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Improvement of Intestinal Absorption of Pglycoprotein Substrate by Dtartaric Acid
Drug Metab. Pharmacokinet. 21 (5): 424428 (2006).
Regular Article Improvement of Intestinal Absorption of Pglycoprotein Substrate by Dtartaric Acid Aiko IIDA*, Mikio TOMITA, Yoko IDOTA, Yusuke TAKIZAWA and Masahiro HAYASHI Department of Drug Absorption and Pharmacokinetics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: The purpose of the present experiment was to examine the eects of Dtartaric acid (TA) on intestinal drug absorption under both in situ and in vitro experimental conditions. In the in vitro diusion chamber experiments, TA (10 mM) added to the mucosal side of rat colon signicantly decreased rhoda mine123 (Rho 123) transport from the serosal to mucosal side. Since TA has been shown to change the integrity of the epithelial tight junctions in rat colon at low pH conditions, resulting in improved paracel lular drug transport, the eect of TA on membrane resistance was examined at pH 7.4 in the present study. It was found that membrane resistance, an indicator of paracellular integrity, did not change at pH 7.4. In the in situ loop method, TA (20 mM) increased the absorption of Rho123 in both ileum and colon but not in jejunum. TA (20 mM) also increased the absorption of daunorubicin in the ileum, but TA (20 mM) did not change the expression level of Pglycoprotein (Pgp). TA (20 mM) signicantly inhibited ex cretion of i.v.administered Rho123 and daunorubicin into the ileal lumen. In conclusion, for the rst time we demonstrated that TA increases the intestinal absorption of Pgp substrates Rho123 and daunorubi cin, possibly by modulating the Pgp function without changing the expression level of Pgp in the rat in testine. Key words: Dtartaric acid; Pglycoprotein; jejunum; ileum; colon; absorption improvement tion of catechin either by increasing the transepithelial transport resulting from the changed integrity of tight junctions or by inhibiting the eux of catechin from the cell to lumen resulting from the modulation of Pgp function. If TA has an eect to increase the intestinal absorp tion of Pgp substrates other than catechin, this compound may have a great impact on the improvement of drug therapy. To evaluate this possibility, we exam ined the eects of TA on the intestinal absorption in both the in vitro and in situ experimental conditions. Our results demonstrated for the rst time that TA increases intestinal absorption of Pgp substrates possibly by directly modulating the Pgp function.
Introduction Tartaric acid is a dicarboxylic acid considered to have no pharmacological eect except for shortening intesti nal transit time at larger doses.1) For this reason, this organic acid has been widely used as a drug excipient in the pharmaceutical industry. In our previous study, we demonstrated that Dtartar ic acid (TA) has an eect of changing the integrity of the epithelial tight junctions in rat colon at low pH.2) This signies that use of TA at low pH possibly enhances intestinal absorption of drugs such as hydrophilic macromolecules. In the rat, oral administration of TA together with a low molecular weight polyphenol, catechin, signicantly increased the serum concentra tion of catechin glucuronide, indicating that TA increased the intestinal absorption of catechin.3) It was also suggested that tea catechins are substrates of Pglycoprotein (Pgp).4,5) These results suggest the possibility that TA may increase the intestinal absorp
Materials and Methods Materials: Dtartaric acid, rhodamine 123 (Rho123), daunorubicin, verapamil and FITCdextran 40000 (FD40) were purchased from Sigma Aldrich Co. Ltd. All other reagents were of analytical grade or higher.
