Effects of E. Coli lipopolysaccharide on the pharmacokinetics of ipriflavone and its metabolites, M1 and M5, after intravenous and oral administration of ipriflavone to rats: Decreased metabolism of ipriflavone due to decreased expression of hepatic CYP1A2 and 2C11

Effects of E. coli Lipopolysaccharide on the Pharmacokinetics of Ipriflavone and Its Metabolites, M1 and M5, after Intravenous and Oral Administration of Ipriflavone to Rats: Decreased Metabolism of Ipriflavone Due to Decreased Expression of Hepatic CYP1A2 and 2C11 HYE J. CHUNG,1 HEE E. KANG,1 EUN J. BAE,1 INCHUL LEE,2 SANG G. KIM,1 MYUNG G. LEE1 1

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea

2

Department of Diagnostic Pathology, College of Medicine, University of Ulsan, Asan Foundation, Asan Medical Center, Seoul, South Korea

Received 14 September 2007; revised 7 December 2007; accepted 1 January 2008 Published online 3 March 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21343

ABSTRACT: It was reported that ipriflavone was primarily metabolized via hepatic CYP1A1/2 and 2C11 in rats. In the present study, the expression of CYP1A2 and 2C11 decreased in the liver, but increased in the intestine in rats pretreated with E. coli lipopolysaccharide (ECLPS; an animal model of inflammation). Thus, pharmacokinetic parameters of ipriflavone and its metabolites, M1 and M5, were evaluated in ECLPS rats. After intravenous administration (20 mg/kg) to ECLPS rats, the AUC of ipriflavone was significantly greater (26.7% increase) and CLNR of ipriflavone was significantly slower (19.9% decrease) than in the controls. This could have been due to decreased expression of hepatic CYP1A2 and 2C11 compared to the controls. After oral administration (200 mg/kg) to ECLPS rats, the AUC of ipriflavone was also significantly greater (130% increase) than in the controls. Although the expression of intestinal CYP1A2 and 2C11 increased in ECLPS rats, contribution of this increase to the significantly greater AUC of ipriflavone after oral administration of ipriflavone to ECLPS rats was not considerable. This could have also been due to a significantly decreased expression of hepatic CYP1A2 and 2C11 in ECLPS rats. The formation of M1 and M5 could be mediated via CYP1A2 and/or 2C11 in rats. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5024–5036, 2008

Keywords: ipriflavone; M1, and M5; E. coli lipopolysaccharide; pharmacokinetics; expression of hepatic and intestinal CYP1A2 and 2C11; rats

INTRODUCTION Ipriflavone (7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one) is a derivative of naturally occurring

Hye J. Chung’s present address is Center for Chemoinformatics, Life Sciences Research Division, Korea Institute of Science and Technology, Seoul, South Korea. Correspondence to: Myung G. Lee (Telephone: þ82-2-8807855; Fax: þ82-2-889-8693; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 5024–5036 (2008) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association

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isoflavone. It 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.1 Ipriflavone is therefore expected to inhibit bone resorption in animal models of experimental osteoporosis and in osteoporotic patients.2 Thus, ipriflavone has been used orally in the treatment of osteoporosis.3 However, still some controversies exist in the use of ipriflavone in postmenopausal women; ipriflavone is considered as a second-line preventive therapy in

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postmenopausal women.4 Ipriflavone is extensively metabolized in rats, dogs, and humans and undergoes an extensive first-pass metabolism.5 Seven metabolites of ipriflavone (M1–M7) have been identified in animals and humans.3,6 Ipriflavone is metabolized primarily in the liver by oxidation of the isopropyl group or hydroxylation of the b-ring followed by phase II glucuronidation or sulfation. Ipriflavone is present in human plasma and urine in very small quantities and the most frequent metabolites are M1, M2, and M5, suggesting that the pharmacological action of ipriflavone is presumed to be represented by the total potencies of unchanged ipriflavone and its metabolites.7,8 M1 and M2 circulate in blood as conjugated forms, but M5 and ipriflavone are recovered as unconjugated forms, suggesting that M5 may greatly contribute to the action of ipriflavone.3,6,7 Recently, Chung et al.9 reported that ipriflavone was primarily metabolized via hepatic microsomal cytochrome P450 (CYP) 1A1/2 and 2C11, but not via CYP2B1/2, 2D1, 2E1, and 3A1/2, in male Sprague–Dawley rats. Lipopolysaccharide (LPS), an active component in the outer membrane of Gram-negative bacteria, has been used as a classic model of inflammation in rats, since Gram-negative sepsis initiates a systemic inflammatory response after systemic cytokine release.10–13 Changes in the expression and mRNA levels of hepatic CYP enzymes have been reported in rats pretreated with Escherichia coli LPS (ECLPS). For example, the expression and mRNA level of hepatic CYP2C11 decreased in male rats with Fisher 344 or Sprague–Dawley strain 24 h after intraperitoneal injection of ECLPS at a dose of 1.0 mg/kg.14–17 To our knowledge, no studies on changes in the expression and mRNA level of hepatic CYP1A1/2 and those of intestinal CYP1A1/2 and 2C11 in rats pretreated with ECLPS have yet been reported. Although pharmacokinetic changes for some drugs have been previously reported in rats pretreated with LPS (ECLPS or Klebsiella pneumoniae LPS, KPLPS),18 changes with respect to hepatic and intestinal CYP isozyme changes have received little attention except chlorzoxazone (primarily metabolized to 6-hydroxychlorzoxazone via hepatic CYP2E1) in rats pretreated with ECLPS.11 No studies on pharmacokinetic changes of ipriflavone, M1, and M5 after intravenous or oral administration of ipriflavone to rats pretreated with ECLPS with respect to changes in the expression of hepatic and intestinal CYP1A2 and 2C11 have yet been reported. DOI 10.1002/jps

