Pharmacokinetics of ipriflavone and its two metabolites, M1 and M5, after the intravenous and oral administration of ipriflavone to rat model of diabetes mellitus induced by streptozotocin

European Journal of Pharmaceutical Sciences 38 (2009) 465–471

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Pharmacokinetics of ipriflavone and its two metabolites, M1 and M5, after the intravenous and oral administration of ipriflavone to rat model of diabetes mellitus induced by streptozotocin Dae Y. Lee a,1 , Hye J. Chung a,b , Young H. Choi a , Unji Lee c , So H. Kim d , Inchul Lee e , Myung G. Lee b,∗ a

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, South Korea Center for Chemoinformatics, Life Sciences Research Division, Korea Institute of Science and Technology, 66, Haegiro, Dongdaemun-Gu, Seoul 130-741, South Korea College of Pharmacy, Ewha Womans University, 11-1, Daehyun-Dong, Seodaemun-Gu, Seoul 120-750, South Korea d College of Dentistry and Research Institute of Oral Science, Kangnung National University, 120, Daehangno, Gangneung, Gangwon-do 210-702, South Korea e Department of Diagnostic Pathology, College of Medicine, University of Ulsan, Asan Foundation, Asan Medical Center, 388-1, Pungnap-Dong, Songpa-Gu, Seoul 138-736, South Korea b c

a r t i c l e

i n f o

Article history: Received 30 May 2009 Received in revised form 25 August 2009 Accepted 6 September 2009 Available online 15 September 2009 Keywords: Ipriflavone M1 and M5 Diabetes mellitus induced by streptozotocin Pharmacokinetics CYP1A subfamily and 2C11 Rats

a b s t r a c t Ipriflavone was reported to be primarily metabolized via hepatic cytochrome P450 (CYP) 1A1/2 and 2C11 in male Sprague–Dawley rats. The protein expression and/or mRNA levels of hepatic CYP1A subfamily and 2C11 was reported to be increased and decreased, respectively, in diabetic rats induced by streptozotocin (DMIS rats). Thus, the pharmacokinetic parameters of ipriflavone and its two metabolites, M1 and M5, were compared after the i.v. (20 mg/kg) and p.o. (200 mg/kg) administration of ipriflavone to control and DMIS rats. After both i.v. and p.o. administration of ipriflavone to DMIS rats, the AUCs of ipriflavone were significantly smaller (by 31.7% and 34.2% for i.v. and p.o. administration, respectively) than controls. The faster Clnr (smaller AUC) of i.v. ipriflavone could have been due to the faster hepatic Clint (because of an increase in the protein expression and/or mRNA level of hepatic CYP1A subfamily) and the faster hepatic blood flow rate than controls. The smaller AUC of p.o. ipriflavone in DMIS rats could have mainly been due to the faster intestinal Clint (because of an increase in the intestinal CYP1A subfamily) than controls. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Ipriflavone (7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one), a derivative of naturally occurring isoflavones, reduces renal excretion of calcium, enhances calcium-stimulated calcitonin secretion in the presence of estrogen, and improves osteopenia induced by low calcium and low vitamin D diet (Takenaka et al., 1982). It is therefore expected to inhibit bone resorption in animal models of experimental osteoporosis and in osteoporotic patients (Kakai et al., 1992). Thus, it has been orally used in the treatment of osteoporosis (Reginster, 1993). Ipriflavone was extensively metabolized in rats, dogs, and humans and undergoes an extensive first-pass metabolism (Ferenc and István, 1995). Seven metabolites of ipriflavone (M1–M7) have been identified in animals and humans (Reginster, 1993; Rohatagi

∗ Corresponding author. Tel.: +82 2 8807855; fax: +82 2 8898693. E-mail address: [email protected] (M.G. Lee). 1 Present address: Research Laboratory, Dong-A Pharmaceutical Company, Ltd., 47-5, Sanggal-Dong, Giheung-Gu, Yongin 446-905, South Korea. 0928-0987/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2009.09.008

et al., 1997). Ipriflavone was mainly metabolized by oxidation of the isopropyl group or hydroxylation of the ␤-ring followed by phase II glucuronidation or sulfation in the liver. The very small quantities of ipriflavone was present in human plasma and urine and the most frequent metabolites were M1, M2, and M5, suggesting that the pharmacological action of ipriflavone was presumed to be represented by the total potencies of unchanged ipriflavone and its metabolites (Ondelli et al., 1991). M1 and M2 circulated in blood as conjugated forms, but M5 and ipriflavone were recovered as unconjugated forms, suggesting that M5 might greatly contribute to the action of ipriflavone (Ondelli et al., 1991; Reginster, 1993; Rohatagi et al., 1997). After the intravenous (i.v.), oral (p.o.), intraportal, intragastric, and intraduodenal administration of ipriflavone at a dose of 50 mg/kg (as a solid dispersion of ipriflavone and polyvinylpyrrolidone (SIP) to increase the dissolution rate and the extent of absolute oral bioavailability (F) of ipriflavone) to rats, the unabsorbed fraction from the gastrointestinal tract for up to 24 h was approximately 6% of the p.o. dose, F was 24%, hepatic firstpass effect after absorption into the portal vein was less than 6%, and intestinal first-pass effect was 75% of the p.o. dose (Kim and Lee, 2002).