Received; January 13, 2006, Accepted; August 22, 2006 *To whom correspondence should be addressed : Aiko IIDA, Department of Drug Absorption and Pharmacokinetics, School of Pharmacy and Life Science, 14321 Horinouchi, Hachioji, Tokyo, 1920392 Japan. Tel. {81426763168, Fax. {81426763142, Email: aiaiädadahotmail. com 424
Dtartaric
Acid Improves Intestinal Drug Absorption
Animals and experimental design: Male Wistar rats (eight weeks old) were purchased from Japan SLC Ltd. (Shizuoka, Japan). All of the animal experiments were performed according to the guideline of Tokyo Univer sity of Pharmacy and Life Science. The animals were fasted for 1820 hours before starting the experiment. Water was freely given while fasting. Measurement of transepithelial transport of Rho123 and membrane resistance (Rm) in the in vitro diusion chamber methods: The methods used in this study followed those previously described by Tomita et al..6) The transepithelial transport of Rho123 in rat colon was examined using diusion chamber method. The serosal and mucosal reservoirs were lled with 5 mL Krebs Henseleit bicarbonate buer (KHBB) solution, which was continuously circulated and oxygenated by mixed gas (95z O2 W 5z CO2 ) to maintain tissue viability at pH 7.4 and 379C throughout the experiments. After 20 min, the KHBB solution was exchanged with 5 mL KHBB solution containing Rho123 in the serosal side (donor side) and with 5 mL KHBB solution without Rho123 in the mucosal side (acceptor side). In the same experiments, Rho123 was added to the mucosal side (donor side) but not to the serosal side (acceptor side). To examine the eect of TA, the mucosal reservoir was lled with KHBB solution containing TA (5 or 10 mM). The pH of KHBB solution containing TA was adjusted to 7.4 using sodium hydroxide. The samples were taken from the acceptor side at intervals of 10 min. Permea tion clearance (CLp) was obtained as follows. CLp(dQ W dt) W (A~C0 ) The dQ W dt is the transport rate ( mg W min) and cor responds to the slope of the regression line between the transport amounts and time. C0 is the initial concentra tion in the donor chamber ( mg W mL), and A is the area of the membrane (0.64 cm2 ). Simultaneously, the Rm of the rat colon was calculat ed from the membrane potential dierence measured under the load of a small external current (0.1 mA and 0.01 mA) according to Ohm's law.7) Absorption experiments using the in situ loop method: Intestinal absorption was evaluated by the method described in our previous report.8) A 5mL of KHBB solution containing Rho 123 (26 mM), daunorubicin (26 mM), or FD40 (0.1z) with or without TA or verapamil was administered into the loop. One milliliter was then sampled from the loop at 0 and 30 min after administration. The residue was collected at 60 min. The concentrations of Rho123 or daunorubicin as a model compound of Pgp substrate, and FD40 as a volume indicator were measured by a uorescent spec trophotometer (HITACHI FP6500, Tokyo, Japan). The absorption clearance was calculated by the method
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described in our previous report.8) Western blotting: An in situ ileum loop (18 cm length) was prepared in each rat and KHBB solution with or without TA (20 mM) (pH 7.4) administered into the loop (5.0 mL). At 60 min after administration, the crude membrane fractions were prepared from the ileum using magnesium chloride precipitation.9,10) Brie y, the ileal luminal contents were thoroughly washed out with a sucient amount of icecold saline and the ileum was then divided into several parts of the same length. The ileum's mucosal surface was scraped o with a slide glass. The mucosa collected was homogenized in a buer containing 0.05 mg W mL phenylmethylsulfonyluoride, 300 mM mannitol, 12 mM Tris, and 5 mM EGTA (pH 7.1) with a tissue homogenizer. The 10 mM magnesium chloride aqueous solution was added to the homogenate. The homogenate was centrifuged at 3,000 g for 10 min. The supernatant was then centrifuged at 42,000g for 30 min. The pellet was resuspended in 300 mM mannitol, 20 mM HEPES, 10 mM Tris, and 4mM magnesium chloride (pH 7.4). The protein expression levels of Pgp in crude membrane fraction were evaluated by Western blotting. Western blotting using C219 monoclonal antibody (Alexis) for Pgp was performed as reported previously.10) Excretion experiments: Rats were anesthetized with pentobarbital (50 mg W kg i.p. injection) and axed supine under the heater to maintain their body tempera ture. Cannulation (silicone tubing, Silascon} Kaneka Medix Co.) was made at a jugular vein for administra tion and sampling. Also, the ileal lumen (7 cm length) was ushed with saline prewarmed at 379 C, and the proximal end of the lumen was catheterized with an inow glass cannula, which was connected to the perfusion system. The distal end of the ileum was also catheterized with an outow glass cannula to collect intestinal euents serially. The single perfusion of KHBB solution into the ileal lumen was started at a rate of 1 mL W min. After a 10 min perfusion, saline contain ing Rho123 (260 mM) or daunorubicin (520 mM) was administered by i.v. injection as a bolus with a volume of 2.8 mL W kg, followed by a saline injection at the same volume via the cannula inserted at the jugular vein. Blood samples (300 mL) were drawn before and 1.5, 3, 5, 10, 20, 30 and 60 min after administration. The intestinal euent samples were then collected every 10 min. Excretions of Rho123 and daunorubicin from blood to ileal lumen were expressed as total excreted amounts for 60 min. Whole blood samples were centrifuged for 5 min at 12,000 rpm. After centrifuge, the supernatant of samples (100 mL) were dissolved into puried water in order to make the volume sucient for assay. Statistical analysis: All the results were expressed by
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Fig. 1. Eect of Dtartaric acid on rhodamine123 permeation clearance (CLp) in the serosal to mucosal direction (a) and in the mucosal to serosal direction (b) and membrane resistance (Rm) through rat colonic epithelia measured using in vitro diusion chamber method at pH 7.4. Symbol: ç Rm of each condition. Data represent mean}S.E. (n3¿23). N.S.: not signicant, *: pº0.05 compared with control.
