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Kinetic Analysis of Glycyrrhetic Acid, an Active Metabolite of Glycyrrhizin, in Rats: Role of Enterohepatic Circulation
Kinetic Analysis of Glycyrrhetic Acid, an Active Metabolite of Glycyrrhizin, in Rats: Role of Enterohepatic Circulation JUNICHI KAWAKAMI', YOSHIKAZU YAMAMURA, TOMOFUMI SANTA, HAJIMEKOTAKI, KATSUYOSHI UCHINO, YASUFUMI SAWADA, AND TATSUJI IGA Received November 20, 1991,from the Department of Pharmacy, University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Accepted for publication August 2, 1992. Hongo, Bunkyo-ku, Tokyo 773, Japan. Abstract 0 The role of enterohepaticcirculationof glycyrrheticacid (GA) in rats was determined by kinetic analysis of GA. The concentrations of
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GA in the plasma of the control rat (without bile duct cannulation) during the first 5 h after intravenous (iv) administration of GA (2, 5, 10, and 20 mg/kg) were similar to those in the bile duct-cannulated rat at each dose. No significant difference was obsewed in the values of the terminal
half-life,the total body clearance, the distribution volume at steady state, the area under the cuwe of concentration in plasma versus time, and the mean residence time in each dose between both groups. When GA (2, 5, 10, and 20 mg/kg) was administered iv to the bile duct-cannulated rat, excretion of unchanged GA in bile was < 1% of each dose, that of the acid-hydrolyzed products was 14-1 6%, and that of GA-3-Oglucuronide was only 1-2%. In the control rat, a secondary peak of GA concentration was observed 12 h after iv administration of GA (20 mg/kg). The enterohepatic circulation of GA was confirmed by the linked-rat method in which bile of the donor rat after iv administration of GA (20 mg/kg) was allowed to flow directly into the duodenum of the recipient rat. GA was found in the plasma of the recipient rat after 6 h, and its concentration reached the maximum (-0.5 pg/mL) fi-12 h after dosing the donor rat. Comparing the area under the curve of concentration in plasma versus time in the recipient and the bile duct-cannulated rats, the estimated degree of the enterohepatic circulation in the overall disposition of GA was at most 3.5%.
Glycydizin
Glycyrrhetic acid
on
Glycyrrhetic acid (GA) is a triterpenoid and is used clinically for the treatment of patients with Addison's disease1.2 and neurodermatitiss because of its antiallergic and antiinflammatory effects.4.5 GA is also the active metabolite of glycyrrhizin, which is one of the main ingredients of Leguminosae Glycyrrhizae Radix and which has been used for the treatment of chronic hepatitis,Gs inflammation, and allergic disorder.9 Furthermore, GA has a potentiating effect on the action of aldosterone,lO an inhibiting effect on aldosterone metabolism,ll and produces side effects such as pseudoaldosteronism.12-17 Therefore, it is important to clarify the disposition of GA, especially its pharmacokinetic characteristics. However, little literature is available on the pharmacokinetics of GA. Ishida et al.15 reported that the disappearance of GA from plasma in rats after intravenous (iv) administration was dose dependent in the dose range 2-12.5 mg/kg, and they suggest that the dose dependency of GA was due to the saturation in the elimination process. Iveson et al.19 reported that tritium-labeled GA administered intraduodenally to rats was readily absorbed from the intestinal tract, large amounts of this radioactive material were excreted in bile, and three conjugates of GA were identified from the bile of rats orally or intraperitoneally dosed with GA. This latter finding suggests that GA is subject to enterohepatic circulation. Based on the studies of enterohepatic circulation of GA with tritium-labeled GA, Parke20 showed that the radioactive material was found partly in bile when pooled rat bile after iv administration of tritium-labeled GA was introduced intraduodenally into another rat. However, detailed studies of 0022-3549/93/0300-030 1$02.50/0 0 1993, American Pharmaceutical Association
Glycyrrhetic acid 3-0-glucuronide
the nature of this absorbed radioactive material and of the time courses of GA and the degree of the enterohepatic circulation have not been carried out. In this study, we investigated the plasma pharmacokinetic behavior of GA to elucidate the role of the enterohepatic circulation in the overall disposition of GA with two groups of rats (i.e., with and without bile duct cannulation).
Experimental Section Chemicals-GA used in this study was plant test grade (Nacalai (GA-Glu; Tesque, Inc., Kyoto, Japan). Glycyrrhetic-3-0-glucuronide mp, 243-244 "C),was synthesized in our laboratory according to the method of Hirooka et al.21 p-Glucuronidase (E. COW was obtained from Boehringer Mannheim (Germany). 3-Hydroxy-11-oxolean-12en-3-0-methyloic acid (GA-Me), used as the internal standard for GA-Glu assay, was synthesized according to the method of Hashimoto et al.22 Capric acid, used as the internal standard for GA-Glu assay, was obtained from Tokyo Kasei (Tokyo, Japan). 4-Bromomethyl-7methoxycoumarin, used as the fluorescent agent for GA-Glu assay, and tetra-n-amylammonium bromide, used as the counter ion for GA-Glu assay, were obtained from Wako Pure Chemical Industries (Osaka, Japan). All other reagents were commerciallyavailable and of analytical grade. Animals-Male Wistar rats weighing 280-320 g (Japan Laboratory Animals Company, Tokyo, Japan) were used. Animals were fed on commercial food pellets and tap water ad libitum. Journal of Pharmaceutical Sciences / 301 Vol. 82, No. 3, March 7993
Intravenous Administration Study-Rats were fasted for 18 h before use. Each rat was held on its back to a board and, under light anesthesia with ether, rats were cannulated with polyethylene tubing in the femoral artery and vein, the urinary bladder, and the bile duct. In the control rat, no bile duct cannulation was performed. The animals were allowed to recover from anesthesia for at least 1.5 h after the surgery. GA was dissolved in dimethyl sulfoxide (dosing volume: 0.15 mL/300 g body weight) because of its low solubility in aqueous solution. GA(2-, 5-, lo-, or 20-mgkg dose) was administered iv to the control and the bile duct-cannulated rats. Blood samples (-0.3 mL each) were collected through the cannula from the artery into heparinized glass tubes at 1, 5, 15,30, 45, 60, 90, and 120 min after dosing of 2 m&g, and at 1,5,15,30,60,120, and 180 min after dosing of 5,10,and 20 mglkg. In another experiment, GA (ZO-rngkg dose) was administered iv to the control rat, and blood samples were collected in