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Yun and Lee19 reported that osteoporosis may result from disequilibrium between structural demand for key minerals and their biologic demand during maladaptive states of inflammation; clinical observation demonstrates coincidence of systemic osteoporosis with periods of systemic inflammation. Clowes et al.20 reported that an acute or chronic imbalance in immune system due to infection or inflammation could contribute to systemic (or local) bone loss and increase in the risk of bone fracture. Thus, we examined ipriflavone for this study. The aim of this study was to report pharmacokinetic changes of ipriflavone, M1, and M5 after intravenous or oral administration of ipriflavone to rats pretreated with ECLPS with respect to changes in the expression of hepatic and intestinal CYP1A2 and 2C11. Changes in the expression of hepatic and intestinal CYP1A2 and 2C11 were also reported.

EXPERIMENTAL Chemicals Ipriflavone, M1, and M5 were donated by Research Laboratory of Sam Chun Dang Pharmaceutical Company (Seoul, South Korea). Polyethylene glycol 400 (PEG 400) was a product from Duksan Chemical Company (Seoul, South Korea). Testosterone [an internal standard for the highperformance liquid chromatographic (HPLC) analysis of ipriflavone], phenytoin (an internal standard for the HPLC analysis of M1 and M5), b-actin, dimethylacetamide (DMA), the reduced form of b-nicotinamide adenine dinucleotide phosphate (NADPH; as a tetrasodium salt), tris(hydroxymethyl)aminomethane (Tris)-buffer, ethylenediamine tetraacetic acid (EDTA), and ECLPS (serotype 0127; B8) were purchased from Sigma–Aldrich Corporation (St. Louis, MO). Rabbit polyclonal anti-rat CYP1A2 and 2C11 antibodies were products from Detroit R&D, Inc. (Detroit, MI). Horseradish peroxidaseconjugated goat anti-rabbit IgG was supplied from Zymed Laboratories (San Francisco, CA). Other chemicals were of reagent grade or HPLC grade.

Animals The protocol for this animal study was approved by the Institute of Laboratory Animal Resources

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of the Seoul National University, Seoul, South Korea. Male Sprague–Dawley rats, 6–8 weeks old and weighing 230–300 g, were purchased from the Taconic Farms, Inc. (Samtako Bio Korea, O-San, South Korea). They were maintained in a clean room (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University) at a temperature of 20–238C with 12-h light (07:00–19:00) and dark (19:00–07:00) cycles and a relative humidity of 50%  5%. Rats were housed in metabolic cages (Tecniplast, Varese, Italy) under filtered pathogen-free air and with food (Agribrands Purina Korea, Pyeongtaek, South Korea) and water available ad libitum. Treatment of ECLPS Rats were randomly divided into two groups, control rats and rats pretreated with ECLPS. ECLPS (dissolved in 0.9% NaCl-injectable solution to produce a concentration of 1 mg/mL) at a dose of 1 mg/kg was administered intraperitoneally to rats (rats pretreated with ECLPS16). An equal volume of 0.9% NaCl-injectable solution was injected into the controls.

Preliminary Study The following preliminary study was performed 24 h after intraperitoneal injection of ECLPS at a dose of 1 mg/kg or 0.9% NaCl-injectable solution (n ¼ 5, each) to measure kidney and liver function. A 24-h urine sample was collected for the measurement of creatinine level. A plasma sample was collected for the measurement of total protein, 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.

Preparation of Rat Hepatic and Intestinal Microsomes The procedures used for the preparation of rat hepatic microsomal fractions were similar to reported methods.9 Rats pretreated with ECLPS