Fig. 2. Absorption clearance (CL) of rhodamine123 (a) and daunorubicin (b) in the absence (open column) or presence of either Dtartaric acid (20 mM) (closed column) or verapamil (1 mM) (dotted column) from the loops of rat jejunum, ileum and colon. Data represent mean}S.E. (n4¿6). *: pº0.05, ***: pº0.001 compared with control.
mean value}standard error (Mean}SE). Statistical signicance between two groups was analyzed using Dunnett's test, and P value less than 0.05 was consi dered to be signicantly dierent. Results Eects of TA on Rho123 transport and membrane resistance (Rm) using in vitro diusion chamber method: The eects of TA (10 mM) on permeation clearance (CLp) of Rho123 in the serosal to mucosal direction (S to M) across rat colonic epithelia at pH 7.4 were measured. The results indicated that, when TA was added to the mucosal side, TA (10 mM) signicantly decreased the Rho123 excretion (Fig. 1a). However, TA (5 mM) did not show such eects. No marked change was observed in Rm in the presence of TA at pH 7.4 (Fig. 1a). The eects of TA (10 mM) on CLp of Rho123 in the mucosal to serosal direction (M to S) through rat colonic epithelia at pH 7.4 were also measured. The results indicated that TA (10 mM) signicantly in creased the Rho123 absorption. However, TA (5 mM) did not show such eects (Fig. 1b). No marked change
was observed in Rm in the presence of TA at pH 7.4 (Fig. 1b). Absorption site dependency of the eects of TA and verapamil on absorption clearance of Pgp substrates using in situ loop method: We examined the eects of TA (20 mM) on the absorption of rhodamine123 and daunorubicin from the loop of rat jejunum, ileum and colon. TA (20 mM), added to the lumen of the loop, signicantly increased the absorption of Rho123 both in ileum and colon but not in jejunum (Fig. 2a). On the other hand, TA (10 mM) did not change the absorption clearance of Rho123 (data not shown). In ileum but not in jejunum and colon, TA (20 mM) signicantly increased the absorption of daunorubicin (Fig. 2b). Eects of verapamil on the absorption of Rho123 in rat jejunum, ileum and colon were examined using the in situ loop method. Verapamil (1 mM), added to the lumen of the loop, signicantly increased the absorption of Rho123 and daunorubicin both in jejunum and ileum but not in colon (Fig. 2). Eect of TA in mucosal side on the luminal excretion of i.v.administered Pgp substrates: Total excreted amounts of Rho123 and daunorubicin intravenously
Dtartaric
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Fig. 3. Total excreted amount of rhodamine123 (a) and daunorubicin (b), intravenously administered via the jugular vein, to the ileum in the absence (open column) or presence of Dtartaric acid (20 mM) (closed column) or presence of verapamil (1 mM) (dotted column). Data represent mean}S.E. (n3¿6). *: pº0.05, ***: pº0.001 compared with control.
Fig. 4. Representative results of Western blotting for ileum Pgp after administration of 20 mM of Dtartaric acid into the ileal lumen.
administered via the jugular vein to the ileal lumen were measured. When TA (20 mM) or verapamil (1 mM) was applied to the ileal lumen, excretion of Rho123 was signicantly inhibited (Fig. 3a). Also, excretion of daunorubicin tended to be decreased by TA (20 mM) or verapamil (1 mM) (Fig. 3b). Eect of TA on expression of Pgp protein: In the ileum, TA (20 mM) did not change the expression of Pgp protein as measured with Western blotting (Fig. 4). Discussion In the rst series of experiments, we examined the eects of TA on Rho123 transport across rat colonic epithelium using the in vitro diusion chamber method. It was found that TA (10 mM), added to the mucosal side at pH 7.4, signicantly decreased the Rho123 trans port from the serosal to mucosal side (S to M) (Fig. 1a), but increased the transport from the mucosa to serosal side (M to S) (Fig. 1b). In colon, TA (10 mM) at low pH (pH 34) has been reported to change the integrity of tight junctions and to signicantly decrease Rm, an indicator of the integrity of tight junctions.2) To examine the similar TA eect at neutral pH, we measured Rm in the presence of TA (10 mM) at pH7.4. The results indicated that TA (10 mM) did not change Rm at pH 7.4 (Fig. 1a, b), suggesting that TA changes the integrity of tight junctions at low pH but not at neutral pH. It was suggested that the
