Dose-Dependent Pharmacokinetics of Paeoniflorin in Rats

Shiro IsHIUA,Yoko SAKIYA,and Tsutomu ICHIKAWA Faculty of PharmaceuticalSciences, Tokushima Bunri University, Yamashiro-cho,Tokushima-shi 770, Japan

Key words : Paeoniflorin; Dose-dependentpharmacokinetics ; Rat ; Biliary ; Renal clearance; Renal plasma flow

Summary

The pharmacokinetics of paeoniflorin (PAE) was examined after an iv dose of 20, 40, or 100mg/kg in rats. The decline of plasma concentration was biexponential after each dose. Dose, however, had a marked effect on the pharmacokinetics, with a greater than proportional increase in AUC at 100mg/ kg, even though the increase was proportional at the dose range from 20 to 40mg/kg. There was also significant decrease of the steady-state distribution volume (Vdss), and total body (CLtot), and renal (CLR) clearances at the high dose (p<0.01 or 0.05), without a significant difference between the two low doses. Plasma unbound fraction (0.75) was constant over the observed range of plasma PAE concentrations (0.8-700pg/ml). The decrease of Vdss was ascribed to the dose-dependent Kp values in liver, kidney, gastrointestinal tract, and skin. The significant decrease of biliary clearance (CLB) at 100mg/ kg as compared with that at 20 and 40mg/kg contributed to the decrease of CLtot. It was suggested that the decreases of CLB and CLR may be a conse quence of saturable transport from plasma into liver, and the significant decrease of glomerular rate (p<0.05) at the higher steady-state plasma level than at the lower plasma levels might be owing to a decreasing effect of PAE on renal plasma flow, because transport from liver to bile did not appear to be saturable, and the excretion into seemed to occur only by glomerular filtration. Thus, the pharmacokinetics of PAE in rats was dose dependent. Introduction

Peony root or its extract is an important crude drug which is used as a herbal medicine or a constituent of a number of herbal medicines. Paeoniflorin (PAE) is the main com ponent of peony root') and has many pharmacological effects, e.g., anti-allergic 2), sedative, anti-convulsive 3), analgesic 3,4), anti -hypertensive5>, muscle relaxant 5,6), and anti-inflam matory') effects. It relieves or prevents gastric ulcers induced by stress'). These pharma cological effects are more potent than those of other monoterpene glucoside components (albiflorin, oxypaeoniflorin, and benzylpaeoniflorin) present in peony root3'5,7> Haradaa> reported that the therapeutic effects of peony root result from PAE. Accordingly, the pharmacological effects of peony root preparations have been studied by using PAE, mainly after intraperitoneal administration to rats and mice in the dose range of 0. 5---2g/kg3,5,7> However, no data on the pharmacokinetic characteristics have been reported so far. The purpose of this study was to examine the pharmacokinetic behavior of paeoniflorin after an iv dose of 20, 40, or 100mg/kg in rats with and without cannulated bile duct.

Materials and Methods

Materials PAE, inulin, and mannitol were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). p- sodium salt (PAH-Na) was obtained from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). Acetonitrile (CH3CN) was of liquid chromatographic reagent grade (Wako Pure Chemical Ind., Ltd.). All other reagents were commercial products of analytical grade.

Animals Male Wistar rats (weighing 240-260g), fasted for 20 to 24h prior to the experiments, were used throughout. Animals were lightly anesthetized with ether in all surgical procedures. The right femoral vein and left femoral artery were cannulated with PE-50 polyethylene tubing for iv drug administration and blood sampling, respectively. For the biliary and urinary excretion studies, a bile fistula and urinary bladder cannula were used to collect samples of bile and urine, respectively. For measurement of the renal clearance (CLR) and glomerular filtration rate (GFR) at a steady-state plasma level of PAE, the left femoral artery, right femoral vein, and both right and left ureters were cannulated by the method of Shim et all). For measurement of renal plasma flow rate (RPF), the left renal vein was also cannulated. Cannulated rats were kept in restraining cages with free access to water under normal housing conditions prior to the experiments. After 1h of recovery from anesthesia, the drug was given. The boody temperature was kept at 37`C throughout the experiments by means of a heat lamp.