and the controls were fasted overnight with free access to water. The livers of rats pretreated with ECLPS and the controls (n ¼ 5, each) were homogenized (Ultra-Turrax T25; Janke & Kunkel, IKA-Labortechnik, Staufeni, Germany) in 15 mL of ice-cold buffer of 0.154 M KCl/50 mM Tris–HCl in 1 mM EDTA (pH 7.4). The homogenate was centrifuged (10000g, 30 min) and the supernatant fraction was further centrifuged (100000g, 90 min). The microsomal pellet was resuspended in the buffer of 0.154 M KCl/50 mM Tris–HCl in 1 mM EDTA (pH 7.4). Liver microsomal preparations were stored at 708C (Model DF8517; Ilshin Laboratory Company, Seoul, South Korea) until used. Protein content was measured using a reported method.21 The procedures used for the preparation of intestinal microsomal fractions were similar to reported methods22 with a minor modification. The small intestines of overnight fasted rats pretreated with ECLPS (n ¼ 4) and the controls (n ¼ 5) were cut and flushed with ice-cold washing solution containing ice-cold 0.9% NaCl-injectable solution plus reducing agent, dithiothreitol (1 mM). The tissue was then cut open lengthwise to flush any remaining fecal material away with the washing solution. Opened intestine was placed into ice-cold solution A containing 8 mM KH2PO4, 5.6 mM Na2HPO4, 1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, and 0.04 mg/mL phenylmethylsulfonyl fluoride (PMSF), and washed twice with it. The intestinal strips were then blot dried and scraped, and the scraped mucosal cells were put into ice-cold solution B containing 8 mM KH2PO4, 5.6 mM Na2HPO4, 1.5 mM EDTA, 0.5 mM dithiothreitol, and 0.04 mg/mL PMSF. Cells were collected by centrifugation (900g, 5 min) and washed twice with 12 mL of homogenization buffer containing 0.154 M KCl/50 mM Tris–HCl (pH 7.4), 1 mM EDTA, and 0.04 mg/mL PMSF. The cells were resuspended in 5 mL of the homogenization buffer and homogenized. Then, the homogenate was sonicated to disrupt the cell membranes. After low-speed (15000g, 15 min) centrifugation at 48C, the supernatant was collected, and the fat layer and pellet were discarded. The intestinal microsomes were pelleted by high-speed centrifugation (90000g, 60 min) at 48C. The resulting intestinal microsomes were resuspended in the buffer of 0.154 M KCl/50 mM Tris–HCl in 1 mM EDTA (pH 7.4). Intestinal microsomal preparations were stored at 708C until used. Protein content was also measured using a reported method.21

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Immunoblot Analysis

Pretreatment of Rats for Intravenous or Oral Studies

The procedures used were similar to reported methods.23,24 Microsomal proteins were separated by 7.5% sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose paper. A nitrocellulose paper was incubated with polyclonal rabbit anti-rat CYP1A2 or 2C11 antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Immunoreactive protein was visualized through incubation with an enhanced chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, United Kingdom).25 An equal loading of protein was verified by b-actin immunoblotting with a goat anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Scanning densitometry was performed with a Image Scan & Analysis System (Alpha-Innotech Corporation, San Leandro, CA).

In the early morning of the next day after starting the treatment with ECLPS or 0.9% NaCl-injectable solution, the carotid artery (for blood sampling) and the jugular vein (for drug administration for intravenous study only) were cannulated with a polyethylene tube (Clay Adams, Parsippany, NJ) while each rat was under light ether anesthesia.27 A heparinized 0.9% NaClinjectable solution (15 units/mL), 0.3 mL, was used to flush the cannula to prevent blood clotting. Both cannulas were exteriorized to the dorsal side of the neck, where each cannula was terminated with a long silastic tube (Dow Corning, Midland, MI). Both silastic tubes were covered with a wire sheath to allow free movement of the rats. Then, each rat was housed individually in a metabolic cage (Daejong Scientific Company, Seoul, South Korea) and allowed to recover from anesthesia for 4–5 h before beginning the experiment. Hence, the rats were not restrained in the present study.

Measurement of Vmax, Km, and CLint for the Disappearance of Ipriflavone in Hepatic and Intestinal Microsomal Fractions of Rats Pretreated with ECLPS and the Controls

Intravenous Study

The procedures used were similar to reported methods.9 The Vmax (the maximum velocity) and Km (the 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 microsomal fractions (equivalent to 0.5 mg protein for hepatic microsomes and 1.5 mg protein for intestinal microsomes, since the intestinal microsomes have a low activity), a 5-mL aliquot of dimethylsulfoxide containing 0.5, 1, 2, 5, 10, 20, or 50 mM ipriflavone, and a 50-mL aliquot of 0.1 M phosphate buffer (pH 7.4) containing 1 mM NADPH in a final volume of 0.5 mL by adding 0.1 M phosphate buffer (pH 7.4) in a thermomixer (Thermomixer 5436; Eppendorf, Hamburg, Germany) kept at 378C and 500 revolutions/min (rpm). All of the above microsomal incubation conditions were linear. The reaction was terminated by addition of 1 mL of acetonitrile containing 1 mg/mL of testosterone (an internal standard) after 15 min incubation. The kinetic constants (Km and Vmax) for the disappearance of ipriflavone were calculated using a nonlinear regression method.26 The CLint for the disappearance of ipriflavone was calculated by dividing the respective Vmax by the respective Km. DOI 10.1002/jps

Ipriflavone (dissolved in DMA: PEG 400 ¼ 50: 50, v/v) at a dose of 20 mg/kg was infused (total infusion volume of 2 mL/kg) over 1 min via the jugular vein of rats pretreated with ECLPS (n ¼ 11) and the controls (n ¼ 10). A blood sample (approximately 0.22 mL) was collected via the carotid artery at 0 (control), 1 (at the end of the infusion), 5, 15, 30, 60, 120, 240, 360, 480, 600, and 720 min after the start of the intravenous infusion of ipriflavone. A heparinized 0.9% NaClinjectable solution, 0.3 mL, was used to flush each cannula immediately after each blood sampling. Blood samples were immediately centrifuged and two 50-mL aliquots of each plasma sample were stored at 708C until used for the HPLC analysis of ipriflavone, M1, and M5.28 At the end of the experiment (24 h), each metabolic cage was rinsed with 10 mL of distilled water and the rinsings were combined with the 24-h urine sample. After measuring the exact volume of the combined urine sample, two 50-mL aliquots of the combined urine sample were stored at 708C until used for the HPLC analysis of ipriflavone, M1, and M5.28 At the same time (24 h), each rat was exsanguinated and sacrificed by cervical dislocation. Then, the abdomen was opened and the entire gastrointestinal tract (including its contents and feces) of each rat was removed, transferred into a beaker containing 100 mL of methanol (to facilitate the

