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Nonlinear absorption of methylprednisolone by absorptive and secretory transporters

International Journal of Pharmaceutics 387 (2010) 1–6

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International Journal of

Pharmaceutics

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /i j p h a r

Nonlinear absorption of methylprednisolone by absorptive and secretory transporters

Mikio Tomita ?,Atsuko Watanabe,Ikue Fujinaga,Tomoko Yamakawa,Masahiro Hayashi

Department of Drug Absorption and Pharmacokinetics,School of Pharmacy,Tokyo University of Pharmacy and Life Sciences,1432-1Horinouchi,Hachioji,Tokyo 192-0392,Japan

a r t i c l e i n f o Article history:

Received 1June 2009

Received in revised form 5October 2009Accepted 22October 2009

Available online 31 October 2009Keywords:

Methylprednisolone Nonlinear absorption Rat intestine

Caco-2cell monolayers

a b s t r a c t

The intestinal absorption rate constant of methylprednisolone (MP)evaluated by the loop method increased signi?cantly with increasingly higher concentrations of the drug up to 500?M in a nonlinear fashion but did not increase further at higher concentrations.Mucosal-to-serosal directed permeation of MP across rat jejunal sheets also increased in a nonlinear fashion in a low concentration range (100–150?M),followed by a decrease as the concentration increased further,whereas serosal-to-mucosal directed permeation decreased in a concentration-dependent manner.Vectorial transport of MP across Caco-2cell monolayers was observed,with greater transport in the basolateral-to-apical direction at 37?C.These observations suggest that MP is taken up in the intestinal epithelial cells by a carrier-mediated transport mechanism.The absorptive and secretory clearance of MP increased and decreased with P-glycoprotein (P-gp)inhibitors,respectively.These results strongly suggest that MP is secreted into the intestinal lumen predominantly by P-gp.We conclude that intestinal transport of MP involves P-gp or some other transporters in both the absorptive and secretory directions,and complex nonlinear intestinal absorption characteristics can be ascribed to the existence of multiple transport mechanisms.

1.Introduction

The bioavailability of drugs after oral administration depends nonlinearly on concentration in some cases,which shows a dose-dependent increase and/or a decrease.Understanding the mechanism causing such nonlinear behavior would be helpful in the development of orally active drugs,as well as in their clin-ical applications.Nonlinear phenomena in intestinal absorption can be caused by several factors.A decrease of bioavailability with increase of dose can be accounted for by limited solubility of the drug in the intestinal lumen or by capacity-limited permeation across the intestinal epithelial membranes,such as specialized carrier-mediated transport mechanism.Recent studies on drug transport in the intestine have demonstrated the presence of many carrier-mediated transport systems for various drugs as well as nat-ural compounds.On the other hand,a dose-dependent increase of bioavailability can be mainly ascribed to saturable ?rst-pass metabolism in the liver and/or gastrointestinal tract.The ?nd-ing of drug-metabolizing enzymes such as the cytochrome P-450isozyme CYP3A,in the small intestine has contributed to a mech-anistic understanding of alterations of drug bioavailability arising from causes other than hepatic metabolism (Ducharme et al.,1995;

?Corresponding author.Tel.:+810426763168;fax:+810426763142.E-mail address:tomita@ps.toyaku.ac.jp (M.Tomita).Watkins et al.,1987).Another possible cause of dose-dependent changes in bioavailability is the contribution of an intestinal lumi-nal secretory system.P-glycoprotein (P-gp),which was originally found in multidrug-resistant tumor cells as an anticancer drug ef?ux pump,is also present in the luminal membrane of intestinal epithelial cells (Thiebaut et al.,1987)and acts as a secretory trans-porter into the intestinal lumen from the cells (Hsing et al.,1992),in addition to its role as a component of the blood–brain barrier,transferring drugs out of the brain (Tsuji et al.,1992).P-gp thus functions as an absorption barrier to various drugs,resulting in a lower absorption rate than would be expected from the lipophilic-ity of the drug molecules.If such secretory transporters,including P-gp,become saturated at high doses of drugs,an apparent increase of bioavailability would be expected at high drug concentrations.