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D.Y. Lee et al. / European Journal of Pharmaceutical Sciences 38 (2009) 465–471

Ipriflavone was reported to be primarily metabolized via hepatic cytochrome P450 (CYP) 1A1/2 and 2C11, but not via CYP2B1/2, 2D1, 2E1, and 3A1/2, in male Sprague–Dawley rats (Chung et al., 2006). Compared to controls, the protein expression and/or mRNA levels of hepatic CYP1A subfamily and 2C11 increased and decreased, respectively, in male Sprague–Dawley rats with diabetes mellitus induced by streptozotocin (DMIS rats) (Ma et al., 1989; Yamazoe et al., 1989; Shimojo et al., 1993; Kim et al., 2005; Sindhu et al., 2006; Ahn et al., 2008). In DMIS rats, the protein expression of intestinal CYP1A1/2 significantly increased and decreased, respectively, compared to controls (our unpublished data). Compared to controls, the activity of intestinal aryl hydrocarbon hydroxylase (a CYP1A1 marker) also increased in DMIS rats (Al-Turk et al., 2008). Thus, it could be expected that the pharmacokinetics of ipriflavone, M1, and M5 after the i.v. and p.o. administration of ipriflavone are altered in DMIS rats. The increased risk of osteoporosis for patients with type 1 diabetes has long been known. For example, a number of studies on adults and children have shown that low bone mineral density, a known risk factor for osteoporosis and bone fracture, was more prevalent in patients with type 1 diabetes than that in the general population (Tuominen et al., 1999; Valerio et al., 2002; Liu et al., 2003; Kemmis and Stuber, 2005). However, no pharmacokinetic studies on ipriflavone, M1, and M5 in patients with diabetes mellitus with respect to CYP isozyme changes in the liver and intestine have yet been reported. Thus, ipriflavone was chosen in this study using DMIS rats as an animal model. The aim of this study is to report the pharmacokinetic changes of ipriflavone, M1, and M5 after the i.v. (20 mg/kg) and p.o. (200 mg/kg) administration of ipriflavone to DMIS rats with respect to changes in the protein expression and/or mRNA levels of CYP1A subfamily and 2C11 in the rat liver and intestine. 2. Methods and materials 2.1. Chemicals Ipriflavone, M1, and M5 were donated from Research Laboratory of Samchundang Pharmaceutical Company (Seoul, South Korea). Polyethylene glycol 400 (PEG 400) was a product from Duksan Chemical Company (Seoul, South Korea). Testosterone (internal standard for the high-performance liquid chromatographic (HPLC) analysis of ipriflavone), phenytoin (internal standard for the HPLC analysis of M1 and M5), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), streptozotocin, and the reduced form of ␤-nicotinamide adenine dinucleotide phosphate (NADPH; as a tetrasodium salt) were purchased from Sigma–Aldrich Corporation (St. Louis, MO). Other chemicals were of reagent or HPLC grade. 2.2. Animals The protocols for the animal studies were approved by Animal Care and Use Committee of College of Pharmacy, Seoul National University, Seoul, South Korea. Male Sprague–Dawley rats (7–8 weeks old, weighing 250–300 g) purchased from Charles River Company Korea (Orient, Seoul, South Korea) were maintained in a clean-room (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University) at a temperature of 20–23 ◦ C with 12 h light/dark cycles (lights on 07:00 and lights off 19:00), and a relative humidity of 50 ± 5%. The rats were housed in metabolic cages (Tecniplast, Varese, Italy) under filtered pathogen-free air, and with food (Samyang Company, Seoul, South Korea) and water available ad libitum.