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extraction of ipriflavone, M1, and M5), and cut into small pieces using scissors. After stirring with a glass rod for 1 min, two 50-mL aliquots of the supernatant were collected from each beaker and stored at 708C until used for the HPLC analysis of ipriflavone, M1, and M5.28

Oral Study Ipriflavone (the same solution used in the intravenous study) at a dose of 200 mg/kg was administrated orally (total oral volume of approximately 2 mL/kg) using a feeding tube to rats pretreated with ECLPS (n ¼ 7) and the controls (n ¼ 11). A blood sample was collected at 0, 30, 60, 120, 240, 360, 480, 600, 720, 840, 960, 1080, 1200, and 1440 min after oral administration of ipriflavone. Other procedures were similar to those for the intravenous study.

Measurement of Rat Plasma Protein Binding of Ipriflavone Using Equilibrium Dialysis Protein binding of ipriflavone to fresh plasma from rats pretreated with ECLPS and the controls (n ¼ 5, each) was determined using equilibrium dialysis.29 Plasma (1 mL) was dialyzed against 1 mL of isotonic Sørensen phosphate buffer (pH 7.4) containing 3% (w/v) dextran (‘‘the buffer’’) to reduce volume shift30 in a 1 mL dialysis cell (Spectrum Medical Industries, Los Angeles, CA) using a Spectra/Por 4 membrane (mol. wt. cutoff of 12,000–14,000 Da; Spectrum Medical Industries). To reduce equilibrium time between ‘‘the buffer’’ and plasma compartments, ipriflavone was spiked into the plasma side.31 After 8 h incubation, two 100-mL aliquots were removed from each compartment and stored at 708C until used for the HPLC analysis of ipriflavone.28 Kim et al.29 reported that binding of ipriflavone to 4% human serum albumin was constant, 96.6%  0.407%, at ipriflavone concentrations ranging from 1 to 200 mg/mL. Thus, an ipriflavone concentration of 5 mg/mL was arbitrarily chosen for this plasma protein binding study.

HPLC Analysis of Ipriflavone, M1, and M5 Concentrations of ipriflavone, M1, and M5 in the above biological samples were determined by a reported HPLC method.28 A 100-mL aliquot of acetonitrile containing 1 mg/mL of testosterone

(an internal standard for ipriflavone) or 50 mg/mL of phenytoin (an internal standard for M1 and M5) was added to deproteinize32 a 50-mL aliquot of biological sample. After vortex-mixing and centrifugation, a 50-ml aliquot of the supernatant was injected directly onto a reversed-phase (C18) HPLC column. The mobile phase, 0.05 M acetate buffer (pH 3):acetonitrile:methanol (40:35:25, v/v/v) for ipriflavone or 0.05 M acetate buffer (pH 3): acetonitrile:methanol (60:35.5:4.5, v/v/v; phosphoric acid was added to adjust pH of 2) for M1 and M5, was run at a flow-rate of 1.5 mL/min. An ultraviolet detector at 254 nm monitored the column eluent. The retention times of testosterone (an internal standard) and ipriflavone were approximately 6 and 12 min, respectively. The retention times of phenytoin (an internal standard), M1, and M5 were approximately 4.7, 8, and 9.6 min, respectively. The detection limits of ipriflavone, M1, and M5 in rat plasma sample 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%.

Pharmacokinetic Analysis The total area under the plasma concentration– time curve from time zero to time infinity (AUC) or the last measured time, 20 h, in plasma (AUC0–20 h for M1 and M5 after oral administration) was calculated using the trapezoidal rule-extrapolation method.33 The area from the last datum point to time infinity (for the calculation of AUC) was estimated by dividing the last measured plasma concentration by the terminal-phase rate constant. Standard methods34 were used to calculate the following pharmacokinetic parameters using a noncompartmental analysis (WinNonlin1; Pharsight Corporation, Mountain View, CA); the time-averaged total body (CL), renal (CLR), and nonrenal (CLNR) clearances, the terminal half-life, the first-moment of AUC (AUMC), the mean residence time (MRT), the apparent volume of distribution at steady state (Vss), and the extent of absolute oral bioavailability ( F). 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),

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assuming that kidney function was stable during the experimental period. The CLCR was calculated by dividing the total amount of creatinine excreted in the urine over 24 h by the AUC0–24 h of creatinine in plasma.

Statistical Analysis A p-value < 0.05 was deemed to be statistically significant using the t-test between the two means for the unpaired data. Data are expressed as means  standard deviations (SDs) except medians (ranges) for Tmax.