Plasma disposition PAE was dissolved in 5 % glucose solution. PAE (20, 40, or 100mg/kg) (0. 5ml) was given to rats with and without biliary fistulization, followed by 0.5m1 of 5 % glucose solution. After dosing, blood samples (300pl each) were collected in heparinized polyethylene centrifuge tubes at 1, 2, 3, 5, 10, 20, 30, 60, and 90min. Further, samples were taken at 120min in the case of 40mg/kg, and at 120, 150, 180, and 210min in the case of 100 mg/kg. Rats were given blood (1.5m1), obtained from other rats, through the left femoral artery cannula immediately after each sampling at 10 and 120min. Plasma was harvested immediately and frozen at -201C until analysis.

Biliary and urinary excretions Bile was sampled during 0-20, 20-40, 40-50, 50-70, 70-80, 80.100min, 100min-4h and 4--6, 6.24, and 24-26h following an iv dose of 20, 40, or 100mg/kg. In the control rats (without biliary fistulization) and bile duct cannulated rats, urine samples were collected during 0---1, 1-2, 2-6, 6-24, and 24---26h following an iv dose of 20, 40, or 100mg/kg. Bile and urine volumes were measured and the samples were stored at -20C until analysis.

Intestinal absorption Intestinal absorption was examined by the in situ recirculating perfusion technique of Karino et al10'. The recirculating solution (isotonic phosphate buffer solution, pH6.5, containing 0.34mg/ml of PAE and 0.32mg/ml of inulin) warmed at 371C was started at a perfusion flow rate of 5ml/min. Starting at 5min (lag time) after the bigining of the perfusion, 0.5ml aliquots of the perfused solution were sampled at 0, 15, 30, 45, and 60min. Since inulin is a high molecular compound and is regarded as nonabsorbable in the lumen, the solution volume was determined from inulin concentration difference in the perfusate between 0min and each sampling time after the lag time.

Tissue-to-plasma partition coefficient (Kp) The Kp values were determined at 30min after 40mg/kg iv dosing or at 10, 30, 60, and 90min after 100mg/kg iv dosing to rats with biliary fistulization. After removal of blood samples, animals were killed by injection of saturable KC1 solution through the femoral artery cannula. Immediately, the abdomens of the animals were opened, and perfused with cold physiological saline via the venous trunk just inferior to the renal veins and portal vein until the effusate become colorless. Blood-free brain, lung, heart, liver, kidney, spleen, gastrointestinal (GI) tract, skin, muscle, and adipose tissue samples at 30min after dosing or blood-free liver, kidney, GI tract, and skin at 10, 60, and 90min after the 100mg/kg dose were quickly excised, rinsed well with cold physiological saline, blotted, and weighed. All the samples were stored at -20C until required. Each tissue sample was homogenized with two volumes of physiological saline in an ice-bath before the determination of PAE. The volume of each tissue was calculated from the wet tissue weight on the assumption that the tissue density is 1. 0.

Plasma protein binding Plasma protein binding of PAE was determined by an technique using a membrane cone (Amicon Centriflo ultrafiltration membrane cone, type CF-25). Plasma was obtained by centrifugation of the fresh blood obtained from several rats. One milliliter of plasma containing PAE (1-800µg) was applied to the membrane cone after incubation at 37C for 5min. The applied plasma and its filtrate were analyzed for PAE. The adsorption of the drug on the membrane and the leakage of macromolecular components of plasma were negligible.