eect of TA (10 mM) to change the transport of Rho123 is not attributable to the changes in the integrity of tight junction, at least at pH7.4. Considering the fact that Rho123 is a Pgp substrate, a possible explanation for the TAinduced decrease in Rho 123 excretion is that TA modulates Pgp function. Although TA also increased Rho123 absorption in the diusion chamber experiments, this eect is attributable to eects neither on tight junction nor Pgp function. Further experiments are necessary to clarify the mechanism of this eect. To further examine the eects of TA, we used the in situ loop method in the rat intestine. TA (20 mM) signicantly increased the absorption of Rho123 both in the ileum and colon (Fig. 2a), supporting the in vitro results. In this experiment, 20 mM TA was necessary to in crease the absorption, whereas in the in vitro experiment 10 mM TA inhibited Rho123 transport from the serosal to mucosal side. The dierence in the eective concen trations of TA may possibly be attributable to the dierent experimental conditions. To examine whether or not TA increases the absorp tion of Pgp substrate other than Rho123 (Fig. 2a), we obtained the eect of TA on the transport of daunorubi cin (Fig. 2b), because it has been reported that excretion of daunorubicin depends more strongly on Pgp than Rho123.11) In colon, TA increased the absorption of Rho123 but not of daunorubicin (Fig. 2a, b). The result that verapamil did not increase the absorption of daunorubi cin in the colon (Fig. 2b) indicates that the role of Pgp is small in the colon. Also, it is suggested that the eect of TA on Rho123 absorption is not only attributable to the inhibition of Pgp, but also to the activation of the absorption transporters for Rho123. It can also be considered that an excretion mechanism other than Pgp is inhibited by TA. It has been reported that Rho123 is
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transported by a transporter, a family of an organic cation transporter that transports Rho123 from the serosal side into the cell.12) Further experiments are necessary to evaluate these possibilities. We have found that Dtartaric acid eectively inhibit ed the Rho123 transport from the serosal to mucosal side in rat colon, although Ltartaric acid was ineective (unpublished observation). Such a structuredependent dierence in the action of TA supports the suggestion that TA is transported by a specic membrane trans porter. This transporter may be the Na{dependent dicarboxylate transporter which has been found in both the small intestine and kidney.1315) TA did not increase the Pgp substrate absorption in jejunum. Such a regional dierence may possibly be due to the dierent distribution of dicarboxylate transporter in the intes tine. The possibility that the inhibitory eect of TA is due to inhibition of Pgp protein expression was ruled out by the Western blotting method (Fig. 4), indicating that TA directly modulates the Pgp function. To further conrm this possibility, we administered Rho123 and daunorubicin intravenously and deter mined the amount of Rho123 and daunorubicin excret ed to the ileal lumen (Fig. 3). TA (20 mM) signicantly inhibited the excretion of Rho123. Because of the relatively large standard error, the dierence was not signicant, but TA also inhibited daunorubicin excretion. These results, together with the in vitro diusion chamber experiments, support the suggestion that the eect of TA is to inhibit excretion of Pgp. Also, the results obtained by in situ excretion and in vitro experiments in the ileum are consistent with those shown by absorption experiments in the in situ ileal loop method. In conclusion, for the rst time we demonstrated that TA increases the intestinal absorption of Pgp sub strates, Rho123 and daunorubicin, possibly by modulat ing the Pgp function without changing the expression of Pgp protein in the rat ileum and colon. Our results suggest the usefulness of TA as a pharmaceutical excipient to increase the absorption of Pgp substrates in the intestine.
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Acknowledgments: The authors thank Miss Akiko Muto, Miss Keiko Sato, Miss Kanae Takehi, Mr. Takuya Obata, Miss Asako Fukushima, and Mr. Tsuyoshi Kuga for technical assistance.
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References
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Spiller, G. A., Story, J. A., Furumoto, E. J., Chezem, J. C. and Spiller, M.: Eect of tartaric acid and dietary ber from sundried raisins on colonic function and on
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bile acid and volatile fatty acid excretion in healthy adults. Br. J. Nutr., 90: 803807 (2003). Hayashi, M., Sakai, T., Hasegawa, Y., Nishikawahara, T., Tomioka, H., Iida, A., Shimizu, N., Tomita, M. and Awazu, S.: Physiological mechanism for enhancement of paracellular drug transport. J. Control Release., 62: 141148 (1999). Yamashita, S., Sakane, T., Harada, M., Sugiura, N., Koda, H., Kiso, Y. and Sezaki, H.: Absorption and metabolism of antioxidative polyphenolic compounds in red wine. Ann. N.Y. Acad. Sci., 957: 325328 (2002). Vaidyanathan, J. B. and Walle, T.: Cellular uptake and eux of the tea avonoid (|) epicatechin3gallate in the human intestinal cell line Caco2. J. Pharmacol. Exp. Ther., 307: 745752 (2003). Kitagawa, S., Nabekura, T. and Kamiyama, S.: Inhibi tion of Pglycoprotein function by tea catechins in KB C2 cells. J. Pharm. Pharmacol., 56: 10011005 (2004). Tomita, M., Ohkubo, R. and Hayashi. M.: Lipopolysac charide transport system across colonic epithelial cells in normal and infective rat. Drug Metab. Pharmacokinet., 19: 3340 (2004). Yamashita, S., Saitoh, H., Nakanishi, K., Masada, M., Nadai, T. and Kimura,T.: Eects of diclofenac