RESULTS Preliminary Study Body weight, plasma chemistry data, CLCR, and relative liver and kidney weights in rats pretreated with ECLPS and the controls are listed in Table 1. For comparison, the ranges in normal (albino) rats35,36 are also listed in Table 1. In rats pretreated with ECLPS, plasma levels of GOT (24.7% increase) and GPT (17.7% increase) became significantly higher and relative liver weight became significantly heavier (19.1% increase) than in the controls. The GOT and GPT levels for both groups of rats were in the reported ranges in normal (albino) rats.35,36 Other parameters listed in Table 1 were comparable between two groups of rat. The above data suggest that kidney function was not seriously impaired in rats pretreated with ECLPS. Consistent with the

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kidney histology, no significant findings were detected in the kidneys of both groups of rat. However, liver function seemed to be somewhat impaired in rats pretreated with ECLPS based on the liver histology; mild hepatocelluar degeneration was observed in the livers of rats pretreated with ECLPS, but no significant findings were observed in the livers of the controls. Expression of Hepatic and Intestinal CYP1A2 and 2C11 in Rats Pretreated with ECLPS and the Controls Levels of hepatic (Fig. 1A) and intestinal (Fig. 1B) CYP1A2 and 2C11 were measured for both groups of rats using the Western blot analysis. The expression of hepatic CYP1A2 and 2C11 significantly decreased (87.6% and 60.7% decrease, respectively) in rats pretreated with ECLPS compared to the controls. Similar result on hepatic CYP2C11 has also been reported in other studies.14–17 Whereas, the expression of intestinal CYP1A2 and 2C11 significantly increased (184% and 273% of the controls, respectively) in rats pretreated with ECLPS compared to the controls.

Measurement of Vmax, Km, and CLint for the Disappearance of Ipriflavone in the Hepatic and Intestinal Microsomal Fractions The Vmax, Km, and CLint for the disappearance of ipriflavone in hepatic microsomal fractions of both groups of rats are listed in Table 2. In rats pretreated with ECLPS, the Vmax became

Table 1. Body Weight, Plasma Chemistry Data, CLCR, and Relative Liver and Kidney Weights in Control Rats and Rats Pretreated With ECLPS Parameter Body weight (g) Plasma Total protein (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 ¼ 5)

ECLPS (n ¼ 5)

304  28.2

281  8.22

5.70  0.579 3.76  0.351 14.8  5.07 54.2  4.82 22.6  2.97 2.68  0.329 3.20  0.233 0.737  0.0982

5.68  0.311 3.62  0.217 20.1  5.77 67.6  6.07a 26.6  2.30b 2.80  0.878 3.81  0.354b 0.784  0.0317

Normal (Albino) Rats

4.70–8.15 2.70–5.10 5.00–29.0 45.7–80.8 17.5–30.2 5.24 4.00 0.800

For comparison, the ranges in normal (albino) rats are also listed. GOT, glutamate oxaloacetate; GPT, glutamate pyruvate transaminase; IU, International unit; CLCR, creatinine clearance.  Values expressed as mean  SD. a ECLPS was significantly different ( p < 0.01) from control. b ECLPS was significantly different ( p < 0.05) from control. DOI 10.1002/jps

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Figure 1. Immunoblot analyses of CYP1A2, 2C11, and b-actin were carried out with hepatic (A) and small intestinal (B) microsomal proteins prepared from rats pretreated with ECLPS and the controls. Representative blots are shown (left panel) and the relative levels of each protein (right panel) were measured by a scanning densitometry of the immunoblot bands. Each lane was loaded with 10 and 25 mg of hepatic and small intestinal microsomal proteins, respectively. Bars represent standard deviation (n ¼ 3–5). Significant difference from the controls, p < 0.05 and p < 0.01.

significantly slower (58.4% decrease) than in the controls, suggesting that the maximum velocity for the disappearance (primarily metabolism) of ipriflavone in the liver was significantly slower by pretreatment of ECLPS. In rats pretreated with ECLPS, the Km of the liver became significantly lower (45.2% decrease) than in the controls, suggesting that the affinity for ipriflavone of the enzyme(s) in the liver increased compared to the controls. As a result, in rats pretreated with ECLPS, the CLint for the disappearance of

ipriflavone became significantly slower (20.8% decrease) than in the controls, suggesting that metabolism of ipriflavone in the liver significantly decreased in rats pretreated with ECLPS. Protein content in the liver microsomes of rats pretreated with ECLPS (11.4  2.72 mg/g liver) was significantly smaller (29.6% decrease) than in the controls (16.2  1.73 mg/g liver). The Vmax, Km, and CLint for the disappearance of ipriflavone in the intestinal microsomal fractions of both groups of rats are also listed in

Table 2. Km, Vmax, and CLint for the Disappearance of Ipriflavone after Incubation of Ipriflavone With Hepatic and Intestinal Microsomal Fractions of Control Rats and Rats Pretreated With ECLPS Hepatic Microsomes Parameter Vmax (nmol/min/mg protein) Km (mM) CLint (mL/min/mg protein)

Control (n ¼ 5) 0.543  0.135 9.82  2.27 0.0553  0.00496

Intestinal Microsomes

ECLPS (n ¼ 5) a

0.226  0.0814 5.38  2.401b 0.0438  0.00990b

Control (n ¼ 5)

ECLPS (n ¼ 4)

0.0221  0.0181 6.24  5.31 0.00357  0.000323

0.0876  0.0373b 20.2  10.2b 0.00449  0.000809

Vmax, maximum velocity; Km, apparent Michaelis–Menten constant; CLint, intrinsic clearance.  Values expressed as mean  SD. a ECLPS was significantly different ( p < 0.01) from control. b ECLPS was significant different ( p < 0.05) from control. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 11, NOVEMBER 2008

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Table 2. In rats pretreated with ECLPS, the Vmax became significantly faster (296% increase) than in the controls, suggesting that maximum velocity for the disappearance of ipriflavone (primarily metabolism) in the intestine was faster by pretreatment of ECLPS. In rats pretreated with ECLPS, the Km became significantly higher (224% increase) than in the controls, suggesting that the affinity for ipriflavone of the enzyme(s) in the intestine decreased compared to the controls. Thus, the CLint of the intestine became comparable between two groups of rats.