the same manner as described above at 4,6,8,10, 12,14, 18, and 24 h after dosing. Blood samples were immediately centrifuged at 1620 x g for 5 min, and plasma was separated. After each time of blood sampling, a n equivalent volume of blood collected from other rats was transfused through the venous cannula. The body temperature was maintained at 37-38 "C with a heat lamp. Urine and bile samples were collected for 24 h. All samples were stored at -20 "C until analysis. Enterohepatic Circulation Study-The degree of enterohepatic circulation was estimated by a linked-rat method.23Paired rats, the donor and recipient, were anesthetized with ether, and then one end of a polyethylene tubing was inserted into the bile duct of the donor rat, and the opposite end was inserted into the upper duodenum of the recipient one. After an animal recovered from anesthesia, GA (20 mgkg) was injected rapidly through the cannula inserted into the femoral vein. Blood samples (-0.3 mL each) were collected through the cannula inserted into the femoral artery at 2, 4, 6,8, 10, 12, 14, 18, and 24 h after injection. Urine samples of both rats and bile samples of the recipient rat were collected for 24 h through the bile duct and the urinary bladder cannulas, respectively. Blood transfusion and maintenance of body temperature in both rats and treatments of blood, bile, and urine samples were performed in the same way as described above. Determination of GA Concentration-GA concentration in biological fluids was determined by high-performance liquid chromatography (HPLC). To 0.1 mL of plasma, urine, or bile were added 1mL of 0.1 M acetate buffer (pH 5.0) and 5 mL of dichloromethane. The mixture was shaken for 10 rnin and then centrifuged at 1620 x g for 5 min. Then, 4 mL of the organic phase was transferred to another tube, and 100 pL of ethanol, containing GA-Me (10 pg/mL) as the internal standard, was added. The mixture was evaporated at 40 "C. The residue was dissolved in 50 pL of acetonitrile, then an aliquot (20 pL) was injected into the HPLC column. A Shimadzu HPLC system was used with a ODS 5C,, (Senshu Scientific Company Ltd., Tokyo, Japan) column (5 pm particles, 250 x 6 mm id.). The mobile phase consisted of acetonitrile and 0.2% acetic acid (9:1, vlv). The column temperature was held at 40 "C, the flow rate was 1.0 mL/min, and UV detection was set at 254 nm. Coefficientsofvariation for the assay were 3.3 and 2.7% for 0.5- and BO-pg/mLsamples (n = 4), respectively. The quantitative limits of GA in plasma, bile, and urine were 100 ng/mL each. The HPLC chromatograms of GA in extracts of plasma, bile, and urine are shown in Figure 1. Determination of GA-Glu Concentration-The concentration of GA-Glu in biological fluids was determined by HPLC with fluorescence detection according to the method of Yamamura et al.24 This method includes ion-pair formation using tetra-n-amylammonium bromide, extraction with ethyl acetatex-hexane mixture (5:1) from salt-saturated aqueous phase, and labeling with 4-bromomethyl-7methoxycoumarin. The quantitative limits of GA-Glu in plasma, bile, and urine were 1, 2.5 and 2.5 pg/mL, respectively. Determination of Hydrolyzed Products-Preliminary experiments on hydrolysis of GA metabolites in bile and urine samples were carried out as follows: One milliliter of 0.1 M acetate buffer (pH 5.0) was added to 0.2 mL of each sample, and the aqueous phase was washed once with 8 mL of dichloromethane to remove GA in the samples. Then, to 0.1 mL of the upper aqueous phase, which was transferred to another tube, was added 0.5 mL of the same acetate buffer containing 20 IU of P-glucuronidase or 0.1 mL of 4 or 10 M hydrochloric acid solution. The sample containing P-glucuronidase was placed in an incubator at 37 "C for 24 h and that containing 302 I Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
t
I
I
0
8
16
I
24
0
I
I
8
16
24
0
1
8
16
24
Retention Time ( m i d
Figure 1-Chromatograms obtainedfrom: (a) rat plasmacontaining1 pg of GA (upper trace) and blank rat plasma (lower trace); (b) rat bile containing 1 pg of GA (upper trace) and blank rat bile (lower trace); (c) rat urine containing 1 pg of GA (upper trace) and blank rat urine (lower trace). Key: (Peak 1) GA; (Peak 2) GA-GIu (internal standard).
hydrochloric acid was placed in boiling water or in a water bath at 80 "C for 10,20,30,40,50, or 60 min. The liberated GA was analyzed in the same manner as described above. GA was liberated from bile and urine samples by incubation with hydrochloric acid but not P-glucuronidase. The amount of liberated GA from the bile sample supplemented with 10 M hydrochloric acid gradually declined in boiling water after 10 min. In the sample supplemented with 4 M hydrochloric acid, the amount of liberated GA became relatively constant within 30 min after incubation in boiling water, but not a t 80 "C (Figure 2). Therefore, suitable conditions for the hydrolysis were incubation with 4 M hydrochloric acid for 40 min in boiling water, and the procedure for determination of hydrolyzed products in bile and urine was established as follows: To 0.2 mL of bile and urine was added 8 mL of dichloromethane. The mixture was vigorously shaken for 10 min then centrifuged at 1620 x g for 5 min. Hydrochloric acid (4 M, 0.1 mL) was added to 0.1 mL of the aqueous phase, and then the mixture was placed in a boiling water bath for 40 min. The GA liberated by acid hydrolysis was analyzed in the same manner as described for GA above. Pharmacokinetic Analysis-The concentration of GA in plasma for individual animals after iv administration of GA to the control 0.2
1
U 0 ru 0
0.1
0.0 0
40
20
60
Time (min) Figure 2-Hydrolysis profiles of GA metabolites in various acidities and temperature. Key: (0)10 M HCI; (A)4 M HCI at 80 "C;(W) 4 M HCI in boiling water. Each point and vertical bar represent mean ? SD of three experiments.
and bile duct-cannulatedrats were fitted to the equation: C, = A x e-" + B x e - @ , where C,, is the drug concentration at time t , A and B are ordinate axis intercepts, and a and p are the corresponding first-order disposition rate constants. The terminal half-life was calculatedby dividing 0.693 by p. The area under the curve (AUC)of the concentration of GA in plasma versus time (plasma concentration-time curve) and the area under the first moments curve (AUMC) were calculated by Ala + Blp and AIa2 + B/p2,respectively.26 The total body clearance (CL,,,) was calculated from the ratio of the dose to AUC, and the mean residence time (MRT) was calculated by dividing AUMC by AUC. The steady-state volume of distribution (Vd,,) was calculated as the product of CL,, and MRT.
