female rats. We also observed 167% faster CLR, 30.9%
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Percentage of spiked corydaline remaining in hepatic microsomes (%)
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Figure 3. Mean arterial plasma concentration–time profiles of corydaline following its 1-min intravenous administration at a dose of 1.1 mg/kg to male and female rats (n ¼ 7 each). Vertical bars represent SD.
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Table 1. Pharmacokinetic parameters of corydaline after its intravenous administration (1.1 mg/kg) to male and female rats. Parameters
Male rats (n ¼ 7)
Female rats (n ¼ 7)
Body weight (g) AUC0–8 h (mg min/mL) AUC (mg min/mL) Terminal half-life (min) CL (mL/min/kg) CLR (mL/min/kg) CLNR (mL/min/kg) Vss (mL/kg) MRT (min) Ae0–24 h (% of dose) GI24 h (% of dose)
310 ± 11.6 105 ± 10.7 112 ± 11.0 188 ± 35.1 9.90 ± 1.02 0.0214 ± 0.00681 9.88 ± 1.02 1150 ± 289 116 ± 25.1 0.221 ± 0.0831 0.275 ± 0.109
227 ± 4.88*** 139 ± 29.4* 164 ± 41.3** 246 ± 36.6* 7.04 ± 1.48** 0.0571 ± 0.0242** 6.99 ± 1.48** 1250 ± 291 183 ± 50.6** 0.874 ± 0.465** 0.588 ± 0.335*
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Figure 2. Mean percentages of the spiked amounts of corydaline, 2 mM (A) and 20 mM (B), remaining in hepatic microsomes from male and female rats (n ¼ 4 each) at 10, 20, 30, 45, and 60 min after the start of incubation. Vertical bars represent SD. Significant difference from male rats, ***p50.001.
longer terminal half-life, 57.8% longer MRT, 295% greater Ae0–24 h, and 114% greater GI24 h values in females than in male rats. Pharmacokinetics of corydaline after its oral administration to male and female rats The mean arterial plasma concentration–time profiles of corydaline after its oral administration at a dose of 4.5 mg/kg to male and female rats are shown in Figure 4. The relevant pharmacokinetic parameters are listed in Table 2. Note that the AUC0–8 h, AUC, and Cmax values of corydaline in female rats were 695%, 793%, and 466% greater than in male rats, respectively. Moreover, the F value of corydaline in female rats (55.7%) was considerably greater than that in male rats
Data are expressed as mean ± SD. The value was significantly different from male rats (*p50.05, **p50.01, ***p50.001).
(9.14%). The GI24 h value was 815% greater in female rats compared to male rats, but the values in male and female rats were both negligible at 51% of the oral dose. Pharmacokinetics of corydaline after oral administration of DA-9701 to male and female rats The mean arterial plasma concentration–time profiles of corydaline after oral administration of DA-9701 at a dose equivalent to 4.5 mg/kg corydaline to male and female rats are shown in Figure 5. The relevant pharmacokinetic parameters are listed in Table 3. Note that the AUC0–8 h, AUC, and Cmax values of corydaline in female rats were 398%, 501%, and 143% greater than in male rats, respectively. As a result, the F value of corydaline in female rats (29.7%) was also considerably greater than that in male rats (7.22%). The Ae0– 24 h value was 475% greater in female rats compared to that in male rats, but the values of both male and female rats were negligible at 50.1% of the oral dose.
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Figure 4. Mean arterial plasma concentration–time profiles of corydaline following its oral administration at a dose of 4.5 mg/kg to male (n ¼ 7) and female (n ¼ 8) rats. Vertical bars represent SD.
Figure 5. Mean arterial plasma concentration–time profiles of corydaline following oral administration of 328 mg/kg DA-9701 (equivalent to 4.5 mg/kg corydaline) to male (n ¼ 8) and female (n ¼ 9) rats. Vertical bars represent SD.