Adrenocortical steroid hormones are widely used in the treat-ment of various in?ammatory and immunologic diseases due to their broad pharmacological activities (Fauci et al.,1976).They have been considered to be well-absorbed from the gastrointestinal tract by passive diffusion because of their relatively high lipophilic-ity (Georgitis et al.,1982).Therefore,detailed information has not been available on their mechanism of absorption in spite of their expanded clinical use since the 1950s.Recently,several reports have described that various endogenous steroid hormones inter-act with P-gp as substrates or modulators (Rao et al.,1994;Wolf and Horwitz,1992).Although P-gp was ?rst found to be one of the most important factors involved in the multidrug resistance

2M.Tomita et al./International Journal of Pharmaceutics387 (2010) 1–6

(MDR)of tumor cells Juliano and Ling,1976,it is regarded to serve as a kind of transporter that secretes endogenous steroid hormones into blood(Borst et al.,1993).Intestinal P-gp was also found to be expressed on the luminal membrane(Hsing et al.,1992)and capa-ble of restricting the absorption of various compounds(Tsuji and Tamai,1996).These?ndings imply that intestinal P-gp may sig-ni?cantly interfere with the absorption of some steroid hormones. Therefore,the intestinal absorption of various steroid hormones should be reevaluated in detail.

In the present study,we examined the absorptional behavior including the absorptive and secretory transport system of methyl-prednisolone(MP)in the rat jejunal membrane in situ,in vitro and in Caco-2cell monolayers,we reveal the participation of its mul-tiple transport mechanisms.Studies on MP metabolism have been limited to the liver(Lu et al.,2008;Villikka et al.,2001),and none have involved the small intestine.Thus,the intestinal metabolism of MP can be considered to have been ignored.The transport mech-anism can be exclusively examined using the above-mentioned experimental systems.

2.Materials and methods

2.1.Materials

Methylprednisolone,verapamil and?uorescein isothiocyanate dextran40,000(FD40)were purchased from Sigma–Aldrich Chem-ical Co.(St.Louis,MO,USA).All other chemicals were commercial products of reagent grade.

2.2.Measurement of intestinal absorption by the in situ loop method

Male Wistar/ST rats(8-weeks old)were purchased from Japan SLC Ltd.(Shizuoka,Japan).All of the experiments with the animal were performed according to the guidelines of Tokyo Univer-sity of Pharmacy and Life Sciences.The animals were fasted for 18h before starting the experiments.Water was freely given while fasting.Rats were anesthetized with somnopentyl(pento-barbital sodium,50mg/kg).All the experiments were performed under Somnopentyl-induced anesthesia,and body temperature was maintained at37?C with a heat lamp.Intestinal absorption of methylprednisolone(MP)was evaluated by the loop method (Tomita et al.,2009).The jejunum of male Wistar/ST rats weigh-ing200–250g(Japan SLC,Hamamatsu,Japan)was exposed by a midline abdominal incision,and two L-shaped glass cannulas(i.d. 2mm,o.d.4mm)were inserted through small slits at the proxi-mal and distal ends(7cm).The proximal end and distal end of the cannulas were inserted into a point5cm and12cm from the Treitz ligament,respectively.Each cannula was secured by ligation with a silk suture,and the intestine was returned to the abdominal cav-ity to maintain its integrity.A4cm portion of Tygon tubing(i.d. 3mm,o.d.5mm)was attached to the exposed end of each can-nula,and a10ml hypodermic syringe?tted with a connecting tube and containing perfusion solution warmed at37?C was attached to the proximal cannula.To clear the gut,saline was passed slowly through it to the distal cannula and discarded until the ef?uent was clear.The remaining perfusion solution was carefully expelled from the intestine by means of air pumped through the syringe,and 5ml of MP solution was immediately introduced into the intestine. The distal cannula was connected to a10ml syringe?tted with a three-way stopcock.At15,30,45and60min after administration of a drug solution,a0.5ml aliquot of luminal solution was removed through the attached syringe.The test solution was composed of 126mM NaCl,5.0mM KCl,1.4mM CaCl2,3.5mM NaHCO3,4.85mM NaH2PO4·2H2O,0.95mM Na2HPO4and2g/L d(+)-glucose at pH6.5,and the solution was gassed with95%O2/5%CO2before and dur-ing the transport experiment.MP concentrations in the solution used in this study were100–1000?M.MP was soluble in the above buffer solution containing1%dimethylsulfoxide to make1000?M or more.The absorption rate constant was evaluated from the slope of decline of the concentration in the luminal?uid with time.Here, the change in volume of water in the intestinal lumen was cor-rected with a measurement of the change in concentration of an unabsorbable marker,?uorescein isothiocyanate dextran40,000 (FD-40),administered simultaneously with MP.The concentration of FD-40used in this study was0.1%.