Results Intravenous Administration of GA-Figure 3 shows plasma concentration-time courses of GA after iv administration of four different doses of GA to the control rat. The concentration of GA in plasma declined biexponentially with each dose. Similar results were obtained in the bile ductcannulated rat (Figure 4). GA-Glu, the conjugated metabolite of GA, was not detected in any plasma sample. The results of pharmacokinetic analysis of the concentration of GA in plasma of the control rat are summarized in Table I. There were no significant differences in the values of t,,2 in the terminal phase and Vd,,among doses (paired test, p < 0.05). The values of CL,, and MRT after 5-, lo-, and 20-mgkg doses were not significantly different. However, the CLtotafter the 2-mgikgdose was greater than that after the other doses. The value of AUC increased linearly with dose in the range of 5 to 20 mg/kg. Pharmacokinetic parameters of GA in the bile duct-cannulated rat are summarized in Table 11. No significant difference in pharmacokinetic parameters for each dose was observed between the bile duct-cannulated and the control rats. Biliary and Urinary Excretions-Biliary and urinary excretion data of GA and its metabolites after iv administration of GA to the bile duct-cannulated rat are summarized in Table 111. The mean excretion ratios of GA in bile were only 0.1-0.7% of dose and those in urine were negligible. The excretion ratios of the hydrolyzed products that were obtained by treatment with hydrochloric acid were 14-17% in bile and <1%in urine. GA-Glu was excreted in bile and its ratio was 1-2%. No dose dependency
0.1
0
~ _ _ _ _
~
AUC, wg. h/mL CL,, mUh/kg
min Vd,,, mUkg MRT, min
5 ~~
10
20
39.3 2 8.2 262 t 51 4 3 + 15 172 2 15 37 t 4
85.9 t 8.0 234 f 23 41 -e 11 180 2 41 45 f 9
~
4.5 f 0.7 455 t 76 33t9 187 f 44 21 2 4
19.2 f 4.2 272 t 72 4 8 k 12 174 30 33 t 7
*
f
SD of four rats.
Table 11-Pharmacokinetic Parameters of GA after iv Administration of GA to Bile Duct-Cannulated Rats"
t,/2, min
Vd,,, mUkg MRT, min a
3
2 _____
Data are expressed as mean
-
2
Dose, mg/kg
Parameter
AUC, pg h/mL CLtot,mUh/kg
Time (h) Figure %Time courses of concentration of GA in plasma of control rats after iv administrationof GA. Each point and vertical bar represent mean c S D of four rats. The solid lines represent the predicted concentrations obtained from fitting biexponential functions to the mean data. Key: (A) 20-mg/kg dose; (A) lO-mg/kg dose; (0)5-mg/kg dose; (0)2-mglkg dose.
3
2
Table I-Pharmacoklnetic Parameters of GA after iv Administration of GA to Control Rats'
500 1
1
1
Time (h) Figure &Time courses of concentration of GA in plasma of bile duct-cannulatedrats after iv administrationof GA. Each point and vertical bar represent mean t S D of four rats. The solid lines represent the predicted concentrations obtained from fitting biexponential functions to the mean data. Key as in Figure 3.
Parameter
0
\
L
1 a 1
Dose, mglkg 2
5
5.0 + 0.9 414 f 75 27k4 1 3 6 2 23 19t2
21.7 2.2 233 f 23 51 t 11 174 t 13 3825
Data are expressed as mean
*
f
10
20
43.9 c 5.9 231 +. 29 3928 156 t 28 42 -+ 3
84.9 2 8.6 237 t 25 46 f 8 182 2 12 42 f 7
SD of four rats.
was observed in the excretion pattern of these compounds. Urinary excretion ratios of GA and the hydrolyzed products in the control rat were similar to those in the bile ductcannulated rat (data not shown). Enterohepatic Circulation Study-The plasma concentration-time course of GA for times later than 4 h after iv administration of GA (20 mgikg) to the control rat is shown in Figure 5. The concentration of GA in plasma after 6 h declined more slowly and the second peak was observed at 12 h after dosing, suggesting the presence of enterohepatic circulation of GA. To estimate the degree of enterohepatic circulation, linkedrat experiments were performed. Plasma concentration-time course data of GA in the recipient rat (Figure 6) indicate that GA was found in plasma after 6 h, and its concentration reached the maximum (-0.5 pg/mL) during the 8-12-h periods after administration to the donor rat. The results of Journal of Pharmaceutical SciencesI 303 Vol. 82, No. 3, March 1993
Table Ill-Blliary and Urinary Excretions of GA and Metabolites for 24 h after iv Administration of GA to Bile Dud-Cannulated Rats'
Urine, Yo
Bile, %
Dose, mgikg 2 5 10 20
GA
Hydrolysate
GA
H ydrolysate
0.68 f 0.40 0.29 f 0.13 0.21 f 0.06 0.08f 0.02
16.3 f 2.2 16.7 f 3.2 15.4 2 6.3 14.3 f 4.5
0.025 0.01 0.06 5 0.05 0.06 0.03 0.02 50.02
0.042 0.03 NDb 0.03f 0.02 NDb
*
'Data are expressed as mean f SD of four rats; values represent percent of dose.
Total, % 17.0 2 2.6 17.1 f 3.4 15.72 6.4 14.4f 4.5
Bile, % GA-GIu
0.99 f 0.59 0.94 2 0.24 1.09 f 0.62 1.96 f 1.41
ND, Not detected.
Table IV-Blliary and Urinary Excretlons of GA and Metabolites for 24 h in Recipient Rats'
Material GA
Hydrolysate
Amount (77) Excreted in: Bile Urine 0.03f 0.02 1.36 2 0.02
0.00f 0.00 NDb
a GA (20mglkg) was given iv to the donor rat; the total amount of excretion of GA and hydrolysate in the recipient rat was 1.392 0.01%; GA-GIu excreted in bile of the recipient rat was 0.02 f 0.01%; values represent percent of dose; data are expressed as mean f SD of three rats. Not detected.
Discussion Time (h) Flgure %Time courses of concentration of GA in plasma of control rats after iv administration of GA (20 mglkg). Each point and vertical bar
represent mean
2 cu
f
SD of three rats.