sodium and disodium ethylenediaminetetraacetate on electrical parameters of mucosal membrane and their relation to the permeability enhancing eects in the rat jejunum. J. Pharm. Pharmacol., 39: 621626 (1987). Iida, A., Tomita, M. and Hayashi, M.: Regional Dier ence in Pglycoprotein Function in Rat Intestine. Drug Metab. Pharmacokinet., 20: 100106 (2005). Stieger, B. and Murer, H.: Heterogeneity of brushbord er membrane vesicles from rat intestine prepared by a precipitation method using Mg W EGTA. Eur. J. Biochem., 135: 95101 (1983). Hsing, S., Gatmaitan, Z. and Arias, I. M.: The function of Gp170, the multidrugresistance gene product, in the brushborder of rat intestinal mucosa. Gastroenterol., 102: 879885 (1992). Wang, E. J., Casciano, C. N., Clement, R. P. and Johnson, W. W.: Active transport of uorescent Pglycoprotein substrates: evaluation as markers and interaction with inhibitors. Biochem. Biophys. Res. Commun., 289: 580585 (2001). Troutman, M. D. and Thakker, D.: Rhodamine 123 requires carriermediated inux for its activity as a P glycoprotein substrate in Caco2 cells. Pharm. Res., 20: 11921199 (2003). Pajor, A. M.: Citrate transport by the kidney and intes tine. Semin Nephrol., 19: 195200 (1999). Pajor, A. M.: Sodiumcoupled transporters for Krebs cycle intermediates. Annu Rev Physiol., 61: 663682 (1999). Kimmich, G. A., Randles, J. and Bennett, E.: Sodium dependent succinate transport by isolated chick intestinal cells. Am J Physiol., 260: 11511157 (1991).
Regular Article Improvement of Intestinal Absorption of Pglycoprotein Substrate by Dtartaric Acid Aiko IIDA*, Mikio TOMITA, Yoko IDOTA, Yusuke TAKIZAWA and Masahiro HAYASHI Department of Drug Absorption and Pharmacokinetics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: The purpose of the present experiment was to examine the eects of Dtartaric acid (TA) on intestinal drug absorption under both in situ and in vitro experimental conditions. In the in vitro diusion chamber experiments, TA (10 mM) added to the mucosal side of rat colon signicantly decreased rhoda mine123 (Rho 123) transport from the serosal to mucosal side. Since TA has been shown to change the integrity of the epithelial tight junctions in rat colon at low pH conditions, resulting in improved paracel lular drug transport, the eect of TA on membrane resistance was examined at pH 7.4 in the present study. It was found that membrane resistance, an indicator of paracellular integrity, did not change at pH 7.4. In the in situ loop method, TA (20 mM) increased the absorption of Rho123 in both ileum and colon but not in jejunum. TA (20 mM) also increased the absorption of daunorubicin in the ileum, but TA (20 mM) did not change the expression level of Pglycoprotein (Pgp). TA (20 mM) signicantly inhibited ex cretion of i.v.administered Rho123 and daunorubicin into the ileal lumen. In conclusion, for the rst time we demonstrated that TA increases the intestinal absorption of Pgp substrates Rho123 and daunorubi cin, possibly by modulating the Pgp function without changing the expression level of Pgp in the rat in testine. Key words: Dtartaric acid; Pglycoprotein; jejunum; ileum; colon; absorption improvement tion of catechin either by increasing the transepithelial transport resulting from the changed integrity of tight junctions or by inhibiting the eux of catechin from the cell to lumen resulting from the modulation of Pgp function. If TA has an eect to increase the intestinal absorp tion of Pgp substrates other than catechin, this compound may have a great impact on the improvement of drug therapy. To evaluate this possibility, we exam ined the eects of TA on the intestinal absorption in both the in vitro and in situ experimental conditions. Our results demonstrated for the rst time that TA increases intestinal absorption of Pgp substrates possibly by directly modulating the Pgp function.
Introduction Tartaric acid is a dicarboxylic acid considered to have no pharmacological eect except for shortening intesti nal transit time at larger doses.1) For this reason, this organic acid has been widely used as a drug excipient in the pharmaceutical industry. In our previous study, we demonstrated that Dtartar ic acid (TA) has an eect of changing the integrity of the epithelial tight junctions in rat colon at low pH.2) This signies that use of TA at low pH possibly enhances intestinal absorption of drugs such as hydrophilic macromolecules. In the rat, oral administration of TA together with a low molecular weight polyphenol, catechin, signicantly increased the serum concentra tion of catechin glucuronide, indicating that TA increased the intestinal absorption of catechin.3) It was also suggested that tea catechins are substrates of Pglycoprotein (Pgp).4,5) These results suggest the possibility that TA may increase the intestinal absorp
Materials and Methods Materials: Dtartaric acid, rhodamine 123 (Rho123), daunorubicin, verapamil and FITCdextran 40000 (FD40) were purchased from Sigma Aldrich Co. Ltd. All other reagents were of analytical grade or higher.