2.3.Transport experiments with diffusion chambers

Rat jejunum tissue sheets were prepared as described previ-ously(Eto et al.,2006).Tissue preparation,consisting of the mucosa and most of the muscularis mucosa,was made by removing the submucosa and tunica muscularis with?ne forceps.Tissue sheets were mounted vertically in a diffusion chamber that provided an exposed area of0.64cm2.The volume of the bathing solution on the mucosal and serosal sides was5ml,and the solutions tempera-ture was maintained at37?C in a water-jacketed reservoir.The test solution was composed of126mM NaCl,5.0mM KCl,1.4mM CaCl2, 3.5mM NaHCO3,4.85mM NaH2PO4·2H2O,0.95mM Na2HPO4and 2g/L d(+)-glucose at pH6.5,and the solution was gassed with95% O2/5%CO2before and during the transport experiment.The con-centration of MP in test solution on each side was determined as described above.Verapamil was used as a speci?c P-gp inhibitor (Bansal et al.,2009;Tomita et al.,2008;Panchagnula et al.,2005; Varma and Panchagnula,2005).To examine the inhibitory effect of verapamil,the mucosal reservoir was?lled with test solutions of containing various concentrations of verapamil.The samples were taken from the acceptor side at intervals of30min.Permeation clearance was obtained as follows:

Permeation clearance=

d Q/d t

A×C0

d Q/d t is th

e rate o

f transport(?g/min)determined from the slope of the linear regression line between the transport amount and time. C0is the initial concentration in the donor chamber(?g/ml),and A is the area of the membrane(0.64cm2).

Simultaneously,the membrane resistance(Rm)of the rat jejunum tissue sheets was calculated from the membrane poten-tial difference measured under the load of a small external current (0.1mA and0.01mA)according to Ohm’s law using a Short Circuit Current Amplifer(CEZ-9100,Nihon Koden,Japan).The change in Rm during the transport experiment(2h)was maintained within 20%of the initial value(about50 cm2).

2.4.Cultivation of Caco-2cell monolayers

Caco-2cells were grown in Dulbecco’s modi?ed Eagle’s medium containing10%fetal bovine serum and1%non-essential amino acids,2mM l-glutamine,100units/ml penicillin G and100?g/ml streptomycin,as described previously(Tomita et al.,2002). The cells were passaged upon reaching80%con?uency using trypsin–EDTA and plated at densities of1:5in170cm2T?asks. Caco-2cells(passage numbers40–66)were seeded at a density of 60,000cells/cm2on snapwell polycarbonate membranes of Snap-well(1cm2,0.4?m pore size,Costar,Cambridge,MA,USA).The medium was changed the day after seeding and every other day thereafter.For the transport experiments,Caco-2cells were grown on Snapwell microporous polycarbonate membranes(Costar,Bed-ford,MA)and cultured for about3weeks before use for transport experiments.

M.Tomita et al./International Journal of Pharmaceutics387 (2010) 1–63

2.5.Transport experiments with Caco-2cell monolayers

The transport experiments with Caco-2cells grown on Snapwell membranes were performed as described(Nagira et al.,2006).The con?uent cells were washed with Modi?ed Krebs Ringer Phosphate Bicarbonate Buffer(MKRPB)(0.645mM CaCl2·2H2O, 4.83mM KCl,1.214mM KH2PO4,1.205mM MgSO4·7H2O,113.3mM NaCl, 10.18mM Na2HPO4,5mM d-glucose and16.96mM NaHCO3,pH 7.4,osmolarity was315mOsm/kg),and1.5ml and2.6ml of MKRPB were added on the apical and basolateral sides,respectively,of a cell insert.To measure apical-to-basolateral or basolateral-to-apical?ux,a test compound was included in the apical and basolateral side,respectively.At the designated time,0.1ml of basolateral or0.2ml of apical side solution was withdrawn and replaced with an equal volume of MKRPB.The perme-ation of MP at increasingly higher concentrations of MP across Caco-2cell monolayers was measured in both the apical-to-basolateral and basolateral-to-apical directions,and permeation clearance was obtained as the slope of the time course of appearance of intact MP in either the basal or apical bathing solu-tion.