Pharmacokinetics of Ipriflavone, M1, and M5 after Intravenous Administration of Ipriflavone to Rats For the intravenous administration of ipriflavone at a dose of 20 mg/kg to rats pretreated with ECLPS and the controls, the mean arterial plasma concentration–time profiles of ipriflavone, M1, and M5 are shown in Figure 2A–C, respectively, and relevant pharmacokinetic parameters are listed in Table 3. In rats pretreated with ECLPS, changes in pharmacokinetic parameters of ipriflavone compared to the controls are as follows; the AUC became significantly greater (26.7% increase), while the CL (18.6% decrease) and CLNR (19.9% decrease) became significantly slower than in the controls. Pharmacokinetic parameters of both M1 and M5 were comparable between two groups of rats except significantly lower Cmax of M1 (32.0% decrease) and significantly smaller (29.8% decrease) MP ratio of M1 (AUCM1/AUCipriflavone) in rats pretreated with ECLPS.

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Pharmacokinetics of Ipriflavone, M1, and M5 after Oral Administration of Ipriflavone to Rats For the oral administration of ipriflavone at a dose of 200 mg/kg to both groups of rats, the mean arterial plasma concentration–time profiles of ipriflavone, M1, and M5 are shown in Figure 3A–C, respectively, and relevant pharmacokinetic parameters are listed in Table 4. Absorption of ipriflavone from the rat gastrointestinal tract was rapid; ipriflavone was detected in plasma from the first blood sampling time (15 min) for both groups of rat. In rats pretreated with ECLPS, changes in pharmacokinetic parameters of ipriflavone compared to the controls are as follows; the AUC and percentage of the ipriflavone dose excreted in the 24-h urine as unchanged drug (Ae0–24 h) became significantly greater (130% and 434% increase, respectively) than in the controls. Because the Ae0–24 h of ipriflavone was almost negligible, contribution of Ae0–24 h changes to the pharmacokinetic changes of ipriflavone in rats pretreated with ECLPS could be almost negligible. In rats pretreated with ECLPS, changes in pharmacokinetic parameters of M1 and M5 compared to the controls are as follows; the AUC0–20 h, Cmax, and Ae0–24 h of M1, and MP ratio of M1 (AUCM1, 0–20 h/AUCipriflavone, 0–20 h) were significantly smaller (39.9% decrease), lower (47.4% decrease), smaller (62.7% decrease), and smaller (65.1% decrease), respectively, and the AUC0–20 h, Cmax, and Ae0–24 h of M5, and percentage of the dose recovered from the gastrointestinal tract (including its contents and feces) as unchanged drug at 24 h (GI24 h) of M5, and MP ratio of M5 (AUCM5, 0–20 h/AUCipriflavone, 0–20 h)

Figure 2. Mean arterial plasma concentration–time profiles of ipriflavone (A), M1 (B), and M5 (C) after 1 min intravenous infusion of ipriflavone at a dose of 20 mg/kg to control rats (*; n ¼ 10) and rats pretreated with ECLPS (*; n ¼ 11). Bars represent standard deviation. DOI 10.1002/jps

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Table 3. Pharmacokinetic Parameters of Ipriflavone, M1, and M5 after 1 min Intravenous Infusion of Ipriflavone at a Dose of 20 mg/kg to Control Rats and Rats Pretreated With ECLPS Parameter Body weight (g) Ipriflavone AUC (mg 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 (mg min/mL) Terminal half-life (min) Cmax (mg/mL) Tmaxa (min) Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) AUCM1/AUCipriflavone ratio (%) M5 AUC (mg min/mL) Terminal half-life (min) Cmax (mg/mL) Tmaxa (min) Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) AUCM5/AUCipriflavone ratio (%)

Control (n ¼ 10)

ECLPS (n ¼ 11)

283  11.6

257  19.8b

892  157 301  90.2 171  91.5 3600  1950 23.1  4.10 0.0169  0.0172 23.1  4.34 0.0648  0.0611 1.17  1.43

1130  268c 289  73.5 156  52.9 3030  1560 18.8  4.71c 0.00581  0.00276 18.5  4.86c 0.0315  0.0126 0.411  0.588

91.7  21.6 258  117 0.962  0.238 15 (15) 24.0  11.2 2.34  1.56 10.5  2.86

80.2  19.6 276  144 0.654  0.161b 30 (15–60) 24.2  7.76 2.97  2.58 7.37  2.00b

514  237 205  65.7 2.76  0.736 30 (30–60) 6.13  1.21 0.453  0.341 58.3  24.4

587  251 167  112 2.80  0.653 60 (30–240) 7.09  1.90 0.633  0.491 52.9  19.1

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 nonrenal clearance; Ae0–24 h, percentage of the dose excreted in the 24-h urine; GI24 h, percentage of the dose recovered from the gastrointestinal tract (including its contents and feces) at 24 h; Cmax, maximum plasma concentration; Tmax, time to reach Cmax.  Values expressed as mean  SD. a Median (ranges). b ECLPS was significantly different ( p < 0.01) from control. c ECLPS was significantly different ( p < 0.05) from control.

were significantly smaller (43.4% decrease), lower (49.9% decrease), smaller (80.4% decrease), greater (82.1% increase), and smaller (67.3% decrease), respectively. Rat Plasma Protein Binding of Ipriflavone Protein binding values of ipriflavone to fresh plasma from rats pretreated with ECLPS and the controls were 97.1%  3.08% and 98.7%  0.229%, respectively; they were not significantly different.