CLR, GFR, and RPF at steady-state plasma level of PAE (1) CLR and GFR : The priming doses of PAE were 1.68, 3.36, 16.80, or 134.40mg/kg for the sustaining doses of 4.72, 9.44, 47.20, or 378. 16mg/h/kg, respectively. Inulin (to estimate the glomerular filtration rate) was primarily dosed at 53. 4mg/kg for the sustaining dose of 96.0mg/h/kg. PAE and inulin were dissolved in 5 % glucose solution containing 3 mannitol (to obtain a suitable urine flow) and injected through the femoral vein cannula. The sustaining solution was infused (infusion pump model KN ; Natsume Seisakusho Co., Tokyo, Japan) at a rate of 3m1/h. Constant urine flow (ca. 0.02m1/min), and the steady state plasma concentrations of the drug (ca. 5, 10, 50, or 400pg/ml) and inulin (ca. 20 pg/ml) were obtained within 10min after the initiation of infusion in each case. A point 15min after the initiation of infusion was taken as 0 time, and urine was collected over 10min intervals for 30min through each ureter cannula and blood samples (300µl each) were withdrawn through the femoral artery cannula at the midpoint of each urine collection interval. The control experiment was similarly carried out by using only inulin. (2) RPF by the p-aminohippuric acid (PAH) method" : PAE and PAH (as PAH-Na) were dissolved in the same solution as in the case of (1) and infused at a rate of 3m1/h by using a priming dose of the drug and PAH. The priming dose of the drug was 3.36 or 134.40mg/kg with an infusion rate of 9.44 or 378.16mg/h/kg, respectively, and that of PAH was 100mg/kg with an infusion rate of 120mg/h/kg. Steady-state plasma levels of the drug (ca. 13 or 350µg/ml) and PAH (ca. 85µg/ml in arterial plasma and ca. 20mg/ml in urine) were achieved within 10min after the initiation of infusion. Urine samples were collected via the cannulas from 15min after the start of infusion and blood samples (300µl each) were taken from both femoral artery and renal vein cannulas according to the same procedures as in the case of (1). The control experiments were carried out by the same procedure as described above using only PAH. Blood samples collected were centrifuged to obtain plasma.

Determination method (1) PAE : HPLC conditions The apparatus used was a Hitachi liquid chromatograph, model 655, with a Zorbax ODS column (250 X 4mm i.d.) and a spectrophotometric detect or operating at 240nm. The mobile phase consisted of CH3CN-0. 2M NaH2PO4 solution (3 : 17v/v). The column was maintained at room temperature and the mobile phase flow rate was 1.0ml/min. For quantitative calculation, a recording integrator (model 3390 A, Yokogawa-Hewlett Packard Co., Tokyo, Japan) was employed. Extraction method Samples of 100µl for plasma, blood, filtrates of plasma, and perfused solution, 20-100µl for bile and urine, and 300r600µ1 for tissue homogenates were shaken vigorously with 5ml of MeOH-AcOEt (1 : 4v/v) for 20min. This extraction was repeated again, and the combined MeOH-AcOEt extracts were evaporated to dryness at 37C under a nitrogen stream. The residue was transferred to a 10-ml test tube by washing with MeOH, and dried under a nitrogen stream. Each residue was dissolved in 500µl of MeOH, and 5-'-10pl of this solution was injected into the liquid chromatograph. Standards were prepared by adding various amounts of PAE to the pooled plasma, blood, bile, and urine (0.2.1000 µg/ml) and to the pooled tissue homogenates (0.2-150pg/ml). Concentrations of PAE in samples were determined from the slope of calibration plots of the peak height versus drug concentration. The intra-and interday coefficients of variation (CV) for PAE at 0.2 pg/ml were less than 10% and those at 150 or 1000µg/ml were less than 3%. The limit of detection of PAE in the assay was 0.1µg/ml with a CV of 10% in all ceses. (2) Inulin and PAH : Inulin in the perfused solution, plasma (100µl each) and urine (50µl), and PAH in plasma (100µl) and urine (50µl) were determined by the methods of Tsuji et all'). and Bratton and Marshall"), respectively.