2.3. Induction of diabetes mellitus in rats by streptozotocin injection Rats were randomly divided into two groups, DMIS and control rats. Freshly prepared streptozotocin at a dose of 45 mg (1 ml)/kg was injected once via the tail vein to the overnight-fasted rats (Kim et al., 2005). An equal volume of the citrate buffer (pH 4.5) was injected into the control rats. On the seventh day after the i.v. administration of streptozotocin (DMIS rats) or the citrate buffer, pH 4.5 (control rats), blood glucose levels were measured using the Medisense Optium kit (Abbott Laboratories, Bedford, MA) and rats with blood glucose levels higher than 250 mg/dl were selected as being diabetic. 2.4. Preliminary study The following preliminary study was performed at the seventh day to measure the liver and kidney functions in DMIS and control rats (n = 5, each). A 12 h urine sample was collected for the measurement of creatinine levels. A plasma sample was also collected for the measurement of total proteins, albumin, urea nitrogen, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and creatinine levels (analyzed by Green Cross Reference Lab., Seoul, South Korea). The whole kidney and liver of each rat were excised, rinsed with 0.9% NaCl-injectable solution, blotted dry with tissue paper, and weighed. Small portions of each organ were fixed in 10% neutral phosphate-buffered formalin and then processed for routine histological examination with hematoxylin–eosin staining. 2.5. Measurement of Vmax , Km , and Clint for the disappearance of ipriflavone in rat hepatic and intestinal microsomes The procedures used for preparation of hepatic and intestinal microsomes were similar to a reported method (Choi et al., 2008). Protein contents in hepatic and intestinal microsomes were measured using a reported method (Bradford, 1976). The Vmax (the maximum velocity) and Km (apparent Michaelis–Menten constant; the concentration at which the rate is one-half of Vmax ) for the disappearance of ipriflavone were determined after incubating the above microsomes (equivalent to 0.5 and 1.5 mg proteins for hepatic and intestinal microsomes, respectively), 5 ␮l of DMSO containing final ipriflavone concentrations of 1, 2, 5, 10, and 50 ␮M, and 50 ␮l of 1 mM NADPH. The volume was adjusted to 0.5 ml by adding 0.1 M phosphate buffer (pH 7.4), and the components were incubated in a thermomixer [Eppendorf, Germany; kept at 37 ◦ C and 500 oscillations/min (opm)]. Incubation times were 5, 15, 30, and 45 min, respectively. All of the above microsomal incubation conditions were in the linear range of the reaction rate. After 15 min (for hepatic microsomes) and 45 min (for intestinal microsomes) incubation, two 50 ␮l of reaction mixture were collected and then the reaction was terminated by addition of 100 ␮l of acetonitrile. The kinetic constants (Km and Vmax ) for the disappearance of ipriflavone were calculated using a non-linear regression method (Duggleby, 1995). The intrinsic clearance (Clint ) for the disappearance of ipriflavone was calculated by dividing the Vmax by the Km . 2.6. i.v. and p.o. administration of ipriflavone The procedures used for the pretreatment of rats including the cannulation (early in the morning on the seventh day) of the carotid artery (for blood sampling) and the jugular vein (for drug administration in the i.v. study) were similar to a reported method (Kim and Lee, 2002). Ipriflavone (dissolved in DMA:PEG 400 = 50:50,

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Table 1 Mean (±S.D.) body weight, blood glucose level, 12 h urine volume, plasma chemistry data, creatinine clearance (Clcr ), and relative liver and kidney weight in DMIS and control rats. For comparison, the ranges in normal (Arbino) rats are added. Parameter Body weight (g) Initiala Finalb Blood glucose (mg/dl) Urine volume (ml/12 h/kg) Plasma Total proteins (g/dl) Albumin (g/dl) Urea nitrogen (mg/dl) GOT (IU/L) GPT (IU/L) Clcr (ml/min/kg) Liver weight (% of body weight) Kidney weight (% of body weight)

Control (n = 5)

DMIS (n = 5)

252 294 116 29.9

± ± ± ±

19.2 30.5 8.69 12.9

248 223 441 179

± ± ± ±

8.37 4.47c 43.1c 74.4c

5.86 3.82 13.1 59.0 24.2 3.25 3.20 0.704

± ± ± ± ± ± ± ±

0.230 0.148 2.43 5.83 1.92 1.45 0.194 0.506

4.98 3.26 27.1 142 66.0 4.07 4.16 1.06

± ± ± ± ± ± ± ±

0.507c 0.351c 5.87c 45.8c 11.9c 1.50 0.247c 0.0805c

Normal (albino) rats

50.0–315

4.70–8.15 2.70–5.10 5.00–29.0 45.7–80.8 17.5–30.2 5.24 4.00 0.80

GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; IU, international unit; Clcr , creatinine clearance. a Measured just before treatment. b Measured just before experiment. c Significantly different (P < 0.05) from control.