'1
0.75
0
Time (h) Flgure &Time courses of concentration of GA in the plasma of the recipient rats after an iv administration of GA (20mg/kg) to donor rats. Each point and vertical bar represent mean f SD of three rats. the biliary and urinary excretion in the recipient rat are summarized in Table IV.The total (sum of GA and hydrolyzed products) biliary excretion ratio for 24 h in the recipient rat was 1.4% of dose given to the donor rat, and the urinary excretion ratio was ~0.1%.To evaluate the degree of the enterohepatic circulation of GA, the AUC in the recipient rat was compared with that in the bile duct-cannulated rat. The AUC (0-24 h) in the recipient rat was 2.94 pg h/mL. This value corresponded to 3.5% of the AUC (0-m) in the bile duct-cannulated rat (84.9 pg * h/mL). The urinary excretion ratios of GA in the donor rat were 0.02 2 0.01% [mean 2 standard deviation (SD), n = 31 and hydrolysis products were not detected. These results were the same as those in the bile duct-cannulated rat (Table 111). 304 1 Journal of Pharmaceutical Sciences Vol. 62, No. 3,March 1993
The disappearance of GA from plasma of the control rat (without bile duct cannulation) showed linearity in the dose range of 5 to 20 m&g after iv administration. The reason why the value of CL,, following the 2-mg/kg dose was larger than that after the other doses is not evident from the results of the present study; further study will be necessary to elucidate this mechanism. On the other hand, Ishida et al.18 reported that the disappearance of GA from plasma of rats became much slower with increases in the iv dose of GA (2,5, and 12.5 mg/kg). This discrepancy between the results of Ishida et al.18 and our results on the disappearance of GA from plasma may have arisen because of the difference in experimental method (e.g., the analytical procedure or the restraint state of rats; that is, kept in restraining cages or back of rat on a board during experiments). The method used by Ishida et al.,lB which was based on extraction with methanol, did not include internal standard as an analytical carrier. The similarity of the pharmacokinetic parameters of GA between control and bile duct-cannulated rats suggests that the enterohepatic circulation of GA was negligible during the first 3 h after administration of GA. The plasma concentration-time profile of GA in the recipient rat well reflects the appearance of the second phase in the plasma concentrationtime profile in the control rat shown in Figure 5. Further, in the linked-rat experiments, GA was not detected in the recipient rat during the first 4 h after dosing to the donor rat, in agreement with the CLtot values in both rats with and without bile duct cannulation (Tables I and 11).In comparison with the AUCs in the recipient and the bile duct-cannulated rat, the contribution of the enterohepatic circulation of GA in the overall disposition of GA was estimated to be at most 3.5%.The excreted amounts of the unchanged GA in bile and urine after iv administration of GA (2-20 mg/kg) to the bile duct-cannulated rat was < 1% in each dose, suggesting that most of GA was metabolized. The metabolites of GA in the bile from rats dosed with GA have been identified19 as three conjugates: glycyrrhetyl-3-O-glucuronide, glycyrrhetic acid 3-5-hydrogen sulfate, and probably GA-Glu. However, the contribution of GA-Glu might be minor, because only 1-2% of the dose was detected in the bile after iv dosing with GA. On the other hand, 16-17% of the dose was excreted in bile as the hydrolyzed products. Therefore, it is likely that glycyrrhetic
acid 3-0-hydrogen sulfate and glycyrrhetyl-3-0-glucuronide were partly hydrolyzed to GA, probably in the intestinal tract. Concerning the disposition of GA after dosing of glycyrrhizin, the level of GA in plasma after glycyrrhizin iv dosing (10 mgkg) to rats was negligibly small, and the biliary excretion ratios of unchanged glycyrrhizin and its metabolite (GA) for 34 h was 98.2 and 0.03% of dose, respectively.24 Further, although the incubation studies of glycyrrhizin with an intestinal bacterial mixture obtained from fresh human feces have revealed that glycyrrhizin was hydrolyzed to GA,2637 the hydrolysis rate of the compound may be extremely slow in the in vivo state. From these findings and the results of enterohepatic circulation of GA in the present study, it is suggested that the contribution of the enterohepatic circulation of GA after iv administration of glycyrrhizin was relatively low. In fact, we reported that the concentrations of GA in plasma of patients with liver disease receiving chronic doses of glycyrrhizin (80 mg/day, iv) were ~ 3 0 0 ndmL.28 In conclusion, disappearance of GA from plasma of rats with and without bile cannulation was linear for the first 3 h following 5-20-mgkg doses. The results from the linked-rat experiments indicate that GA is subject to a relatively low degree of enterohepatic circulation in rats.
References and Notes 1. Groen, J.; Pelser, H.; Willebrands, A. F.; Kamminga, C. E. N . Engl. J. Med. 1951,244,471475. 2. Cdvert, R. J. Lancet 1954,1,805-807. 3. Jones, E. C. Postgrad. Med. J. 1960,36,678-682. 4. Finney, R.S.H.; Somers, G. F. J. Pharm. Pharmucol. 1958,10, 613-620. 5. Tangri, K. K.; Seth, P. K.; Parmar, S. S.; Bhargava, K. P. Biochem. Pharmucol. 1965,14, 1277-1281. 6. Hino, K.; Miyakawa, H.; Miyahara, T.; Fujikura, S.; Iwasaki, M.; Takahashi, K. Minophargen Med. Rev. 1981,26,270-277. 7. Kiso, Y.; Tohkin, M.; Hikino, H.; Hattori, M.; Sakamoto, T.; Namba, T. PZanta Medica 1984,50,298-302.
8. Hikino, H. Yakugaku Zusshi 1985,105,109-118. 9. Sotomatsu, S.;Takaishi, Y.; Hiroi, J.; Namikata, A.; Okano, N. Skin Urology 1959,21,138-144. 10. Hart, F.D.; Leonard, J. C. Lancet 1954,I, 804-805. 11. Tamura, Y.; Nishikawa, T.; Yamada, K.; Yamamoto, M.; Kumagai, A. Anneim-Forsch. 1979,29,647-649. 12. Conn, J. W.; Rovner, D. R.; Cohen, E. L. JAMA 1968, 205, 492496. 13. Epstein, M. T.; Espiner, E. A.; Donald, R. A.; Hughes, H. Br. Med. J. 1977,1,488-490. 14. Hoshiai, M.; Kanemoto, N.; Ide, M.; Kobayashi, T. Respir. Circl. 1980,28,169-172. 15. Takada, M.;Iuchi, K.; Yoshida, K.; Iida, H.; Mizumura, Y.; Sugimoto, T.; Nakano, N.; Kato, H.; Yano, S. Biomed. Ther. 1981, 7. . , 771-776. . . - . . -. 16. Shio, H. Clin. Endocrinol. 1982,30,57-62. 17. Terasawa, K.; Bandoh, M.; Tosa, H.; Hirate, J. J. PharmucobioDvn. 1986. 9.95-100. 18. Iihida, S.;'S&iya, Y.; Ichikawa, T.; Awazu, S. Chem. Phurm. Bull. 1989.37,2509-2413. 19. Iveson, P.;Lindup, W. E.; Parke, D. V. Xenobiotica 1971, 1 , 79-95. 20. Parke, D.V.In Symposium on CarbenoxolonSodium; Robson, J.; Sullivan, F., Eds.; Butterworths: London, U.K., 1968;pp 15-24. 21. Hirooka, M.; Morishima, N.; m i , E.; Mori, Y.; Zen, S. Yakugaku Zasshi 1989,109,544-559. 22. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981, 29,1475-1478. 23. Kotaki, H.; Yamamura, Y.; Tanimura, Y.; Saitoh, Y.; Nakagawa, F.; Tamura, Z. J. Phurmucobio-@n. 1984,7,420-425. 24. Yamamura, Y.; Kawakami, J.; Santa, T.; Kotaki, H.; Uchino, K.; Sawada, Y.;Iga, T. J. Chromatom. 1991,567,151-160. 25. Yamaoka, K.YNakagawa, T.; Uno, T. J. Pharmucokinet. BWpharm. 1978,6,547-558. 26. Hattori, M.; Sakamoto, T.; Kobashi, K.; Namba, T. PluntaMedica 1983.48. 38-42. 27. Hattori, M.;Sakamoto, T.; Yam shi, T.; Sakamoto, K.; Konishi, K.; Kobashi, K.; Namba, T. %em. Pharm. Bull. 1985, 33, 210-217. 28. Yamamura, Y.; Kawakami, J.; Santa, T.; Kotaki, H.; Uchino, K,; Sawada, Y.; Iga, T. In The 11 lth Annual Meetin of PhannuceuNojima, S., id.; Pharmaceutical Society of Japan (abstmct N); tical Society of Japan: Tokyo, Japan, 1991;p 70.