Data analysis The AUCo-o.5h was determined by using the trapezoidal method. The residual area beyond 0.5h (AUCo.5--) was estimated as C' /k7., where C' is the concentration at 0.5h and-kz is the rate consant calculated by linear regression analysis for plasma concentration data after 0. 5h. The AUCo--was calculated by adding AUCo-o.oh to AUCo.5h--. Total body clearance (CLtot) and volume of distribution at the steady-state (Vdss) were calculated by means of the following equations : CLtot=dose/AUC, and Vdss=dose •MRT/AUC, where MRT is the mean residence time. The biliary (CLB) and renal (CLR) clearances were calculated from total biliary and urinary excreted amounts divided by AUC, respectively. Metabolic clearance (CLM) in the rats with bile fistula was estimated from CLtot-(CLs+CLR). The intestinal absorption rate constant (ka) was calculated from Eq. 114) k In (Co•Vo/Cst•Vst) (1) a t

Where C and V are the drug concentration in the perfusate and the volume of perfusate, respectively. The subscrips 0 and st represent sampling time 0 and t, respectively. As PAE was not taken up by erythrocytes, the Kp values of liver and kidney, and those of other tissues were corrected by Eqs. 2 and 3 of Chen and Grosses), respectively

K P = CtQ (Qt + CLint,t •fp) (2) t • CP+Vt•kZ•Ct

Kp Q Qt-Ct (3) t•CP+Vt•kz•Ct Where Ct and Cp are tissue and plasma drug concentrations, respectively, CLint,,t and fP are tissue intrinsic clearance and plasma unbound fraction, respectively, Qt is tissue plasma flow, and Vt is tissue volume. The CLint,,t•f0 value was estimated from Eq. 4 :

CLH or CLR= QQt-CLint,t•fp (4) t +CLint,t f Where CLH is hepatic clearance, given by CLM+CLB.It was assumed that the drug is metabolized only in the liver. Values of Qt in brain, heart, lung, spleen, GI tract 16), liver, musclel7), skin18), and adipose tissuel9) were taken from the literature and that in kidney was experimentally determined. Plasma, muscle 17), skin18) and adipose tissue 19) volumes were taken from the literature. Other Vt values were experimentally determined by assu ming a density of 1.0 for each wet tissue. CLR f and GFR were calculated by the method of Arita et a120). Unbound plasma concentration at a steady-state level (Cfs) was estimated as unbound plasma fraction (0.75) x steady-state drug plasma concentration. RPF was calculated by the method of Shim et a19). All means are given with their standard error (mean--!-SEW Student's t test was utilized to determine the significance of differences between the doses, between the rat groups with and without biliary fistulization, and between the CLR,f GFR, or RPF values at various steady-state plasma drug levels.

Results

Pharmacokinetic aspects The plasma disposition of PAE after iv administration of 20, 40, or 100mg/kg in rats with and without biliary fistulization is plotted in Fig. 1. The decline in plasma concentration was biexponential in all cases, but with a much slower terminal disposition at 100mg/kg than at 20 and 40mg/kg in both groups, in which the terminal plasma concentration half life was approximately 20min at the two low doses and approximately 30min at the high dose. Table I shows the pharmacokinetic parameters for both groups. The mean AUC value increased proportionally to administrated dose at the dose range of 20 to 40mg/kg,

Fig. 1 Plasma disposition curves of PAE after intravenous administration of 20, 40, or 100mg/kg to rats Each point represents the mean ± SEM of five to seven rats. A : 20mg/ kg, • : 40mg/kg, • : 100mg/kg. The solid curves were calculated from the mean plasma concentrations using a digital computer by the least-squares method (using the MULTI program")) based on a two-compartment model (A=83.69pg/ml, a= 0.245/min, B=24.65pg/m1, p=0.036/min at 20mg/kg, A=155.12pg/m1, a=0.242/min, B=54.08,ug/ml, p=0.034/min at 40mg/kg, and A=438.25 pg/ml, a=0.205/min, B=311.61pg/ml, p=0.024/min at 100mg/kg in the rats without bile fistulas. A=66.34ug/ml, a=0.259/min, B=26.93pg/ml, p=0.031/min at 20mg/kg, A=157.90pg/m1, a=0. 198/min, B=48.02pg/ml, p=0.030/min at 40mg/kg, and A=412.54,ug/ml, a=0.197/min, B=307.33 pg/ml, p=0.025/min at 100mg/kg in the rats with dile fistulas). Table I . Pharmacokinetic Parameters of PAE in rats