v/v) at a dose of 20 mg (2 ml)/kg was infused for 15 min via the jugular vein of DMIS (n = 8) and control (n = 8) rats. A blood sample (approximately 0.11 ml) was collected via the carotid artery at 0 (control), 1, 5, 15 (end of the infusion), 30, 60, 120, 240, 360, 480, 600, and 720 min after the i.v. administration of ipriflavone. A heparinized 0.9% NaCl-injectable solution (0.3 ml; 5 units/ml) was used to flush the cannula immediately after each blood sampling to prevent blood clotting. A blood sample was immediately centrifuged and 50 ␮l of a plasma sample was stored at −70 ◦ C (Revco ULT 1490 D-N-S; Western Mednics, Ashville, NC) until used for the HPLC analysis of ipriflavone, M1, and M5 (Kim et al., 1997). The preparation and handling of the 24 h urine sample (Ae0–24 h ) and the gastrointestinal tract (including its contents and feces) sample at 24 h (GI24 h ) were similar to a reported method (Kim and Lee, 2002). Ipriflavone (the same solution used in the i.v. study) at a dose of 200 mg (5 ml)/kg was administered orally using a gastric gavage tube to overnight-fasted DMIS (n = 12) and control (n = 9) rats. A blood sample was collected at 0, 30, 60, 90, 120, 240, 360, 480, 600, 720, 840, 960, 1080, 1200, and 1440 min after the p.o. administration of ipriflavone. Other procedures were similar to those for the i.v. study. 2.7. Measurement of rat plasma protein binding of ipriflavone using equilibrium dialysis Protein binding values of ipriflavone to fresh plasma from DMIS and control rats (n = 5, each) were determined using equilibrium dialysis (Kim et al., 1999). Plasma (1 ml) was dialyzed against 1 ml of the isotonic Sørensen phosphate buffer (pH 7.4) containing 3% (w/v) dextran (‘the buffer’) in a 1 ml dialysis cell (Spectrum Medical Industries Inc., Los Angeles, CA) using a Spectra/Por 4 membrane (mw cutoff 12–14 kDa; Spectrum Medical Industries Inc.). Ipriflavone (dissolved in DMSO) was spiked into the plasma side at an ipriflavone concentration of 5 ␮g/ml. After 8 h incubation, two 50 ␮l were removed from each compartment and stored at −70 ◦ C until used for the HPLC analysis of ipriflavone (Kim et al., 1997).

0.05 M acetate buffer (pH 3): acetonitrile: methanol (60:35.5:4.5, v/v/v; phosphoric acid was added to adjust pH 2) was used as the mobile phase. The detection limits of ipriflavone, M1, and M5 in rat plasma samples were 20, 10, and 20 ng/ml, respectively, and the corresponding values in rat urine and tissue homogenate samples were all 50–100 ng/ml. The coefficients of variation (inter- and intra-day) of ipriflavone, M1, and M5 were below 10.9%. 2.9. Pharmacokinetic analysis The total area under the plasma concentration–time curve from time zero to time infinity (AUC) was calculated using the trapezoidal rule – extrapolation method (Chiou, 1978). The area from the last datum point to time infinity was estimated by dividing the last measured plasma concentration by the terminal-phase rate constant. Standard methods (Gibaldi and Perrier, 1982) were used to calculate the following pharmacokinetic parameters using a noncompartmental analysis (WinNonlin 2.1; Pharsight Corporation, Mountain View, CA); the time-averaged total body, renal, and non-renal clearances (Cl, Clr , and Clnr , respectively), the terminal half-life, the mean residence time (MRT), the apparent volume of distribution at steady state (Vss ), and the F (Kim et al., 1993). The peak plasma concentration (Cmax ) and time to reach Cmax (Tmax ) were directly read from the experimental data. Glomerular filtration rate (GFR) was estimated by calculating the creatinine clearance (Clcr ) assuming that kidney function was stable during the experimental period. The Clcr was calculated by dividing the total amount of unchanged creatinine excreted in the urine over 12 h by the AUC0–12 h of creatinine in plasma. 2.10. Statistical analysis A P value <0.05 was deemed to be statistically significant using an unpaired t-test. All data are expressed as means ± deviations (S.D.) except medians (ranges) for Tmax . 3. Results

2.8. HPLC analysis of ipriflavone, M1, and M5 3.1. Preliminary study Concentrations of ipriflavone, M1, and M5 in the samples were determined by a slight modification of a reported HPLC method (Kim et al., 1997); internal standard and the mobile phase for M1 and M5 were changed. Phenytoin was used as internal standard and

Body weight, blood glucose level, 12 h urine output, plasma chemistry data, Clcr , and relative liver and kidney weights in DMIS and control rats are listed in Table 1. For comparison, the ranges

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D.Y. Lee et al. / European Journal of Pharmaceutical Sciences 38 (2009) 465–471 Table 2 Mean (±S.D.) Vmax , Km , and Clint for the disappearance of ipriflavone after the incubation of ipriflavone with hepatic and intestinal microsomes from DMIS and control rats. Parameter Hepatic Vmax (nmol/min/mg protein) Km (␮M) Clint (ml/min/mg protein) Intestinal Vmax (nmol/min/mg protein) Km (␮M) Clint (ml/min/mg protein)

Control (n = 4)

DMIS (n = 4)

0.682 ± 0.178 14.9 ± 4.95 0.0470 ± 0.00693

0.927 ± 0.266 15.7 ± 3.07 0.0590 ± 0.00678a

0.0133 ± 0.00821 7.74 ± 4.32 0.00236 ± 0.000643

0.0339 ± 0.00747a 8.45 ± 1.30 0.00402 ± 0.000731a

Vmax , maximum velocity; Km , apparent Michaelis–Menten constant; Clint , intrinsic clearance. a Significantly different (P < 0.05) from control.