Simultaneously,Rm of the cell was obtained in the same man-ner as in the rat jejunal sheet.The change in the TEER during the transport experiment(2h)was maintained within10%of the initial value(about500 cm2).

2.6.Statistical analysis

All results are expressed as the mean±standard error (mean±S.E.).Statistical signi?cance between the two groups was analyzed using Dunnett’s test.Differences were considered to be signi?cant at a level of P<0.05.

3.Results

3.1.Concentration-dependence of intestinal absorption of methylprednisolone in rats

The relationship between the?rst-order absorption rate con-stant of methylprednisolone(MP)and its concentration was evaluated by the loop method.When100–1000?M MP was per-fused into the intrajejunal loop,the intestinal absorption rate constant was changed nonlinearly as shown in Fig.1.There was an increase in the constant from100to500?M,but not at 1000?M.The absorption rate constant was signi?cantly higher at 500?M than at100?M(P<0.05).This result suggests that at least two nonlinear events are involved in the intestinal absorption of MP.

The transport of MP in rat intestinal tissue was further exam-ined using the diffusion chamber method.Permeation at various concentrations of MP across a rat jejunal sheet was measured in both the mucosal-to-serosal and serosal-to-mucosal directions, and the absorptive and secretory permeation coef?cients were obtained as the slope of the time course of appearance of intact MP in the serosal and mucosal bathing solution,respectively. As is clearly shown in Fig.2,the serosal-to-mucosal permeation coef?cient signi?cantly decreased in a concentration-dependent manner,whereas the mucosal-to-serosal permeation coef?cient increased up to150?M,and at the higher concentrations,tended to decrease.Furthermore,in the presence of verapamil,serosal-to-mucosal transport of100?M MP tended to decrease,whereas the mucosal-to-serosal transport increased signi?cantly(Fig.2).Vec-torial transport of MP across the rat jejunal membrane was not observed above150?M of MP(Fig.2)and verapamil effect is dis-appeared(data not

shown).Fig. 1.Concentration-dependence of jejunal absorption of methylprednisolone in rats.The intestinal absorption rate constant(k a)of methylprednisolone (100–1000?M)was evaluated from the time course of the decrease in the lumi-nal methylprednisolone concentration measured by the in situ loop method.Each datum represents the mean±S.E.(n=3–5for each condition).*Signi?cantly dif-ferent from the value of absorption rate constant at100?M methylprednisolone (P<0.05).N.S.not signi?cantly different from the absorption rate constant at500?M methylprednisolone.

3.2.Transcellular transport of methylprednisolone across Caco-2 cell monolayers

The transport of MP in Caco-2cell monolayers was also examined using the diffusion chamber method.Permeation with increasingly higher concentrations of MP across Caco-2cell monolayers was measured in the apical-to-basolateral and basolateral-to-apical directions.Fig.3indicates that the basolateral-to-apical permeation clearance signi?cantly decreased in a concentration-dependent manner,whereas the apical-to-basolateral permeation clearance increased up to500?M,and at higher concentrations,tended to decrease.The basolateral-to-apical permeation of100?M MP was signi?cantly inhibited by the metabolic inhibitor NaN3(data not shown).In contrast,in

the

Fig.2.Concentration-dependence of methylprednisolone’s clearance across rat jejunal tissue.The permeation of methylprednisolone was evaluated using a dif-fusion chamber mounted with the rat jejunum.Open and closed circles represent the permeation clearance in the serosal-to-mucosal and mucosal-to-serosal direc-tion,respectively.Open and closed triangles represent the serosal-to-mucosal and mucosal-to-serosal transfer of100?M methylprednisolone in the presence of 500?M verapamil,respectively.Each datum represents the mean±S.E.for seven experiments.*Signi?cantly different from the in?ux value in the absence of vera-pamil(P<0.05).