DISCUSSION Pharmacokinetic changes of drugs (compounds) seemed to be dependent on the gender and strain

(CD, Wistar, or Sprague–Dawley rats) of rats, source (E. coli or K. pneumoniae) and dose (50, 250, 500, or 1000 mg/kg) of LPS species, and starting time (2, 6, 10, 24, or 96 h) of experiment after LPS administration.18 In most studies, ECLPS was administered at a dose of 1 mg/kg and experiment was started 24 h after the intraperitoneal injection of ECLPS to male rats.14,17,18 Thus, the same protocol was employed for the present study. After intravenous (at doses of 5–40 mg/kg) and oral (at doses of 50–200 mg/kg) administration of ipriflavone to male Sprague–Dawley rats, the AUC values of ipriflavone were dose-proportional.37 Thus, a 20 mg/kg for the intravenous dose and 200 mg/kg for the oral dose of ipriflavone were arbitrarily chosen for this study.

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Figure 3. Mean arterial plasma concentration–time profiles of ipriflavone (A), M1 (B), and M5 (C) after oral administration of ipriflavone at a dose of 200 mg/kg to control rats (*; n ¼ 11) and rats pretreated with ECLPS (*; n ¼ 7). Bars represent standard deviation. Table 4. Pharmacokinetic Parameters of Ipriflavone, M1, and M5 after Oral Administration of Ipriflavone at a Dose of 200 mg/kg to Control Rats and Rats Pretreated With ECLPS Parameter Body weight (g) Ipriflavone AUC (mg min/mL) Terminal half-life (min) Cmax (mg/mL) Tmaxa (min) CLR (mL/min/kg) Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) F (%) M1 AUC0–20 h (mg min/mL) Cmax (mg/mL) Tmaxa (min) Ae0–24 h (% of the ipriflavone dose) GI24 h (% of ipriflavone dose) AUCM1, 020 h/AUCipriflavone, 020 h ratio (%) M5 AUC0–20 h (mg min/mL) Cmax (mg/mL) Tmaxa (min) Ae0–24 h (% of the ipriflavone dose) GI24 h (% of the ipriflavone dose) AUCM5, 020 h/AUCipriflavone, 020 h ratio (%)

Control (n ¼ 11)

ECLPS (n ¼ 7)

253  7.83

243  11.4b

261  86.2 832  496 0.479  0.187 480 (120600) 0.0292  0.0402 0.00363  0.00404 14.9  7.30 2.93

600  287c 802  408 0.569  0.218 480 (240840) 0.0685  0.0659 0.0194  0.0203b 25.7  6.17c 5.31

82.5  19.5 0.149  0.0497 480 (360–840) 4.18  1.61 0.860  0.442 43.8  12.4

49.6  14.2c 0.0783  0.0235c 480 (60–840) 1.56  0.966c 0.910  0.411 15.3  5.03d

629  229 1.62  0.602 600 (480–840) 2.91  1.08 0.0560  0.0360 321  57.4

356  128b 0.812  0.365c 600 (360–840) 0.569  0.236d 0.102  0.0357b 105  32.2d

AUC, total area under the plasma concentration–time curve from time zero to time infinity; Cmax, maximum plasma concentration; Tmax, time to reach Cmax; CLR, time-averaged renal clearance; Ae0–24 h, percentage of the dose excreted in the 24-h urine; GI24 h, percentage of the dose recovered from the gastrointestinal tract (including its contents and feces) at 24 h; AUC0–20 h, total area under the plasma concentration–time curve from time zero to the last measured time, 20 h, in plasma; F, extent of absolute oral bioavailability.  Values expressed as mean  SD. a Median (ranges). b ECLPS was significantly different ( p < 0.05) from control. c ECLPS was significantly different ( p < 0.01) from control. d ECLPS was significantly different ( p < 0.001) from control. DOI 10.1002/jps