of the parameters reported in normal (albino) rats (Mitruka and Rawnsley, 1981) are also listed in Table 1. Compared to controls, the plasma levels of GOT and GPT were significantly higher (by 141% and 173%, respectively) in DMIS rats, and the values in DMIS rats were higher than the reported ranges in the normal rats (Mitruka and Rawnsley, 1981). However, there were no significant findings in the livers of both groups of rats based on the liver histology. The above data suggest that hepatic function was not seriously impaired in DMIS rats compared to controls. Compared to controls, the plasma level of urea nitrogen was significantly higher (by 107%) in DMIS rats, but was close to the ranges in the normal rats (Mitruka and Rawnsley, 1981). Moreover, the Clcr s were not significantly different between DMIS and control rats. There were no significant findings in the kidneys of both groups of rats based on the kidney histology. The above data also suggest that kidney function was also not seriously impaired in DMIS rats. Although the plasma levels of total proteins and albumin, and relative weights of liver and kidney were significantly different between two groups of rats, the values were in the ranges (or close to) in normal rats. 3.2. Vmax , Km , and Clint for the disappearance of ipriflavone in hepatic and intestinal microsomes from DMIS and control rats The Vmax, Km , and Clint values for the disappearance of ipriflavone in hepatic and intestinal microsomes from both groups of rats are listed in Table 2. The Km s in both hepatic and intestinal microsomes were comparable between two groups of rats, suggesting that the affinity of the enzyme(s) for the ipriflavone was not affected considerably by streptozotocin. However, the Vmax s in DMIS rats were faster (by 35.9%; P = 0.139) and significantly faster (by 155%) in hepatic and intestinal microsomes, respectively,

suggesting that the maximum velocity for the disappearance (primarily metabolism) of ipriflavone was faster than controls. As a result, the Clint s in DMIS rats were significantly faster (by 25.5% and 70.3%, respectively) than controls, suggesting that the hepatic and intestinal metabolism of ipriflavone significantly increased by streptozotocin. 3.3. Pharmacokinetics of ipriflavone, M1, and M5 after the i.v. administration of ipriflavone For the i.v. administration of ipriflavone at a dose of 20 mg/kg to DMIS and control rats, the mean arterial plasma concentration–time profiles of ipriflavone, M1, and M5 are shown in Fig. 1(A)–(C), respectively. The relevant pharmacokinetic parameters are listed in Table 3. Compared to controls, changes in the pharmacokinetics of ipriflavone in DMIS rats are as follow; the AUC was significantly smaller (by 31.7%); and Cl, Clr , and Clnr were significantly faster (by 65.0%, 265%, and 64.5%, respectively). The Ae0–24 h s of ipriflavone were almost negligible (0.0499% and 0.133% of the i.v. dose for control and DMIS rats, respectively). Ipriflavone was below the detection limit in GI24 h for both groups of rats. Following the i.v. administration of ipriflavone, formation of M1 was rapid; M1 was detected in the plasma at the second blood sampling time point (5 min) and rapidly reached Tmax (5 min) for both control and DMIS rats. The pharmacokinetic parameters of M1 listed in Table 3 were comparable (not significantly different) between two groups of rats. Following the i.v. administration of ipriflavone, formation of M5 was also rapid; M5 was detected in the plasma at the second blood sampling time point (5 min) and rapidly reached Tmax (30–60 min) for control and DMIS rats. Compared to controls, changes in the

Fig. 1. Mean arterial plasma concentration–time profiles of ipriflavone (A), M1 (B), and M5 (C) after the i.v. administration of ipriflavone at a dose of 20 mg/kg to DMIS (closed circle; n = 8) and control (open circle; n = 8) rats. Bars represent S.D.

D.Y. Lee et al. / European Journal of Pharmaceutical Sciences 38 (2009) 465–471 Table 3 Mean (±S.D.) pharmacokinetic parameters of ipriflavone, M1, and M5 after the i.v. administration of ipriflavone at a dose of 20 mg/kg to DMIS and control rats. Parameter Body weight (g) Initiala Finalb Blood glucose (mg/dl) Ipriflavone AUC (␮g/min/ml) Terminal half-life (min) MRT (min) Vss (ml/kg) Cl (ml/min/kg) Clr (ml/min/kg) Clnr (ml/min/kg) Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) M1 AUC (␮g/min/ml) Terminal half-life (min) Clr (ml/min/kg) Cmax (g/ml) Tmax (min)d Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) AUCM1 /AUCipriflavone (%) M5 AUC (␮g/min/ml) Terminal half-life (min) Clr (ml/min/kg) Cmax (␮g/ml) Tmax (min)d Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) AUCM5 /AUCipriflavone (%)

Control (n = 8)

DMIS (n = 8)