Results are given as the mean -I SEM of five to seven rats. * : p<0 .05, ** : p<0.01, compared with 20 or 40 mg/kg group. but that at 100mg/kg was greater than the proportional increase of dose in both groups. The mean Vdss, CLtot, CLR, CLB, and CLM values at 100mg/kg were significantly smaller than those at 20 and 40mg/kg (p<0.01 or 0.05). Each parameter (Vdss, CLtot, or CLR) at the same dose showed no significant difference between the two groups. Plasma unbound fraction (0.754±0.008, n=18) was constant over the observed range of plasma PAE con centrations (0.8--700,ug/ml) following an iv dose. The blood-to-plasma concentration ratio RB (0.556±0.025, n=16) was also constant and the hematocrit value determined in this study was 0.43±0.02 (n=5), indicating that PAE is not well taken up by erythrocytes. It was concluded that changes in pharmacokinetic parameters are not related to the plasma protein binding or RB.

Biliary and urinary excretions Table II shows the total cumulative biliary and urinary excretions at the doses of 20, 40, and 100mg/kg. Both the total biliary and urinary excretions showed no significant

Fig. 2 Relationships of bile-to-plasma concentration ratio (Cbile/Cp) against plasma concentration (Cr) and bile-to-liver concentration (Cbile/Ciiver) against liver concentration (Ctiver) of PAE after intravenous administration of 100mg/kg to rats with biliary fistulization Each point represents the mean±SEM of three rats.

Table II . Total cumulative biliary and urinary excretions of PAE

Results are given as the mean±SEM of three rats. a) Per cent of the dose. difference between the doses. Unchanged drug was no longer detectable in bile and urine at 24h following iv administration. Biliary excretion and intestinal absorption (ka=0.00177 ± 0. 00021min-', n=3) of PAE were observed, but enterohepatic cycling made no apparent contribution to the AUC value, because the AUC values at the same dose showed no significant difference between the two groups (Table I ). The relationships of bile-to-plasma concentration ratio (Cbile/CP) against plasma con centration (Cp) and bile-to-liver concentration ratio (Cbile/Cliver) against liver concentration (Cliver) are shown in Fig. 2. The Cbile/Cp ratio decreased with increase of Cp, but the Cbile/ Cliver ratio seemed to be constant with no relation to Cliver . It is suggested that the decrea se of CLB at the highest dose (Table I) may reflect a saturable transport from plasma i nto liver, without a saturable transport from liver to bile.

The Kp values The Kp values of PAE at 30min after a 40 or 100mg/kg iv dose in the rats with bile fistulas are shown in Table M. The Kp values of kidney at both doses were larger than those (<1) of other tissues. The Kp values of liver, kidney, GI tract, and skin were signi ficantly smaller at the high dose than at the low dose (p<0.01 or 0.05). The Kp values of all tissues except for kidney and liver were smaller than 1, corresponding to the inter stitial volume (15% of tissue volume")). The values of the sum of the total tissue distri bution volume (E mean Kp•Vt) and plasma volume amounted to 385.1 and 265.9m1/kg at 40 and100mg/kg iv dose, respectively. These values correspond well to the VdSS values at each dose (Table I ). The sum thus decreased approximately by 31% when dose was

Table M. Tissue-to-plasma partition coefficient (Kp) of PAE at 30min after intravenous administration of 40 or 100mg/ kg in rats with biliary fistulization