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387 (2010) 1–6

Fig.3.Concentration-dependence of methylprednisolone’s permeation clearance across Caco-2cell monolayers.The permeation of methylprednisolone was evalu-ated using a diffusion chamber mounted with Snapwell.Open and closed circles represent the clearance in the basolateral-to-apical and apical-to-basolateral direc-tion,respectively.Open and closed triangles represent the basolateral-to-apical and apical-to-basolateral transfer of200?M methylprednisolone in the presence of500?M verapamil,respectively.Each datum represents the mean±S.E.for seven experiments.*Signi?cantly different from data of in?ux or ef?ux value without verapamil(P<0.05).

presence of verapamil,basolateral-to-apical transport at200?M MP decreased signi?cantly,whereas the apical-to-serosal transport increased signi?cantly.

To determine whether the transport of MP across Caco-2cell monolayers was dependent on temperature dependent,transport ?uxes were measured at4?C and37?C conditions(Fig.4).The per-meation of MP was linear over120min with an initial lag time of a few minutes.The?ux from the basolateral-to-apical side was about six times larger than the reverse?ux(Fig.4).The?ux in at4?C, which is a measure of passive permeability,was signi?cantly lower than that at37?C.Therefore,the transport of MP can be ascribed to active transcellular permeation.

To investigate whether MP interacts with P-gp present in Caco-2 cell monolayers and rat jejunal epithelial cells,the effect of vera-pamil on the transport of MP was examined.Since verapamil as a typical substrate for P-gp was used at a concentration of two times higher than that of MP,the transport of MP was signi?cantly increased in the apical-to-basolateral direction,whereas it was sig-ni?cantly decreased in the basolateral-to-apical direction(Table1).

4.Discussion

The concept that carrier-mediated intestinal absorption and luminal secretion mechanisms as well as intestinal tissue metabolic activity regulate the bioavailability of various drugs has been

estab-Fig.4.Effect of temperature on the polarized permeation of methylprednisolone across Caco-2cell monolayers.The concentration of methylprednisolone used in this study was50?M.Circle and triangle represent the data obtained at37?C and 4?C,respectively.Open and closed symbols represent the data for the basolateral-to-apical and apical-to-basolateral directions,respectively.Each datum represents the mean±S.E.for seven experiments.

lished previously(Benet et al.,1996;Tamai et al.,1997).Such a saturable physiological mechanism may sometimes produce non-linear pharmacokinetic phenomena.The results obtained in the present study represent the?rst evidence to our knowledge that a complex pattern of nonlinear bioavailability of MP is indeed gen-erated by the interaction of intestinal absorptive and secretory transport systems,when MP exists at a concentration beyond linear absorbability in the intestinal luminal?uid.

Intestinal absorption of MP assessed by the in situ loop method in rats exhibited distinctive nonlinearity,which showed a signif-icant,apparently linear,increase in the absorption rate constant up to a drug concentration of500?M when compared with that at100?M,but did not increase further at higher concentration of1000?M(Fig.1).Because the disappearance of intact MP from the intestinal luminal?uid was measured in this experiment,the above observations can be accounted for by saturable secretory and absorptive transport mechanisms,by a saturation of metabolism or by a limitation of solubility.Absorptive-directed(mucosal-to-serosal)?ux of MP across jejunal tissue preparations mounted on a diffusion chamber increased at up to200?M of the drug(Fig.2). This result is consistent with that obtained with the in situ loop method,and both can apparently be explained by the participation of a saturable secretory mechanism.This hypothesis is further sup-ported by the measurement of serosal-to-mucosal?ux using the diffusion chamber,which showed a marked decrease in the per-

Table1

Effects of verapamil on MP transport in the apical-to-basolateral direction(in?ux)and basolateral-to-apical direction(ef?ux)across Caco-2cell monolayers.

Concentration of MP(?M)

50100150200

In?ux(?l/min/cm2)

?verapamil0.556±0.060.694±0.040.678±0.050.932±0.08 +verapamil 1.27±0.07* 1.29±0.05* 1.28±0.12* 1.45±0.03* Ef?ux(?l/min/cm2)

?verapamil 2.83±0.19 3.31±0.05 2.44±0.04 2.32±0.13 +verapamil 1.98±0.01* 1.92±0.07* 1.81±0.04* 1.62±0.03*

The concentrations of verapamil used in the present study were two times higher than the concentration of substrate in all the cases.Data represent the mean±S.E.(n=3–4 for each condition).

*P<0.05compared with no verapamil.