263 ± 12.4 317 ± 14.7 104 ± 20.2

257 ± 20.0 261 ± 19.2c 339 ± 48.9c

1020 ± 176 223 ± 100 131 ± 69.2 2670 ± 1624 20.0 ± 2.82 0.0101 ± 0.0109 20.0 ± 2.82 0.0499 ± 0.0511 BD

697 ± 221c 170 ± 111 119 ± 70.6 3673 ± 2318 33.0 ± 16.7c 0.0369 ± 0.0202c 32.9 ± 16.7c 0.133 ± 0.100 BD

71.8 ± 60.1 39.6 ± 27.2 1.20 ± 0.444 1.60 ± 1.03 5 (5–60) 0.251 ± 0.0677 0.435 ± 0.185 7.15 ± 5.32

44.9 ± 24.9 35.1 ± 49.6 1.70 ± 0.726 1.52 ± 0.432 5 (5–120) 0.248 ± 0.0689 0.734 ± 0.709 6.44 ± 2.84

363 ± 162 67.8 ± 34.0 6.57 ± 10.2 3.14 ± 1.42 30 (30–120) 6.99 ± 10.4 0.124 ± 0.0505 36.6 ± 18.1

230 ± 70.6e 56.7 ± 27.9 5.55 ± 7.56 2.37 ± 0.387 60 (30–240)e 5.50 ± 8.40 0.294 ± 0.109d 33.6 ± 8.18

AUC, total area under the plasma concentration–time curve from time zero to time infinity; MRT, mean residence time; Vss , apparent volume of distribution at steady state; Cl, time-averaged total body clearance; Clr , time-averaged renal clearance; Clnr , time-averaged non-renal clearance; Ae0–24 h , percentage of the dose excreted in the 24 h urine; GI24 h , percentage of the dose recovered from the entire gastrointestinal tract (including its contents and feces) at 24 h; Cmax , peak plasma concentration; Tmax , time to reach Cmax ; BD, below the detection limit. a Measured just before treatment. b Measured just before experiment. c Significantly different (P < 0.05) from control. d Median (ranges).

pharmacokinetics of M5 in DMIS rats are as follows; the AUC was significantly smaller (by 36.6%); Tmax was significantly longer (by 100%); and GI24 h (expressed in terms of the percentages of the i.v. dose of ipriflavone) was significantly greater (by 137%). How-

469

ever, the AUCM5 /AUCipriflavone ratios were comparable between two groups of rats. 3.4. Pharmacokinetics of ipriflavone, M1, and M5 after the p.o. administration of ipriflavone For the p.o. administration of ipriflavone at a dose of 200 mg/kg to DMIS and control rats, the mean arterial plasma concentration–time profiles of ipriflavone, M1, and M5 are shown in Fig. 2(A)–(C), respectively. The relevant pharmacokinetic parameters are listed in Table 4. Gastrointestinal absorption of ipriflavone was rapid; ipriflavone was detected in the plasma at the first or second blood sampling time point (30 or 60 min) for all rats studied. Compared to controls, change in the pharmacokinetic parameter of ipriflavone in DMIS rats is as follows; the AUC was significantly smaller (by 34.2%). After the p.o. administration of ipriflavone, the GI24 h of ipriflavone was greater than that after the i.v. administration; the values were 7.35–9.52% of the oral dose for all rats studied, indicating that gastrointestinal absorption of ipriflavone was not complete in rats as reported in other studies (Kim and Lee, 2002). Following the p.o. administration of ipriflavone, formation of M1 was also rapid; M1 was detected in the plasma at the first blood sampling time point (30 min) and rapidly reached Tmax (120–240 min) for control and DMIS rats. Compared to controls, the pharmacokinetic parameters of M1 listed in Table 4 were also comparable (not significantly different) except terminal half-life between two groups of rats. Following the p.o. administration of ipriflavone, formation of M5 was also rapid; M5 was detected in the plasma from the first blood sampling time point (30 min). Changes in the pharmacokinetic parameters of M5 in DMIS rats compared to controls are as follows; the AUC was significantly smaller (by 41.9%); Clr was significantly faster (by 218%); and GI24 h (expressed in terms of the percentages of the p.o. dose of ipriflavone) was significantly smaller (by 51.1%). However, the AUCM5 /AUCipriflavone ratios were comparable between two groups of rats. 3.5. Rat plasma protein binding of ipriflavone Protein binding values of ipriflavone to fresh plasma from DMIS and control rats were 96.7 ± 0.469% and 97.6 ± 0.761%, respectively; they were not significantly different. Kim et al. (1999) reported that binding of ipriflavone to 4% human serum albumin, similar to the ratio of albumin in rat plasma (Mitruka and Rawnsley, 1981), was independent of ipriflavone con-

Fig. 2. Mean arterial plasma concentration–time profiles of ipriflavone (A), M1 (B), and M5 (C) after the p.o. administration of ipriflavone at a dose of 200 mg/kg to DMIS (closed circle; n = 12) and control (open circle; n = 9) rats. Bars represent S.D.