Table 2. Pharmacokinetic parameters of corydaline after its oral administration (4.5 mg/kg) to male and female rats.
Table 3. Pharmacokinetic parameters of corydaline after oral administration of DA-9701 (328 mg/kg, equivalent to 4.5 mg/kg corydaline) to male and female rats.
Parameters
Male rats (n ¼ 7)
Female rats (n ¼ 8)
Body weight (g) AUC0–8 h (mg min/mL) AUC (mg min/mL) Terminal half-life (min) CLR (mL/min/kg) Cmax (ng/mL) Tmax (min)a Ae0–24 h (% of dose) GI24 h (% of dose) F (%)b
291 ± 12.2 34.7 ± 13.7 41.9 ± 12.2 280 ± 206 0.0141 ± 0.00955 419 ± 233 30 (15–90) 0.0128 ± 0.00875 0.0615 ± 0.0370 9.14
200 ± 9.26*** 276 ± 142*** 374 ± 175*** 324 ± 137 0.0253 ± 0.0511 2370 ± 986*** 30 (15–45) 0.375 ± 0.912 0.563 ± 0.607* 55.7
Data are expressed as mean ± SD. The value was significantly different from male rats (*p50.05, ***p50.001). a Tmax is expressed as median (range). b The extent of absolute oral bioavailability (F) value was calculated by the equation; (AUCoral Doseiv,)/(AUCiv Doseoral).
Protein binding of corydaline to fresh rat plasma from male and female rats The plasma protein binding values of corydaline in male and female rats were 93.7 ± 1.36% (free fraction of 6.26%) and 95.7 ± 0.260% (free fraction of 4.34%), respectively. The values were significantly different between the two groups (p50.05, t-test). Almost complete recovery of corydaline (101 ± 4.70%) from both plasma and buffer sides indicated that non-selective adsorption of corydaline to equilibrium dialysis apparatus was negligible. Disappearance of corydaline in human hepatic microsomal fractions from male pool and female pool The percentages of the spiked amounts of corydaline (2 and 20 mM) remaining after 10-, 20-, 30-, 45-, and 60-min incubations with human liver microsomes from male and
Parameters
Male rats (n ¼ 8)
Female rats (n ¼ 9)
Body weight (g) AUC0–8 h (mg min/mL) AUC (mg min/mL) Terminal half-life (min) CLR (mL/min/kg) Cmax (ng/mL) Tmax (min)a Ae0–24 h (% of dose) GI24 h (% of dose) F (%)b
318 ± 8.84 26.5 ± 10.5 33.1 ± 12.3 252 ± 85.6 0.0166 ± 0.00732 269 ± 159 30 (15–90) 0.0114 ± 0.00440 0.952 ± 1.56 7.22
209 ± 10.2*** 132 ± 31.4*** 199 ± 68.3*** 312 ± 100 0.0136 ± 0.00897 655 ± 227** 60 (15–180) 0.0655 ± 0.0582* 0.639 ± 1.32 29.7
Data are expressed as mean ± SD. The value was significantly different from male rats (*p50.05, **p50.01, ***p50.001). a Tmax is expressed as median (ranges). b The extent of absolute oral bioavailability (F) value was calculated by the equation; (AUCoral Doseiv,)/(AUCiv Dose as corydalineoral).
female pools are shown in Figure 6. Significantly greater remaining values of corydaline in male pool microsomes were observed compared to those in female pool microsomes at 10, 20, and 30 min after the start of incubation with 20 mM corydaline. However, there was no statistically significant difference in recovery of corydaline after 45- and 60-min incubation.
Discussion The doses of corydaline used in this study, 1.1 mg/kg for the intravenous experiments and 4.5 mg/kg for the oral experiments, were determined based on the previous pharmacokinetic study on corydaline in rats (Jung et al., 2014). After intravenous administration of corydaline to female rats, we observed significantly greater AUC compared to male rats (Table 1) due to significantly slower CL and CLNR values.