Journal ofPharmaceutical Sciences I 305 Vol. 82,No. 3, March 1993
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GA in the plasma of the control rat (without bile duct cannulation) during the first 5 h after intravenous (iv) administration of GA (2, 5, 10, and 20 mg/kg) were similar to those in the bile duct-cannulated rat at each dose. No significant difference was obsewed in the values of the terminal
half-life,the total body clearance, the distribution volume at steady state, the area under the cuwe of concentration in plasma versus time, and the mean residence time in each dose between both groups. When GA (2, 5, 10, and 20 mg/kg) was administered iv to the bile duct-cannulated rat, excretion of unchanged GA in bile was < 1% of each dose, that of the acid-hydrolyzed products was 14-1 6%, and that of GA-3-Oglucuronide was only 1-2%. In the control rat, a secondary peak of GA concentration was observed 12 h after iv administration of GA (20 mg/kg). The enterohepatic circulation of GA was confirmed by the linked-rat method in which bile of the donor rat after iv administration of GA (20 mg/kg) was allowed to flow directly into the duodenum of the recipient rat. GA was found in the plasma of the recipient rat after 6 h, and its concentration reached the maximum (-0.5 pg/mL) fi-12 h after dosing the donor rat. Comparing the area under the curve of concentration in plasma versus time in the recipient and the bile duct-cannulated rats, the estimated degree of the enterohepatic circulation in the overall disposition of GA was at most 3.5%.
Glycydizin
Glycyrrhetic acid
on
Glycyrrhetic acid (GA) is a triterpenoid and is used clinically for the treatment of patients with Addison's disease1.2 and neurodermatitiss because of its antiallergic and antiinflammatory effects.4.5 GA is also the active metabolite of glycyrrhizin, which is one of the main ingredients of Leguminosae Glycyrrhizae Radix and which has been used for the treatment of chronic hepatitis,Gs inflammation, and allergic disorder.9 Furthermore, GA has a potentiating effect on the action of aldosterone,lO an inhibiting effect on aldosterone metabolism,ll and produces side effects such as pseudoaldosteronism.12-17 Therefore, it is important to clarify the disposition of GA, especially its pharmacokinetic characteristics. However, little literature is available on the pharmacokinetics of GA. Ishida et al.15 reported that the disappearance of GA from plasma in rats after intravenous (iv) administration was dose dependent in the dose range 2-12.5 mg/kg, and they suggest that the dose dependency of GA was due to the saturation in the elimination process. Iveson et al.19 reported that tritium-labeled GA administered intraduodenally to rats was readily absorbed from the intestinal tract, large amounts of this radioactive material were excreted in bile, and three conjugates of GA were identified from the bile of rats orally or intraperitoneally dosed with GA. This latter finding suggests that GA is subject to enterohepatic circulation. Based on the studies of enterohepatic circulation of GA with tritium-labeled GA, Parke20 showed that the radioactive material was found partly in bile when pooled rat bile after iv administration of tritium-labeled GA was introduced intraduodenally into another rat. However, detailed studies of 0022-3549/93/0300-030 1$02.50/0 0 1993, American Pharmaceutical Association
Glycyrrhetic acid 3-0-glucuronide
the nature of this absorbed radioactive material and of the time courses of GA and the degree of the enterohepatic circulation have not been carried out. In this study, we investigated the plasma pharmacokinetic behavior of GA to elucidate the role of the enterohepatic circulation in the overall disposition of GA with two groups of rats (i.e., with and without bile duct cannulation).
Experimental Section Chemicals-GA used in this study was plant test grade (Nacalai (GA-Glu; Tesque, Inc., Kyoto, Japan). Glycyrrhetic-3-0-glucuronide mp, 243-244 "C),was synthesized in our laboratory according to the method of Hirooka et al.21 p-Glucuronidase (E. COW was obtained from Boehringer Mannheim (Germany). 3-Hydroxy-11-oxolean-12en-3-0-methyloic acid (GA-Me), used as the internal standard for GA-Glu assay, was synthesized according to the method of Hashimoto et al.22 Capric acid, used as the internal standard for GA-Glu assay, was obtained from Tokyo Kasei (Tokyo, Japan). 4-Bromomethyl-7methoxycoumarin, used as the fluorescent agent for GA-Glu assay, and tetra-n-amylammonium bromide, used as the counter ion for GA-Glu assay, were obtained from Wako Pure Chemical Industries (Osaka, Japan). All other reagents were commerciallyavailable and of analytical grade. Animals-Male Wistar rats weighing 280-320 g (Japan Laboratory Animals Company, Tokyo, Japan) were used. Animals were fed on commercial food pellets and tap water ad libitum. Journal of Pharmaceutical Sciences / 301 Vol. 82, No. 3, March 7993
Intravenous Administration Study-Rats were fasted for 18 h before use. Each rat was held on its back to a board and, under light anesthesia with ether, rats were cannulated with polyethylene tubing in the femoral artery and vein, the urinary bladder, and the bile duct. In the control rat, no bile duct cannulation was performed. The animals were allowed to recover from anesthesia for at least 1.5 h after the surgery. GA was dissolved in dimethyl sulfoxide (dosing volume: 0.15 mL/300 g body weight) because of its low solubility in aqueous solution. GA(2-, 5-, lo-, or 20-mgkg dose) was administered iv to the control and the bile duct-cannulated rats. Blood samples (-0.3 mL each) were collected through the cannula from the artery into heparinized glass tubes at 1, 5, 15,30, 45, 60, 90, and 120 min after dosing of 2 m&g, and at 1,5,15,30,60,120, and 180 min after dosing of 5,10,and 20 mglkg. In another experiment, GA (ZO-rngkg dose) was administered iv to the control rat, and blood samples were collected in the same manner as described above at 4,6,8,10, 12,14, 18, and 24 h after dosing. Blood samples were immediately centrifuged at 1620 x g for 5 min, and plasma was separated. After each time of blood sampling, a n equivalent volume of blood collected from other rats was transfused through the venous cannula. The body temperature was maintained at 37-38 "C with a heat lamp. Urine and bile samples were collected for 24 h. All samples were stored at -20 "C until analysis. Enterohepatic Circulation Study-The degree of enterohepatic circulation was estimated by a linked-rat method.23Paired rats, the donor and recipient, were anesthetized with ether, and then one end of a polyethylene tubing was inserted into the bile duct of the donor rat, and the opposite end was inserted into the upper duodenum of the recipient one. After an animal recovered from anesthesia, GA (20 mgkg) was injected rapidly through the cannula inserted into the femoral vein. Blood samples (-0.3 mL each) were collected through the cannula inserted into the femoral artery at 2, 4, 6,8, 10, 12, 14, 18, and 24 h after injection. Urine samples of both rats and bile samples of the recipient rat were collected for 24 h through the bile duct and the urinary bladder cannulas, respectively. Blood transfusion and maintenance