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D.Y. Lee et al. / European Journal of Pharmaceutical Sciences 38 (2009) 465–471 Table 4 Mean (±S.D.) pharmacokinetic parameters of ipriflavone, M1, and M5 after the p.o. administration of ipriflavone at a dose of 200 mg/kg to DMIS and control rats. Parameter Body weight (g) Initiala Finalb Blood glucose (mg/dl) Ipriflavone AUC (␮g/min/ml) Terminal half-life (min) Clr (ml/min/kg) Cmax (␮g/ml) Tmax (min)d Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) F (%) M1 AUC (␮g/min/ml) Terminal half-life (min) Clr (ml/min/kg) Cmax (␮g/ml) Tmax (min)d Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) AUCM1 /AUCipriflavone (%) M5 AUC (␮g/min/ml) Terminal half-life (min) Clr (ml/min/kg) Cmax (␮g/ml) Tmax (min)d Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) AUCM5 /AUCipriflavone (%)

Control (n = 9)

DMIS (n = 12)

285 ± 10.8 277 ± 41.3 91.1 ± 19.3

288 ± 14.4 256 ± 20.7c 283 ± 30.8c

564 ± 171 298 ± 245 0.0588 ± 0.0627 1.32 ± 0.616 720 (480–960) 0.0145 ± 0.0139 9.52 ± 4.93 5.51

371 ± 212c 180 ± 111 0.102 ± 0.143 0.953 ± 0.723 480 (240–720) 0.0180 ± 0.217 7.35 ± 4.26 5.32

113 ± 102 223 ± 101 1.53 ± 1.12 0.272 ± 0.490 240 (120–360) 0.0395 ± 0.0248 0.595 ± 0.368 24.3 ± 25.9

115 ± 135 131 ± 35.2c 1.98 ± 1.35 0.314 ± 0.451 120 (60–360) 0.0671 ± 0.0495 0.287 ± 0.171 27.7 ± 15.0

766 ± 358 140 ± 72.2 7.16 ± 4.83 1.73 ± 0.906 480 (240–720) 2.22 ± 1.93 0.0786 ± 0.0115 144 ± 51.3

445 ± 336c 164.1 ± 78 22.8 ± 10.9c 1.20 ± 1.05 360 (240–720) 4.06 ± 3.99 0.0384 ± 0.0228c 120 ± 67.2

AUC, total area under the plasma concentration–time curve from time zero to time infinity; Clr , time-averaged renal clearance; Cmax , peak plasma concentration; Tmax , time to reach Cmax ; Ae0–24 h , percentage of the dose excreted in the 24 h urine; GI24 h , percentage of the dose recovered from the entire gastrointestinal tract (including its contents and feces) at 24 h; F, extent of absolute p.o. bioavailability. a Measured just before treatment. b Measured just before experiment. c Significantly different (P < 0.05) from control. d Median (ranges).

centrations ranging from 1 to 200 ␮g/ml; the mean value was 96.6 ± 0.407%. Thus, an ipriflavone concentration of 5 ␮g/ml was chosen for this plasma protein binding study. 4. Discussion Kim and Lee (2002) reported that following the i.v. (at doses of 5–40 mg/kg) and p.o. (at doses of 50–200 mg/kg) administration of ipriflavone to male Sprague–Dawley rats, the AUCs of ipriflavone were dose-proportional. Thus, 20 and 200 mg/kg for the i.v. and p.o. doses of ipriflavone, respectively, were chosen in the present study. Following the i.v. administration of ipriflavone, the contribution of the gastrointestinal (including the biliary) excretion of unchanged ipriflavone to its Clnr was almost negligible; ipriflavone was below the detection limit in GI24 h for all rats studied (Table 3). However, below the detection limit of ipriflavone in GI24 h was not likely due to the chemical and enzymatic degradation of ipriflavone in rats’ gastric fluids; ipriflavone was stable for up to 24 h incubation in various buffer solutions having pHs ranging from 1 to 13 (except at pH 8) at an ipriflavone concentration of 10 ␮g/ml, and for up to 3 h incubation in five humans’ gastric juices (pHs of 1.54, 1.84, 3.81, 2.03, and 1.16, respectively) at an ipriflavone concentration of 5 ␮g/ml (Kim et al., 1999). Moreover, it has been reported that the percentages of the i.v. dose of ipriflavone at a dose of 20 mg/kg excreted in the 24 h bile sample as unchanged drug after bile duct cannulation in six rats were only 0.0271 ± 0.0176% (Kim and Lee,