Received; January 13, 2006, Accepted; August 22, 2006 *To whom correspondence should be addressed : Aiko IIDA, Department of Drug Absorption and Pharmacokinetics, School of Pharmacy and Life Science, 14321 Horinouchi, Hachioji, Tokyo, 1920392 Japan. Tel. {81426763168, Fax. {81426763142, Email: aiaiädadahotmail. com 424
Dtartaric
Acid Improves Intestinal Drug Absorption
Animals and experimental design: Male Wistar rats (eight weeks old) were purchased from Japan SLC Ltd. (Shizuoka, Japan). All of the animal experiments were performed according to the guideline of Tokyo Univer sity of Pharmacy and Life Science. The animals were fasted for 1820 hours before starting the experiment. Water was freely given while fasting. Measurement of transepithelial transport of Rho123 and membrane resistance (Rm) in the in vitro diusion chamber methods: The methods used in this study followed those previously described by Tomita et al..6) The transepithelial transport of Rho123 in rat colon was examined using diusion chamber method. The serosal and mucosal reservoirs were lled with 5 mL Krebs Henseleit bicarbonate buer (KHBB) solution, which was continuously circulated and oxygenated by mixed gas (95z O2 W 5z CO2 ) to maintain tissue viability at pH 7.4 and 379C throughout the experiments. After 20 min, the KHBB solution was exchanged with 5 mL KHBB solution containing Rho123 in the serosal side (donor side) and with 5 mL KHBB solution without Rho123 in the mucosal side (acceptor side). In the same experiments, Rho123 was added to the mucosal side (donor side) but not to the serosal side (acceptor side). To examine the eect of TA, the mucosal reservoir was lled with KHBB solution containing TA (5 or 10 mM). The pH of KHBB solution containing TA was adjusted to 7.4 using sodium hydroxide. The samples were taken from the acceptor side at intervals of 10 min. Permea tion clearance (CLp) was obtained as follows. CLp(dQ W dt) W (A~C0 ) The dQ W dt is the transport rate ( mg W min) and cor responds to the slope of the regression line between the transport amounts and time. C0 is the initial concentra tion in the donor chamber ( mg W mL), and A is the area of the membrane (0.64 cm2 ). Simultaneously, the Rm of the rat colon was calculat ed from the membrane potential dierence measured under the load of a small external current (0.1 mA and 0.01 mA) according to Ohm's law.7) Absorption experiments using the in situ loop method: Intestinal absorption was evaluated by the method described in our previous report.8) A 5mL of KHBB solution containing Rho 123 (26 mM), daunorubicin (26 mM), or FD40 (0.1z) with or without TA or verapamil was administered into the loop. One milliliter was then sampled from the loop at 0 and 30 min after administration. The residue was collected at 60 min. The concentrations of Rho123 or daunorubicin as a model compound of Pgp substrate, and FD40 as a volume indicator were measured by a uorescent spec trophotometer (HITACHI FP6500, Tokyo, Japan). The absorption clearance was calculated by the method
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described in our previous report.8) Western blotting: An in situ ileum loop (18 cm length) was prepared in each rat and KHBB solution with or without TA (20 mM) (pH 7.4) administered into the loop (5.0 mL). At 60 min after administration, the crude membrane fractions were prepared from the ileum using magnesium chloride precipitation.9,10) Brie y, the ileal luminal contents were thoroughly washed out with a sucient amount of icecold saline and the ileum was then divided into several parts of the same length. The ileum's mucosal surface was scraped o with a slide glass. The mucosa collected was homogenized in a buer containing 0.05 mg W mL phenylmethylsulfonyluoride, 300 mM mannitol, 12 mM Tris, and 5 mM EGTA (pH 7.1) with a tissue homogenizer. The 10 mM magnesium chloride aqueous solution was added to the homogenate. The homogenate was centrifuged at 3,000 g for 10 min. The supernatant was then centrifuged at 42,000g for 30 min. The pellet was resuspended in 300 mM mannitol, 20 mM HEPES, 10 mM Tris, and 4mM magnesium chloride (pH 7.4). The protein expression levels of Pgp in crude membrane fraction were evaluated by Western blotting. Western blotting using C219 monoclonal antibody (Alexis) for Pgp was performed as reported previously.10) Excretion experiments: Rats were anesthetized with pentobarbital (50 mg W kg i.p. injection) and axed supine under the heater to maintain their body tempera ture. Cannulation (silicone tubing, Silascon} Kaneka Medix Co.) was made at a jugular