of body temperature in both rats and treatments of blood, bile, and urine samples were performed in the same way as described above. Determination of GA Concentration-GA concentration in biological fluids was determined by high-performance liquid chromatography (HPLC). To 0.1 mL of plasma, urine, or bile were added 1mL of 0.1 M acetate buffer (pH 5.0) and 5 mL of dichloromethane. The mixture was shaken for 10 rnin and then centrifuged at 1620 x g for 5 min. Then, 4 mL of the organic phase was transferred to another tube, and 100 pL of ethanol, containing GA-Me (10 pg/mL) as the internal standard, was added. The mixture was evaporated at 40 "C. The residue was dissolved in 50 pL of acetonitrile, then an aliquot (20 pL) was injected into the HPLC column. A Shimadzu HPLC system was used with a ODS 5C,, (Senshu Scientific Company Ltd., Tokyo, Japan) column (5 pm particles, 250 x 6 mm id.). The mobile phase consisted of acetonitrile and 0.2% acetic acid (9:1, vlv). The column temperature was held at 40 "C, the flow rate was 1.0 mL/min, and UV detection was set at 254 nm. Coefficientsofvariation for the assay were 3.3 and 2.7% for 0.5- and BO-pg/mLsamples (n = 4), respectively. The quantitative limits of GA in plasma, bile, and urine were 100 ng/mL each. The HPLC chromatograms of GA in extracts of plasma, bile, and urine are shown in Figure 1. Determination of GA-Glu Concentration-The concentration of GA-Glu in biological fluids was determined by HPLC with fluorescence detection according to the method of Yamamura et al.24 This method includes ion-pair formation using tetra-n-amylammonium bromide, extraction with ethyl acetatex-hexane mixture (5:1) from salt-saturated aqueous phase, and labeling with 4-bromomethyl-7methoxycoumarin. The quantitative limits of GA-Glu in plasma, bile, and urine were 1, 2.5 and 2.5 pg/mL, respectively. Determination of Hydrolyzed Products-Preliminary experiments on hydrolysis of GA metabolites in bile and urine samples were carried out as follows: One milliliter of 0.1 M acetate buffer (pH 5.0) was added to 0.2 mL of each sample, and the aqueous phase was washed once with 8 mL of dichloromethane to remove GA in the samples. Then, to 0.1 mL of the upper aqueous phase, which was transferred to another tube, was added 0.5 mL of the same acetate buffer containing 20 IU of P-glucuronidase or 0.1 mL of 4 or 10 M hydrochloric acid solution. The sample containing P-glucuronidase was placed in an incubator at 37 "C for 24 h and that containing 302 I Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
t
I
I
0
8
16
I
24
0
I
I
8
16
24
0
1
8
16
24
Retention Time ( m i d
Figure 1-Chromatograms obtainedfrom: (a) rat plasmacontaining1 pg of GA (upper trace) and blank rat plasma (lower trace); (b) rat bile containing 1 pg of GA (upper trace) and blank rat bile (lower trace); (c) rat urine containing 1 pg of GA (upper trace) and blank rat urine (lower trace). Key: (Peak 1) GA; (Peak 2) GA-GIu (internal standard).
hydrochloric acid was placed in boiling water or in a water bath at 80 "C for 10,20,30,40,50, or 60 min. The liberated GA was analyzed in the same manner as described above. GA was liberated from bile and urine samples by incubation with hydrochloric acid but not P-glucuronidase. The amount of liberated GA from the bile sample supplemented with 10 M hydrochloric acid gradually declined in boiling water after 10 min. In the sample supplemented with 4 M hydrochloric acid, the amount of liberated GA became relatively constant within 30 min after incubation in boiling water, but not a t 80 "C (Figure 2). Therefore, suitable conditions for the hydrolysis were incubation with 4 M hydrochloric acid for 40 min in boiling water, and the procedure for determination of hydrolyzed products in bile and urine was established as follows: To 0.2 mL of bile and urine was added 8 mL of dichloromethane. The mixture was vigorously shaken for 10 min then centrifuged at 1620 x g for 5 min. Hydrochloric acid (4 M, 0.1 mL) was added to 0.1 mL of the aqueous phase, and then the mixture was placed in a boiling water bath for 40 min. The GA liberated by acid hydrolysis was analyzed in the same manner as described for GA above. Pharmacokinetic Analysis-The concentration of GA in plasma for individual animals after iv administration of GA to the control 0.2
1
U 0 ru 0
0.1
0.0 0
40
20
60
Time (min) Figure 2-Hydrolysis profiles of GA metabolites in various acidities and temperature. Key: (0)10 M HCI; (A)4 M HCI at 80 "C;(W) 4 M HCI in boiling water. Each point and vertical bar represent mean ? SD of three experiments.
and bile duct-cannulatedrats were fitted to the equation: C, = A x e-" + B x e - @ , where C,, is the drug concentration at time t , A and B are ordinate axis intercepts, and a and p are the corresponding first-order disposition rate constants. The terminal half-life was calculatedby dividing 0.693 by p. The area under the curve (AUC)of the concentration of GA in plasma versus time (plasma concentration-time curve) and the area under the first moments curve (AUMC) were calculated by Ala + Blp and AIa2 + B/p2,respectively.26 The total body clearance (CL,,,) was calculated from the ratio of the dose to AUC, and the mean residence time (MRT) was calculated by dividing AUMC by AUC. The steady-state volume of distribution (Vd,,) was calculated as the product of CL,, and MRT.
Results Intravenous Administration of GA-Figure 3 shows plasma concentration-time courses of GA after iv administration of four different doses of GA to the control rat. The concentration of GA in plasma declined biexponentially with each dose. Similar results were obtained in the bile ductcannulated rat (Figure 4). GA-Glu, the conjugated metabolite of GA, was not detected in any plasma sample. The results of pharmacokinetic analysis of the concentration of GA in plasma of the control rat are summarized in Table I. There were no significant differences in the values of t,,2 in the terminal phase and Vd,,among doses (paired test, p < 0.05). The values of CL,, and MRT after 5-, lo-, and 20-mgkg doses were not significantly different. However, the CLtotafter the 2-mgikgdose was greater than that after the other doses. The value of AUC increased linearly with dose in the range of 5 to 20 mg/kg. Pharmacokinetic parameters of GA in the bile duct-cannulated rat are summarized in Table 11. No significant difference in pharmacokinetic parameters for each dose was observed between the bile duct-cannulated and the control rats. Biliary and Urinary Excretions-Biliary and urinary excretion data of GA and its metabolites after iv administration of GA to the bile duct-cannulated rat are summarized in Table 111. The mean excretion ratios of GA in bile were only 0.1-0.7% of dose and those in urine were negligible. The excretion ratios of the hydrolyzed products that were obtained by treatment with hydrochloric acid were 14-17% in bile and <1%in urine. GA-Glu was excreted in bile and its ratio was 1-2%. No dose dependency
0.1
0
~ _ _ _ _
~
AUC, wg. h/mL CL,, mUh/kg
min Vd,,, mUkg MRT, min
5 ~~
10
20
39.3 2 8.2 262 t 51 4 3 + 15 172 2 15 37 t 4
85.9 t 8.0 234 f 23 41 -e 11 180 2 41 45 f 9
~
4.5 f 0.7 455 t 76 33t9 187 f 44 21 2 4
19.2 f 4.2 272 t 72 4 8 k 12 174 30 33 t 7
*
f
SD of four rats.