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Percentage of spiked corydaline remaining in hepatic microsomes (%)
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2 µM of corydaline Male Female 100
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0
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20 µM of corydaline 120
Percentage of spiked corydaline remaining in hepatic microsomes (%)
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0
Male Female
100
* 80
** ***
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0 0
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Figure 6. Mean percentages of the spiked amounts of corydaline, 2 mM (A) and 20 mM (B), remaining in human hepatic microsomes from male pool and female pool (in triplicate) at 10, 20, 30, 45, and 60 min after the start of incubation. Vertical bars represent SD. Significant difference from male, *p50.05, **p50.01, ***p50.001.
Based on negligible Ae0–24 h and GI24 h values (51% of the dose) after intravenous administration of corydaline, the contributions of CLR to the CL and gastrointestinal, including biliary, excretion of unchanged corydaline to its CLNR were negligible. This suggests that most of the administered corydaline was eliminated via non-renal metabolic clearance. Therefore, the significantly greater AUC and slower CLNR in female rats could be due to slower metabolism of corydaline. Corydaline is a drug with an intermediate hepatic extraction ratio, estimated to be 38.9–79.9% (Jung et al., 2014) with a reported equation (Lee & Chiou, 1983). Therefore, hepatic clearance of corydaline depends on the intrinsic clearance of the unbound drug (CLint), free (unbound) fraction in plasma (fu), and hepatic blood flow rate (QH) (Wilkinson & Shand, 1975). Gender differences in rat QH have not been published.
Although the liver weights of female rats were significantly smaller than those of male rats, the values as a percentage of body weight were comparable between the two groups of rats. Therefore, little difference in QH values between male and female is expected, indicating that QH did not contribute to the slower metabolism of corydaline in female rats. We observed a 30.7% reduced free fraction of corydaline in plasma of female rats, as well as slower hepatic CLint compared to males, which could contribute to the slower hepatic clearance in female rats. The major human hepatic enzymes involved in the metabolism of corydaline are CYP2C9 and CYP3A4 (Ji et al., 2011a). They are 77% homologous to rat CYP2C11 and 73% homologous to rat CYP3A1 amino acid sequence (Soucek & Gut, 1992). Rat CYP3A2 also exhibits similar catalytic competence and functional analogies to humans CYP3A4 (Wojcikowski et al., 2012). These hepatic CYP enzymes in rats are expressed in a sex-specific manner; CYP2C11 and CYP3A2 are the male-specific forms expressed 10-fold higher than in females, and CYP3A1 is a male-dominant form (Lewis, 1996; Parkinson et al., 2004). Therefore, the slower metabolism of corydaline in female rats is likely due to the slower hepatic CLint compared to male rats. To confirm slower metabolism of corydaline in female rats, we evaluated in vitro hepatic intrinsic clearance of corydaline in microsomes from male and female rats. However, the metabolism of corydaline in female rats was so slow that the intrinsic clearance of corydaline could not be determined. Instead, we compared hepatic corydaline metabolic activity between male and female rats by measuring disappearance of corydaline in hepatic microsomal fractions within the incubation time. The concentrations of spiked corydaline were determined based on its in vivo hepatic concentrations such that the mean hepatic concentrations of corydaline were approximately 40 mM at 120 min and 13 mM at 360 min after oral administration of DA-9701 at a dose equivalent to 4.5 mg/kg corydaline (unpublished data). We observed significantly slower metabolism in hepatic microsomes of female rats compared to male rats of both low (2 mM) and high (20 mM) concentrations of corydaline (Figure 2), which confirmed the slower hepatic metabolism in female rats. After oral administration of corydaline, AUC and Cmax values in female rats were significantly greater than those in male rats (Table 2). The oral AUC of a drug that is eliminated primarily by the liver without reference to its hepatic extraction ratio is determined by the following equation (Benet & Hoener, 2002): AUCoral ¼ Fabs Fg Dose =ðf u CLint Þ in which Fabs is the fraction of administered drug that is absorbed into the gut wall, Fg is the fraction that passes through the gut wall unchanged, fu is free (unbound) fraction in plasma, and CLint is intrinsic hepatic (primary elimination organ) clearance of the unbound drug. Since absorption of corydaline from the GI tract is almost complete in both male and female rats based on a GI24 h 51% of the dose, there might be no difference in the Fabs between male and female rats. This is supported by the stability of corydaline in the human