vein for administra tion and sampling. Also, the ileal lumen (7 cm length) was ushed with saline prewarmed at 379 C, and the proximal end of the lumen was catheterized with an inow glass cannula, which was connected to the perfusion system. The distal end of the ileum was also catheterized with an outow glass cannula to collect intestinal euents serially. The single perfusion of KHBB solution into the ileal lumen was started at a rate of 1 mL W min. After a 10 min perfusion, saline contain ing Rho123 (260 mM) or daunorubicin (520 mM) was administered by i.v. injection as a bolus with a volume of 2.8 mL W kg, followed by a saline injection at the same volume via the cannula inserted at the jugular vein. Blood samples (300 mL) were drawn before and 1.5, 3, 5, 10, 20, 30 and 60 min after administration. The intestinal euent samples were then collected every 10 min. Excretions of Rho123 and daunorubicin from blood to ileal lumen were expressed as total excreted amounts for 60 min. Whole blood samples were centrifuged for 5 min at 12,000 rpm. After centrifuge, the supernatant of samples (100 mL) were dissolved into puried water in order to make the volume sucient for assay. Statistical analysis: All the results were expressed by
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Fig. 1. Eect of Dtartaric acid on rhodamine123 permeation clearance (CLp) in the serosal to mucosal direction (a) and in the mucosal to serosal direction (b) and membrane resistance (Rm) through rat colonic epithelia measured using in vitro diusion chamber method at pH 7.4. Symbol: ç Rm of each condition. Data represent mean}S.E. (n3¿23). N.S.: not signicant, *: pº0.05 compared with control.
Fig. 2. Absorption clearance (CL) of rhodamine123 (a) and daunorubicin (b) in the absence (open column) or presence of either Dtartaric acid (20 mM) (closed column) or verapamil (1 mM) (dotted column) from the loops of rat jejunum, ileum and colon. Data represent mean}S.E. (n4¿6). *: pº0.05, ***: pº0.001 compared with control.
mean value}standard error (Mean}SE). Statistical signicance between two groups was analyzed using Dunnett's test, and P value less than 0.05 was consi dered to be signicantly dierent. Results Eects of TA on Rho123 transport and membrane resistance (Rm) using in vitro diusion chamber method: The eects of TA (10 mM) on permeation clearance (CLp) of Rho123 in the serosal to mucosal direction (S to M) across rat colonic epithelia at pH 7.4 were measured. The results indicated that, when TA was added to the mucosal side, TA (10 mM) signicantly decreased the Rho123 excretion (Fig. 1a). However, TA (5 mM) did not show such eects. No marked change was observed in Rm in the presence of TA at pH 7.4 (Fig. 1a). The eects of TA (10 mM) on CLp of Rho123 in the mucosal to serosal direction (M to S) through rat colonic epithelia at pH 7.4 were also measured. The results indicated that TA (10 mM) signicantly in creased the Rho123 absorption. However, TA (5 mM) did not show such eects (Fig. 1b). No marked change
was observed in Rm in the presence of TA at pH 7.4 (Fig. 1b). Absorption site dependency of the eects of TA and verapamil on absorption clearance of Pgp substrates using in situ loop method: We examined the eects of TA (20 mM) on the absorption of rhodamine123 and daunorubicin from the loop of rat jejunum, ileum and colon. TA (20 mM), added to the lumen of the loop, signicantly increased the absorption of Rho123 both in ileum and colon but not in jejunum (Fig. 2a). On the other hand, TA (10 mM) did not change the absorption clearance of Rho123 (data not shown). In ileum but not in jejunum and colon, TA (20 mM) signicantly increased the absorption of daunorubicin (Fig. 2b). Eects of verapamil on the absorption of Rho123 in rat jejunum, ileum and colon were examined using the in situ loop method. Verapamil (1 mM), added to the lumen of the loop, signicantly increased the absorption of Rho123 and daunorubicin both in jejunum and ileum but not in colon (Fig. 2). Eect of TA in mucosal side on the luminal excretion of i.v.administered Pgp substrates: Total excreted amounts of Rho123 and daunorubicin intravenously
Dtartaric
Acid Improves Intestinal Drug Absorption
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Fig. 3. Total excreted amount of rhodamine123 (a) and daunorubicin (b), intravenously administered via the jugular vein, to the ileum in the absence (open column) or presence of Dtartaric acid (20 mM) (closed column) or presence of verapamil (1 mM) (dotted column). Data represent mean}S.E. (n3¿6). *: pº0.05, ***: pº0.001 compared with control.