Table 11-Pharmacokinetic Parameters of GA after iv Administration of GA to Bile Duct-Cannulated Rats"
t,/2, min
Vd,,, mUkg MRT, min a
3
2 _____
Data are expressed as mean
-
2
Dose, mg/kg
Parameter
AUC, pg h/mL CLtot,mUh/kg
Time (h) Figure %Time courses of concentration of GA in plasma of control rats after iv administrationof GA. Each point and vertical bar represent mean c S D of four rats. The solid lines represent the predicted concentrations obtained from fitting biexponential functions to the mean data. Key: (A) 20-mg/kg dose; (A) lO-mg/kg dose; (0)5-mg/kg dose; (0)2-mglkg dose.
3
2
Table I-Pharmacoklnetic Parameters of GA after iv Administration of GA to Control Rats'
500 1
1
1
Time (h) Figure &Time courses of concentration of GA in plasma of bile duct-cannulatedrats after iv administrationof GA. Each point and vertical bar represent mean t S D of four rats. The solid lines represent the predicted concentrations obtained from fitting biexponential functions to the mean data. Key as in Figure 3.
Parameter
0
\
L
1 a 1
Dose, mglkg 2
5
5.0 + 0.9 414 f 75 27k4 1 3 6 2 23 19t2
21.7 2.2 233 f 23 51 t 11 174 t 13 3825
Data are expressed as mean
*
f
10
20
43.9 c 5.9 231 +. 29 3928 156 t 28 42 -+ 3
84.9 2 8.6 237 t 25 46 f 8 182 2 12 42 f 7
SD of four rats.
was observed in the excretion pattern of these compounds. Urinary excretion ratios of GA and the hydrolyzed products in the control rat were similar to those in the bile ductcannulated rat (data not shown). Enterohepatic Circulation Study-The plasma concentration-time course of GA for times later than 4 h after iv administration of GA (20 mgikg) to the control rat is shown in Figure 5. The concentration of GA in plasma after 6 h declined more slowly and the second peak was observed at 12 h after dosing, suggesting the presence of enterohepatic circulation of GA. To estimate the degree of enterohepatic circulation, linkedrat experiments were performed. Plasma concentration-time course data of GA in the recipient rat (Figure 6) indicate that GA was found in plasma after 6 h, and its concentration reached the maximum (-0.5 pg/mL) during the 8-12-h periods after administration to the donor rat. The results of Journal of Pharmaceutical SciencesI 303 Vol. 82, No. 3, March 1993
Table Ill-Blliary and Urinary Excretions of GA and Metabolites for 24 h after iv Administration of GA to Bile Dud-Cannulated Rats'
Urine, Yo
Bile, %
Dose, mgikg 2 5 10 20
GA
Hydrolysate
GA
H ydrolysate
0.68 f 0.40 0.29 f 0.13 0.21 f 0.06 0.08f 0.02
16.3 f 2.2 16.7 f 3.2 15.4 2 6.3 14.3 f 4.5
0.025 0.01 0.06 5 0.05 0.06 0.03 0.02 50.02
0.042 0.03 NDb 0.03f 0.02 NDb
*
'Data are expressed as mean f SD of four rats; values represent percent of dose.
Total, % 17.0 2 2.6 17.1 f 3.4 15.72 6.4 14.4f 4.5
Bile, % GA-GIu
0.99 f 0.59 0.94 2 0.24 1.09 f 0.62 1.96 f 1.41
ND, Not detected.
Table IV-Blliary and Urinary Excretlons of GA and Metabolites for 24 h in Recipient Rats'
Material GA
Hydrolysate
Amount (77) Excreted in: Bile Urine 0.03f 0.02 1.36 2 0.02
0.00f 0.00 NDb
a GA (20mglkg) was given iv to the donor rat; the total amount of excretion of GA and hydrolysate in the recipient rat was 1.392 0.01%; GA-GIu excreted in bile of the recipient rat was 0.02 f 0.01%; values represent percent of dose; data are expressed as mean f SD of three rats. Not detected.
Discussion Time (h) Flgure %Time courses of concentration of GA in plasma of control rats after iv administration of GA (20 mglkg). Each point and vertical bar
represent mean
2 cu
f
SD of three rats.