M.Tomita et al./International Journal of Pharmaceutics387 (2010) 1–65

meation coef?cient with an increase in the concentration of MP (Fig.2).The break point observed in Figs.1and2was between 500and150?M in both the experiments,although with the dif-fusion chamber method there is an increase of?ux at a lower concentration(100–150?M)than observed with the in situ loop method(100–500?M).This small difference may be ascribed to the difference in thickness of the unstirred water layer in these exper-imental systems,because the loop method is expected to give a thicker unstirred water layer than that in the isolated jejunal sheet chamber method.

The decrease in the absorption rate constant at higher con-centrations observed with the loop method can be explained by the participation of a saturable absorptive transport mechanism. The decrease in mucosal-to-serosal?ux in the diffusion chamber at concentrations higher than250?M also supports the involve-ment of a saturable absorption mechanism.To con?rm the results above,we used an in vitro method with cultured cells.Caco-2cells, derived from human adenocarcinoma,have been shown to form an intestinal epithelial-like monolayer(Hilgers et al.,1990)and to have many of the small-intestinal membrane transport activi-ties for amino acids(Hu and Borchardt,1992),peptides(Dantzig and Bergin,1990),hexoses(Riley et al.,1991),and monocarboxylic acids(Tsuji et al.,1994)and ef?ux transporter activity such as P-gp (Hilgers et al.,1990;Hsing et al.,1992).As it should be relatively easy to identify specialized transporters in their monolayers,Caco-2cells were used in the present study.

Apical-to-basolateral?ux of MP across the monolayers increased with concentration up to500?M,whereas the basolateral-to-apical clearance signi?cantly decreased in a concentration-dependent manner beyond100?M.These results are also consistent with those obtained using the in situ loop method,and the?ndings both in situ and in vitro can apparently be explained by the participation of a saturable secretory mechanism. The vectorial transport of MP across Caco-2cell monolayers was observed with a higher permeation constant in the basolateral-to-apical direction than in the reverse direction(Fig.4).Accordingly, mechanisms of concentration dependency and vectorial transport may operate in Caco-2cell monolayers similar to those observed in rat intestinal tissues.Here,apical-to-basolateral?ux was signi?cantly higher than the?ux at4?C(Fig.4),supporting the active transport of MP across the monolayers.We have shown the polarized transport of hydrophilic compounds such as?uorescein isothiocyanate dextran4000,?uorescein disulfonic acid and lucifer yellow across rat mucosal tissues by a carrier-mediated transport mechanism(Tomita et al.,2000).

P-gp,an ATP-dependent drug ef?ux pump with broad substrate speci?city,has been demonstrated to have a signi?cant role as an intestinal secretory transporter(Hsing et al.,1992;Hilgers et al.,1990).Accordingly,the nonlinear increase in the absorptive transport of MP observed in the present study can be interpreted in terms of a contribution of P-gp.Several results in the present study support this idea:(1)The serosal-to-mucosal?ux of MP in rat jejunal sheets mounted on a diffusion chamber was suppressed, but the mucosal-to-serosal?ux was increased by verapamil with a high af?nity for P-gp(Fig.2).(2)Apical-to-basolateral transport of MP across Caco-2cell monolayers was signi?cantly increased in the presence of verapamil whereas basolateral-to-apical trans-port was reduced(Fig.3,Table1).(3)Markedly less permeation was observed at4?C when compared with37?C(Fig.4),suggesting that P-gp makes a major contribution to the nonlinear increase in the bioavailability of MP delivered orally administration.

5.Conclusion

In conclusion,MP showed nonlinear intestinal absorption with an increase at the lower concentration ranges and subsequent decrease at the higher concentration range in absorption.Such non-linearity can be explained partly by the operation of absorptive and secretory transporters in the intestine.It is considered that ABC transporter such as P-gp makes a substantial contribution to the secretory transport,though the absorptive transporter has not been identi?ed yet.Clari?cation of the mechanistic and kinetic fea-tures of nonlinear intestinal absorption is important for prediction of drug–drug interactions in clinical use.

Acknowledgments

This research was supported partly by a Grant-in-aid for Sci-enti?c Research(No.18590156)from the Ministry of Education, Science and Culture,Japan and by grants from the Japan health Sci-ences Foundation.The authors thank Miss Kaori Negishi,Mr.Daichi Arai,Miss Miyuki Saeki and Miss Mana Sato for their technical assistance.

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