Fig. 4. Representative results of Western blotting for ileum Pgp after administration of 20 mM of Dtartaric acid into the ileal lumen.
administered via the jugular vein to the ileal lumen were measured. When TA (20 mM) or verapamil (1 mM) was applied to the ileal lumen, excretion of Rho123 was signicantly inhibited (Fig. 3a). Also, excretion of daunorubicin tended to be decreased by TA (20 mM) or verapamil (1 mM) (Fig. 3b). Eect of TA on expression of Pgp protein: In the ileum, TA (20 mM) did not change the expression of Pgp protein as measured with Western blotting (Fig. 4). Discussion In the rst series of experiments, we examined the eects of TA on Rho123 transport across rat colonic epithelium using the in vitro diusion chamber method. It was found that TA (10 mM), added to the mucosal side at pH 7.4, signicantly decreased the Rho123 trans port from the serosal to mucosal side (S to M) (Fig. 1a), but increased the transport from the mucosa to serosal side (M to S) (Fig. 1b). In colon, TA (10 mM) at low pH (pH 34) has been reported to change the integrity of tight junctions and to signicantly decrease Rm, an indicator of the integrity of tight junctions.2) To examine the similar TA eect at neutral pH, we measured Rm in the presence of TA (10 mM) at pH7.4. The results indicated that TA (10 mM) did not change Rm at pH 7.4 (Fig. 1a, b), suggesting that TA changes the integrity of tight junctions at low pH but not at neutral pH. It was suggested that the
eect of TA (10 mM) to change the transport of Rho123 is not attributable to the changes in the integrity of tight junction, at least at pH7.4. Considering the fact that Rho123 is a Pgp substrate, a possible explanation for the TAinduced decrease in Rho 123 excretion is that TA modulates Pgp function. Although TA also increased Rho123 absorption in the diusion chamber experiments, this eect is attributable to eects neither on tight junction nor Pgp function. Further experiments are necessary to clarify the mechanism of this eect. To further examine the eects of TA, we used the in situ loop method in the rat intestine. TA (20 mM) signicantly increased the absorption of Rho123 both in the ileum and colon (Fig. 2a), supporting the in vitro results. In this experiment, 20 mM TA was necessary to in crease the absorption, whereas in the in vitro experiment 10 mM TA inhibited Rho123 transport from the serosal to mucosal side. The dierence in the eective concen trations of TA may possibly be attributable to the dierent experimental conditions. To examine whether or not TA increases the absorp tion of Pgp substrate other than Rho123 (Fig. 2a), we obtained the eect of TA on the transport of daunorubi cin (Fig. 2b), because it has been reported that excretion of daunorubicin depends more strongly on Pgp than Rho123.11) In colon, TA increased the absorption of Rho123 but not of daunorubicin (Fig. 2a, b). The result that verapamil did not increase the absorption of daunorubi cin in the colon (Fig. 2b) indicates that the role of Pgp is small in the colon. Also, it is suggested that the eect of TA on Rho123 absorption is not only attributable to the inhibition of Pgp, but also to the activation of the absorption transporters for Rho123. It can also be considered that an excretion mechanism other than Pgp is inhibited by TA. It has been reported that Rho123 is
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transported by a transporter, a family of an organic cation transporter that transports Rho123 from the serosal side into the cell.12) Further experiments are necessary to evaluate these possibilities. We have found that Dtartaric acid eectively inhibit ed the Rho123 transport from the serosal to mucosal side in rat colon, although Ltartaric acid was ineective (unpublished observation). Such a structuredependent dierence in the action of TA supports the suggestion that TA is transported by a specic membrane trans porter. This transporter may be the Na{dependent dicarboxylate transporter which has been found in both the small intestine and kidney.1315) TA did not increase the Pgp substrate absorption in jejunum. Such a regional dierence may possibly be due to the dierent distribution of dicarboxylate transporter in the intes tine. The possibility that the inhibitory eect of TA is due to inhibition of Pgp protein expression was ruled out by the Western blotting method (Fig. 4), indicating that TA directly modulates the Pgp function. To further conrm this possibility, we administered Rho123 and daunorubicin intravenously and deter mined the amount of Rho123 and daunorubicin excret ed to the ileal lumen (Fig. 3). TA (20 mM) signicantly inhibited the excretion of Rho123. Because of the relatively large standard error, the dierence was not signicant, but TA also inhibited daunorubicin excretion. These results, together with the in vitro diusion chamber experiments, support the suggestion that the eect of TA is to inhibit excretion of Pgp. Also, the results obtained by in situ excretion and in vitro experiments in the ileum are consistent with those shown by absorption experiments in the in situ ileal loop method. In conclusion, for the rst time we demonstrated that TA increases the intestinal absorption of Pgp sub strates, Rho123 and daunorubicin, possibly by modulat ing the Pgp function without changing the expression of Pgp protein in the rat ileum and colon. Our results suggest the usefulness of TA as a pharmaceutical excipient to increase the absorption of Pgp substrates in the intestine.
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Acknowledgments: The authors thank Miss Akiko Muto, Miss Keiko Sato, Miss Kanae Takehi, Mr. Takuya Obata, Miss Asako Fukushima, and Mr. Tsuyoshi Kuga for technical assistance.
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