'1
0.75
0
Time (h) Flgure &Time courses of concentration of GA in the plasma of the recipient rats after an iv administration of GA (20mg/kg) to donor rats. Each point and vertical bar represent mean f SD of three rats. the biliary and urinary excretion in the recipient rat are summarized in Table IV.The total (sum of GA and hydrolyzed products) biliary excretion ratio for 24 h in the recipient rat was 1.4% of dose given to the donor rat, and the urinary excretion ratio was ~0.1%.To evaluate the degree of the enterohepatic circulation of GA, the AUC in the recipient rat was compared with that in the bile duct-cannulated rat. The AUC (0-24 h) in the recipient rat was 2.94 pg h/mL. This value corresponded to 3.5% of the AUC (0-m) in the bile duct-cannulated rat (84.9 pg * h/mL). The urinary excretion ratios of GA in the donor rat were 0.02 2 0.01% [mean 2 standard deviation (SD), n = 31 and hydrolysis products were not detected. These results were the same as those in the bile duct-cannulated rat (Table 111). 304 1 Journal of Pharmaceutical Sciences Vol. 62, No. 3,March 1993
The disappearance of GA from plasma of the control rat (without bile duct cannulation) showed linearity in the dose range of 5 to 20 m&g after iv administration. The reason why the value of CL,, following the 2-mg/kg dose was larger than that after the other doses is not evident from the results of the present study; further study will be necessary to elucidate this mechanism. On the other hand, Ishida et al.18 reported that the disappearance of GA from plasma of rats became much slower with increases in the iv dose of GA (2,5, and 12.5 mg/kg). This discrepancy between the results of Ishida et al.18 and our results on the disappearance of GA from plasma may have arisen because of the difference in experimental method (e.g., the analytical procedure or the restraint state of rats; that is, kept in restraining cages or back of rat on a board during experiments). The method used by Ishida et al.,lB which was based on extraction with methanol, did not include internal standard as an analytical carrier. The similarity of the pharmacokinetic parameters of GA between control and bile duct-cannulated rats suggests that the enterohepatic circulation of GA was negligible during the first 3 h after administration of GA. The plasma concentration-time profile of GA in the recipient rat well reflects the appearance of the second phase in the plasma concentrationtime profile in the control rat shown in Figure 5. Further, in the linked-rat experiments, GA was not detected in the recipient rat during the first 4 h after dosing to the donor rat, in agreement with the CLtot values in both rats with and without bile duct cannulation (Tables I and 11).In comparison with the AUCs in the recipient and the bile duct-cannulated rat, the contribution of the enterohepatic circulation of GA in the overall disposition of GA was estimated to be at most 3.5%.The excreted amounts of the unchanged GA in bile and urine after iv administration of GA (2-20 mg/kg) to the bile duct-cannulated rat was < 1% in each dose, suggesting that most of GA was metabolized. The metabolites of GA in the bile from rats dosed with GA have been identified19 as three conjugates: glycyrrhetyl-3-O-glucuronide, glycyrrhetic acid 3-5-hydrogen sulfate, and probably GA-Glu. However, the contribution of GA-Glu might be minor, because only 1-2% of the dose was detected in the bile after iv dosing with GA. On the other hand, 16-17% of the dose was excreted in bile as the hydrolyzed products. Therefore, it is likely that glycyrrhetic
acid 3-0-hydrogen sulfate and glycyrrhetyl-3-0-glucuronide were partly hydrolyzed to GA, probably in the intestinal tract. Concerning the disposition of GA after dosing of glycyrrhizin, the level of GA in plasma after glycyrrhizin iv dosing (10 mgkg) to rats was negligibly small, and the biliary excretion ratios of unchanged glycyrrhizin and its metabolite (GA) for 34 h was 98.2 and 0.03% of dose, respectively.24 Further, although the incubation studies of glycyrrhizin with an intestinal bacterial mixture obtained from fresh human feces have revealed that glycyrrhizin was hydrolyzed to GA,2637 the hydrolysis rate of the compound may be extremely slow in the in vivo state. From these findings and the results of enterohepatic circulation of GA in the present study, it is suggested that the contribution of the enterohepatic circulation of GA after iv administration of glycyrrhizin was relatively low. In fact, we reported that the concentrations of GA in plasma of patients with liver disease receiving chronic doses of glycyrrhizin (80 mg/day, iv) were ~ 3 0 0 ndmL.28 In conclusion, disappearance of GA from plasma of rats with and without bile cannulation was linear for the first 3 h following 5-20-mgkg doses. The results from the linked-rat experiments indicate that GA is subject to a relatively low degree of enterohepatic circulation in rats.
References and Notes 1. Groen, J.; Pelser, H.; Willebrands, A. F.; Kamminga, C. E. N . Engl. J. Med. 1951,244,471475. 2. Cdvert, R. J. Lancet 1954,1,805-807. 3. Jones, E. C. Postgrad. Med. J. 1960,36,678-682. 4. Finney, R.S.H.; Somers, G. F. J. Pharm. Pharmucol. 1958,10, 613-620. 5. Tangri, K. K.; Seth, P. K.; Parmar, S. S.; Bhargava, K. P. Biochem. Pharmucol. 1965,14, 1277-1281. 6. Hino, K.; Miyakawa, H.; Miyahara, T.; Fujikura, S.; Iwasaki, M.; Takahashi, K. Minophargen Med. Rev. 1981,26,270-277. 7. Kiso, Y.; Tohkin, M.; Hikino, H.; Hattori, M.; Sakamoto, T.; Namba, T. PZanta Medica 1984,50,298-302.
8. Hikino, H. Yakugaku Zusshi 1985,105,109-118. 9. Sotomatsu, S.;Takaishi, Y.; Hiroi, J.; Namikata, A.; Okano, N. Skin Urology 1959,21,138-144. 10. Hart, F.D.; Leonard, J. C. Lancet 1954,I, 804-805. 11. Tamura, Y.; Nishikawa, T.; Yamada, K.; Yamamoto, M.; Kumagai, A. Anneim-Forsch. 1979,29,647-649. 12. Conn, J. W.; Rovner, D. R.; Cohen, E. L. JAMA 1968, 205, 492496. 13. Epstein, M. T.; Espiner, E. A.; Donald, R. A.; Hughes, H. Br. Med. J. 1977,1,488-490. 14. Hoshiai, M.; Kanemoto, N.; Ide, M.; Kobayashi, T. Respir. Circl. 1980,28,169-172. 15. Takada, M.;Iuchi, K.; Yoshida, K.; Iida, H.; Mizumura, Y.; Sugimoto, T.; Nakano, N.; Kato, H.; Yano, S. Biomed. Ther. 1981, 7. . , 771-776. . . - . . -. 16. Shio, H. Clin. Endocrinol. 1982,30,57-62. 17. Terasawa, K.; Bandoh, M.; Tosa, H.; Hirate, J. J. PharmucobioDvn. 1986. 9.95-100. 18. Iihida, S.;'S&iya, Y.; Ichikawa, T.; Awazu, S. Chem. Phurm. Bull. 1989.37,2509-2413. 19. Iveson, P.;Lindup, W. E.; Parke, D. V. Xenobiotica 1971, 1 , 79-95. 20. Parke, D.V.In Symposium on CarbenoxolonSodium; Robson, J.; Sullivan, F., Eds.; Butterworths: London, U.K., 1968;pp 15-24. 21. Hirooka, M.; Morishima, N.; m i , E.; Mori, Y.; Zen, S. Yakugaku Zasshi 1989,109,544-559. 22. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981, 29,1475-1478. 23. Kotaki, H.; Yamamura, Y.; Tanimura, Y.; Saitoh, Y.; Nakagawa, F.; Tamura, Z. J. Phurmucobio-@n. 1984,7,420-425. 24. Yamamura, Y.; Kawakami, J.; Santa, T.; Kotaki, H.; Uchino, K.; Sawada, Y.;Iga, T. J. Chromatom. 1991,567,151-160. 25. Yamaoka, K.YNakagawa, T.; Uno, T. J. Pharmucokinet. BWpharm. 1978,6,547-558. 26. Hattori, M.; Sakamoto, T.; Kobashi, K.; Namba, T. PluntaMedica 1983.48. 38-42. 27. Hattori, M.;Sakamoto, T.; Yam shi, T.; Sakamoto, K.; Konishi, K.; Kobashi, K.; Namba, T. %em. Pharm. Bull. 1985, 33, 210-217. 28. Yamamura, Y.; Kawakami, J.; Santa, T.; Kotaki, H.; Uchino, K,; Sawada, Y.; Iga, T. In The 11 lth Annual Meetin of PhannuceuNojima, S., id.; Pharmaceutical Society of Japan (abstmct N); tical Society of Japan: Tokyo, Japan, 1991;p 70.
Journal ofPharmaceutical Sciences I 305 Vol. 82,No. 3, March 1993