2002). The aforementioned data suggest that the Clnr of ipriflavone could represent its metabolic clearance. Additionally, changes in the Clnr of ipriflavone listed in Table 3 could indicate changes in its hepatic metabolism in rats. Following the i.v. administration of ipriflavone to DMIS rats, the significantly smaller AUC of ipriflavone could have been due to a significantly faster Cl than controls (Table 3). The faster Cl in DMIS rats was mainly due to the significantly faster Clnr than controls; although the Clr of ipriflavone was significantly faster in DMIS rats, its contribution to Cl of ipriflavone was almost negligible, only 0.0505% and 0.112% for control and DMIS rats, respectively (Table 3). Thus, the contribution of the changes in the Clr of ipriflavone to other pharmacokinetic parameters of ipriflavone seemed to be also almost negligible. The faster Clnr of ipriflavone in DMIS rats (Table 3) could have been supported by a significantly faster in vitro hepatic Clint for the disappearance of ipriflavone (Table 2) and the faster hepatic blood flow rate (Sato et al., 1991) than controls, since the free fractions of ipriflavone were comparable between two groups of rats as mentioned earlier. Because ipriflavone is a drug with very close to an intermediate (30–70%) hepatic extraction ratio (hepatic first-pass effect after absorption into the portal vein was found to be 29.4%) in rats (Kim and Lee, 2002). The faster hepatic Clint in DMIS rats was mainly due to an increase in the protein expression and/or mRNA level of CYP1A subfamily, since that of CYP2C11 decreased in DMIS rats (Ma et al., 1989; Yamazoe et al., 1989; Shimojo et al., 1993; Kim et al., 2005; Sindhu et al., 2006; Ahn et al., 2008).

D.Y. Lee et al. / European Journal of Pharmaceutical Sciences 38 (2009) 465–471

Following the i.v. administration of ipriflavone, the Clr of ipriflavone was estimated from the free (unbound to plasma proteins) fractions of the drug in the plasma based on the Clr (Table 3) and plasma protein binding values of ipriflavone. The values thus estimated were 0.467 and 0.316 ml/min/kg for DMIS and control rats, respectively. The 0.316–0.467 ml/min/kg ranges were considerably slower than the Clcr (Table 1). The above data indicate that ipriflavone was mainly reabsorbed in the renal tubules for all rats studied. Following the p.o. administration of ipriflavone to DMIS rats, AUC was also significantly smaller than controls (Table 4). However, this was not likely due to a decrease in the gastrointestinal absorption of ipriflavone in DMIS rats. Based on the linear pharmacokinetics (Kim and Lee, 2002), the mean ‘true’ fractions of the p.o. dose unabsorbed (‘Funabs ’) in this study could be estimated by a reported equation (Lee and Chiou, 1983); the ‘Funabs ’ values thus estimated were 9.52% and 7.53% for control and DMIS rats, respectively. Thus, more than 90% of the p.o. dose of ipriflavone was absorbed for both groups of rats. The intestinal first-pass effect of ipriflavone was 75.0% of the p.o. dose, but the hepatic first-pass effect of ipriflavone after absorption into the portal vein, 29.4%, was equivalent to less than 6% of the p.o. dose considering the 75.0% of intestinal first-pass effect in rats (Kim and Lee, 2002). Thus, the significantly smaller p.o. AUC of ipriflavone in DMIS rats could have mainly been due to an increased metabolism of ipriflavone in the intestine. The smaller AUC of p.o. ipriflavone could have been supported by a significantly faster intestinal Clint (Table 2). This could have mainly been due to a significant increase in protein expression of intestinal CYP1A1/2, because that of CYP2C11 significantly decreased in DMIS rats (our unpublished data). Following both i.v. and p.o. administration of ipriflavone, the metabolic ratios of both M1 (AUCM1 /AUCipriflavone ) and M5 (AUCM5 /AUCipriflavone ) were comparable between two groups of rat (Tables 3 and 4), suggesting that formation of both M1 and M5 was not considerably affected by streptozotocin. Ipriflavone was reported to be metabolized to seven metabolites in rats (Reginster, 1993; Rohatagi et al., 1997). 5. Conclusion Following the i.v. administration of ipriflavone to DMIS rats, its Clnr (AUC) of ipriflavone was significantly faster (smaller) than controls. This could have been due to a faster hepatic Clint (because of an increase in hepatic CYP1A subfamily) and the faster hepatic blood flow rate than controls. Following the p.o. administration to DMIS rats, the AUC of ipriflavone was also significantly smaller than controls. This could have mainly been due to a faster intestinal Clint (because of the increased CYP1A1/2) than controls. Acknowledgement This study was supported in part by the 2008 BK21 Project for Applied Pharmaceutical Life Sciences. References Ahn, C.Y., Bae, S.K., Bae, S.H., Kim, T.R., Jung, Y.S., Kim, Y.C., Lee, M.G., Shin, W.G., 2008. Pharmacokinetics of oltipraz in liver cirrhotic rats with diabetic mellitus. Br. J. Pharmacol. 156, 1019–1028. Al-Turk, W.A., Stohs, S.J., Roche, E.B., 2008. Altered activities of hepatic and extrahepatic microsomal mixed function oxidase enzymes in diabetic and adrenalectomized diabetic rats. Pharmacology 23, 337–345.

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