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After intravenous administration of ipriflavone, contribution of the CLR to the CL of ipriflavone was almost negligible; the Ae0–24 h values of ipriflavone were smaller than 0.0648% of the intravenous dose for all rats studied (Tab. 3). Contribution of the gastrointestinal (including biliary) excretion of unchanged ipriflavone to the CLNR of the drug was also negligible; the GI24 h values of ipriflavone were less than 1.17% of the intravenous dose for both groups of rat (Tab. 3). The small values of GI24 h, less than 1.17%, were not likely due to chemical and enzymatic degradation of ipriflavone in gastric fluids. Kim et al.29 reported that 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 mg/ mL, and for up to 3 h incubation in five human gastric juices (pHs of 1.54, 1.84, 3.81, 2.03, and 1.16, respectively) at an ipriflavone concentration of 5 mg/mL. Moreover, Rohatagi et al.6 reported that percentage of the intravenous dose of ipriflavone at a dose of 20 mg/kg excreted in the 24-h bile juice sample as unchanged ipriflavone after bile duct cannulation was only 0.0271%  0.0176% in six rats. The aforementioned data suggest that ipriflavone is almost completely metabolized after intravenous administration, and the CLNR of ipriflavone could represent the metabolic clearance of the drug. Thus, changes in the CLNR of ipriflavone could represent changes in the metabolism of the drug in rats. After intravenous administration of ipriflavone to rats pretreated with ECLPS, the significantly greater AUC of ipriflavone could have been due to a significantly slower CL of ipriflavone than in the controls (Tab. 3). The slower CL of ipriflavone was attributable to a significantly slower CLNR of ipriflavone than in the controls, since the CLR values of ipriflavone were comparable between two groups of rat (Tab. 3). The slower CLNR of ipriflavone could have been due to a significantly decreased expression of hepatic CYP1A2 and 2C11 in rats pretreated with ECLPS (Fig. 1A). Chung et al.9 reported that ipriflavone was primarily metabolized via hepatic CYP1A1/2 and 2C11 in rats. Kim and Lee37 reported that the hepatic first-pass effect of ipriflavone after absorption into the portal vein was 29.4% based on the AUC difference following intravenous and intraportal administration of the drug at a dose of 20 mg/kg to male Sprague–Dawley rats. Because ipriflavone is close to an intermediate hepatic

extraction ratio drug (30–70%), its hepatic clearance depends on the CLint for the disappearance of ipriflavone, the free (unbound to plasma protein) fraction of ipriflavone in plasma, and the hepatic blood flow rate.38 The significantly slower CLNR of ipriflavone in rats pretreated with ECLPS (Tab. 3) could be supported by the significantly slower CLint for the disappearance of ipriflavone in the liver microsomes (Tab. 2), and slower hepatic blood flow rate39 than in the controls. However, the free fraction of ipriflavone in plasma was comparable between two groups of rat as mentioned earlier. After oral administration of ipriflavone to rats pretreated with ECLPS, the AUC of ipriflavone was also significantly greater than in the controls (Tab. 4). However, this was not likely due to increased gastrointestinal absorption of ipriflavone in rats pretreated with ECLPS. Based on linear pharmacokinetics,37 the mean ‘‘true’’ fraction of the dose unabsorbed ( Funabs) in the present study could be estimated by the following equations:40 0:149 ¼ Funabs þ ð0:0117  0:0293Þ for control rats 0:257 ¼ Funabs þ ð0:00411  0:0531Þ for rats pretreated with ECLPS in which 0.149 (0.0257), 0.0117 (0.00411), and 0.0293 (0.0531) are the GI24 h after oral and intravenous administration, and F, respectively, in control rats (rats pretreated with ECLPS). The Funabs values thus estimated were 25.7% and 14.9% for rats pretreated with ECLPS and the controls, respectively. Thus, percentages of the oral dose of ipriflavone absorbed were approximately 74% and 85% for rats pretreated with ECLPS and the controls, respectively. The above data indicate that absorption rather decreased in rats pretreated with ECLPS. Although, the expression of intestinal CYP1A2 and CYP2C11 significantly increased in rats pretreated with ECLPS (Fig. 1B), the CLint for the disappearance of ipriflavone in the intestinal microsomes were comparable between two groups of rats (Tab. 2). The above data suggest that contribution of increased expression of intestinal CYP1A2 and 2C11 to the significantly greater AUC of ipriflavone after oral administration to rats pretreated with ECLPS could not be considerable. Thus, the significantly greater AUC of ipriflavone after oral administration of ipriflavone to rats pretreated with ECLPS (Tab. 4) could have

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been due to inhibition of hepatic metabolism of ipriflavone caused by decreased expression of hepatic CYP1A2 and 2C11 (Fig. 1A). Although the exact reason is not clear, the AUC difference of ipriflavone after oral administration of ipriflavone to rats pretreated with ECLPS (130% increase; Tab. 4) was greater than 26.7% increase after intravenous administration (Tab. 3). In rats pretreated with ECLPS, the MP ratio of M1 was significantly smaller after both intravenous (Tab. 3) and oral (Tab. 4) administration, and MP ratio of M5 was also significantly smaller than in the controls after oral administration (Tab. 4). This suggests that M1 and M5 could be formed via CYP1A2 and/or 2C11 in rats. In conclusion, in rats pretreated with ECLPS, the expression of both CYP1A2 and 2C11 decreased and increased in hepatic and intestinal microsomes, respectively. After both intravenous and oral administration of ipriflavone to rats pretreated with ECLPS, the AUC of ipriflavone was significantly greater than in the controls (Tab. 3). This could have been due to a significantly decreased expression of hepatic CYP1A2 and 2C11 than in the controls (Fig. 1A). M1 and M5 could be formed via CYP1A2 and/or 2C11 in rats.

5. 6.

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8.

9.

10.

11.

12.

ACKNOWLEDGMENTS This work was supported in part by a grant from the Seoul City Collaborative Project among the Industry, Academy, and Research Institute, Korea.

14.

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