Gender-different PKs of corydaline in rats
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DOI: 10.3109/00498254.2014.988772
intestinal flora and its high permeability through a Caco-2 cell monolayer via passive diffusion (Liu et al., 2013). Based on low F values and intermediate hepatic extraction ratio of corydaline, considerable GI first-pass extraction of corydaline was suspected following its oral administration. However, we could not reveal the possible differences in Fg between male and female rats due to negligible metabolism of corydaline in intestinal microsomal fractions from male and female rats (data not shown). It is obvious that the significantly smaller fu and slower CLint values in female compared to male rats contributed to the greater oral AUC of corydaline in female rats. The AUC and Cmax values of corydaline were also significantly greater in female rats than in male rats following oral administration of DA-9701 (Table 3). In female rats, smaller hepatic first-pass extraction of corydaline as a result of its slower hepatic metabolism than in male rats could result in greater F values following oral administration of corydaline or DA-9701. The considerably greater gender differences in the oral compared to the intravenous AUC could be due in part to the non-linear pharmacokinetic property of corydaline. In female rats, significant difference was observed in the oral exposure (AUC, p50.05; Cmax, p50.001) of corydaline when given equivalent dose as pure corydaline versus as DA-9701 (Tables 2 and 3). This contributed to the smaller gender differences in AUC and Cmax values of corydaline following DA-9701 administration than those of pure corydaline. One of the possible reasons for this phenomenon could be delayed absorption of corydaline in female rats based on the later Tmax following DA-9701 administration. When given as DA-9701, the slowed absorption rate of corydaline could increase its first-pass metabolism. Note that corydaline metabolism is not expected to show a gender difference in humans based on the comparable disappearance of corydaline in human liver microsomes from a male or female pool (Figure 6). The major human hepatic enzymes involved in the metabolism of corydaline, CYP2C9 and CYP3A4, show little gender difference in humans. Reported CYP2C9 activities of human liver microsomes from males and females showed no significant difference (Parkinson et al., 2004), although the CYP3A4 activity of human liver microsomes was slightly greater in females than in males, and a significant gender difference was reported in cryopreserved human hepatocytes (Parkinson et al., 2004). However, the gender difference in CYP3A activity in humans is small compared to the marked sex difference (>10-fold) in rats. In addition, CYP3A4 activity varies considerably within genders due to environmental factors that induce or suppress the enzyme. According to our preliminary study of corydaline metabolism in human liver microsomes, the pharmacokinetic gender differences in rats are likely not replicated in humans. Moreover, factors that affect CYP2C9 or CYP3A4, such as drug-drug interactions, are considered to be more clinically significant in terms of the pharmacokinetics of corydaline.
Conclusions This study revealed gender differences in corydaline pharmacokinetics in rats: AUC and F values were greater in female
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rats as a result of their slower corydaline metabolism. The gender difference in metabolic activity was due primarily to male-specific (CYP2C11 and CYP3A2) or male-dominant (CYP3A1) CYP isozymes involved in corydaline metabolism. However, the gender difference in corydaline pharmacokinetics identified in rats is not expected to occur in humans.
Declaration of interest The authors have declared that there is no conflict of interest. This work was supported by the Global Leading Technology Program of the Office of Strategic R&D Planning (OSP) funded by the Ministry of Knowledge Economy, Republic of Korea (No. 10039321) and by the Research Fund, 2012 of The Catholic University of Korea (M-2012-B0002-00031).
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