Results are given as the mean±SEM of three to five rats. a) Corrected according to the method of Chen and Gross's) b) Plasma, muscle17). skin18). and adipose tissue191 volumes were taken from literature. Other tissue values were determined ex perimetally from the wet tissue weight by assuming a density of 1.0. * : p<0 .05, ** : p<0.01, compared with the 40mg/kg group. Fig. 3 Relationship between tissue-to-plasma partition coefficient (Kp) and plasma concentration of PAE in liver, kidney, gastrointestinal tract, and skin 0 : At 30min after 40mg/kg iv dosing. • : At 10, 30, 60, and 90min after 100mg/kg iv dosing.

changed from 40 to 100mg/kg. The decrease of VdSS at dose of 100mg/kg was approxima tely 33%, corresponding well to the above value of 31%. The Kp values of liver , kidney, GI tract, and skin decreased with increase of Cp (Fig. 3). The decrease of Vd55 at the highest dose (Table I ) may be almost entirely caused by the decrease of distribution volumes of the drug to liver, kidney, GI tract, and skin.

CLR f, GFR, CLR.f/GFR, and RPF at steady-state plasma unbound drug level (Cfs) CLR f, GFR, and CLR,f/GFR are shown in Table N. The mean CLR f and GFR values at the two higher plasma drug levels were significantly smaller than those in the control and at the two lower plasma levels (p<0.05). CLR f/GER was approximately 1 at each plasma level, indicating that PAE is excreted in urine only by glomerular filtration. As shown in Table V, RPF at the high steady-state plasma level was decreased significantly from those in the control and at the low level (p<0.05), suggesting that the decreases Table IV. Renal clearance of plasma unbound drug (CLR), glome rular filtration rate (GFR), and CLR f/GFR ratio at various steady-state unbound plasma levels (Cfs) of PAE

Results are given as the mean -± SEM of three rats. a) Calculated by using the mean plasma unbound fraction value, 0. 75. b) Determined by using inulin. c) The control experiments. * and# (p<0 .05), compared with d), and c) and e), respectively.

Table V. Effect of PAE on renal plasma flow rate (RPF) at various steady-state plasma levels (Cp) in rats

Results are given as the mean±SEM of three rats. a) Determined by the p-aminohippuric acid method11 . b) The control experiment. * : p<0 .05, compared with the control. of CLR f and GFR are caused by the decrease of RPF. The decrease of RPF might cause the decrease of CLR and also Kp in kidney.

Discussion It is well known that water-soluble drugs and metabolites having a larger molecular weight than approximately 350 are generally excreted in bile 23-26). Therefore, the biliary excretion of water-soluble PAE (M.W. 480.47)27) was examined. As biliary excretion and intestinal absorption were observed, enterohepatic cycling may practically occur, however, no influence of enterohepatic cycling on the AUC value was apparent at any dose, as described already. This may be because the biliary excretion amounted to only -18% of administered dose (Table II) and the ka value is low (0. 00177min-') ; for comparison, the ka of salicylic acid in rats is 0. 0112min t10), determined by the same method as that used in this study. The decrease of CLtot at 100mg/kg was ascribed to the dose-dependent CLB, CLR, and CLM. The decrease of CLB may be a consequence of saturable transport of PAE from plasma into liver (Fig. 2), because the Kp in liver decreased with increase of plasma concentration (Fig. 3) and a saturable transport of PAE to isolated hepatocytes was observed (unpublished data). The decrease of Kp in kidney, GI tract, and skin might reflect saturable transport from plasma into these tissues, as in the case of liver. As the total amount of unchanged drug excreted in urine was -41% of the dose (Table II ), it was assumed that PAE is metabolized only in liver, though there is no evidence to supp ort this. The decrease of RPF at the higher steady-state plasma level (Table V) might cau se the decrease of CLR and also Kp in kidney. An influence of PAE on RPF at the high er plasma level was newly found in this study. More study is needed to understand the mechanism of action on RPF and the influence on blood flow of other organs (i.e., liver) of PAE. In summary, the pharmacokinetics of PAE in the rat was dose-dependent. In this study, a decreasing effect of PAE on RPF at a high plasma level was found. This finding may be clinically useful in consecutive administrations of many preparations containing PAE to humans, especially to patients with renal disease.

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