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0022-3565/00/2942-0510$03.00/0THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 294:510–515, 2000 /2576/840577 Hydroxyprolylserine Derivatives JBP923 and JBP485 Exhibitthe Antihepatitis Activities after Gastrointestinal Absorption inRats1 KE-XIN LIU, YUKIO KATO, TAI-ICHI KAKU, TOMOFUMI SANTA, KAZUHIRO IMAI, AKIRA YAGI, TAKASHI ISHIZU, andYUICHI SUGIYAMA Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo (K.L., Y.K., T.S., K.I., Y.S.); Japan BioproductsIndustry Co. Ltd., Tomigaya, Shibuya-ku, Tokyo (T.K.); and Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Gakuencho, 1, Fukuyama, Hiroshima (A.Y., T.I.), Japan Accepted for publication May 1, 2000 This paper is available online at http://www.jpet.org It has been a desire to develop orally effective therapeutic effect on hepatocytes because glutamic-oxaloacetic transam- agents that restore the liver function in chronic injury. Here we inase and lactate dehydrogenase activities in the medium of demonstrated that trans-4-L-hydroxyprolyl-L-serine (JBP923) hepatotoxin-exposed primary cultured hepatocytes were re- and cyclo-trans-4-L-hydroxyprolyl-L-serine (JBP485), which duced by these compounds. When comparing the plasma con- was previously isolated from hydrolysate of human placenta, centration-time profile of JBP923 after its i.v., oral, and portal exhibit potent antihepatitis activity after their oral administra- vein injection, it is suggested that JBP923 is almost completely tion. The increase in bilirubin concentration and activities of absorbed from gastrointestinal lumen, and hepatic first-pass liver cytosolic enzymes in serum caused by ␣-naphthylisothio- removal is minor. JBP923 inhibited the proton-dependent cyanate intoxication in rats were significantly countered both transport of glycylsarcosine in brush-border membrane vesi- after i.v. and oral administration of these dipeptides, whereas cles, suggesting that peptide transport system(s) may recog- glycyrrhizin, which has been used in the treatment of chronic nize JBP923. Thus, these dipeptides are potent antihepatitis hepatitis, is active only after its i.v. administration. Antihepatitis reagents that are still active after oral administration and may activity of dipeptides results, at least partially, from their direct be useful for clinical applications.
Several types of drugs have been used to treat chronic was first isolated from Laennec, a trade name for the hydro- hepatitis and cirrhosis. These include prednisone and aza- lysate of human placenta, as mitogens for a baby hamster thioprine for the treatment of autoimmune chronic hepatitis kidney cell line, and subsequently it has been enantioselec- (Bellary et al., 1995; Czaja, 1999) and interferons for viral tively synthesized by chemical means (Yagi et al., 1998).
hepatitis (Dumoulin et al., 1999; Par et al., 1999; Shiffman et Laennec is produced by Japan Bioproducts Industry Co. Ltd.
al., 1999). To improve the liver function in chronic hepatitis, (Tokyo, Japan) by purification of human placental extracts glycyrrhizin, one of the main constituents of Glycyrrhiza involving dialysis, heat treatment, and hydrolysis. Laennec glabra L, which has antiallergic, anti-inflammatory, and an- has been clinically used to treat chronic hepatic injuries for tihepatitis activities, is frequently used (Nose et al., 1994, over forty years in Japan. Recently we found that Laennec 1996; Wang et al., 1994; Takeda et al., 1996; Arase et al., stimulates liver regeneration and decreases cytosolic enzyme 1997). However, glycyrrhizin is usually administered i.v. be- [glutamic-pyruvic transaminase (GPT), alkaline phospha- cause it is inactive after oral administration. Therefore, for tase (ALP), leucine aminopeptidase (LAP), ␥-glutamyltrans- the treatment of chronic liver injuries, orally effective ther- ferase (␥-GTP)] activities in serum in ␣-naphthylisothiocya- apeutic agents have to be developed.
nate (ANIT)-intoxicated rats (Liu et al., 1995). We have cyclo-trans-4-L-Hydroxyprolyl-L-serine (JBP485) (Fig. 1) found that Laennec also contains trans-4-L-hydroxyprolyl-L-serine (JBP923). Those findings prompted us to synthesize Received for publication February 8, 2000.
1 these dipeptides and investigate their protecting effect on This study was supported in part by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Science and Culture of Japan.
hepatocytes from hepatotoxin treatment.
ABBREVIATIONS: JBP485, cyclo-trans-4-L-hydroxyprolyl-L-serine; JBP923, trans-4-L-hydroxyprolyl-L-serine; ANIT, ␣-naphthylisothiocyanate;
TFA, trifluoroacetic acid; BIL, bilirubin concentration; GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase; LAP, leucine
aminopeptidase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; DMSO, dimethyl sulfoxide; BBMVs, brush-border membrane vesicles;
Gly-Sar, glycylsarcosine; ␥-GTP, ␥-glutamyltransferase.
Dipeptides Orally Exhibiting Antihepatitis Activities
as described previously (Kato et al., 1994). Briefly, isolated hepato-cytes suspended in Williams' medium E supplemented with 5% calfserum, 10⫺9 M insulin, and 10⫺9 M dexamethasone were plated onto24-well plastic dishes coated with type I collagen. The nonattachedcells were removed by washing with fresh culture medium at 3 hafter plating. CCl , first dissolved in dimethyl sulfoxide (DMSO) at 1.0 M, was diluted with fresh medium containing JBP923, JBP485,glycyrrhizin, or 18␤-glycyrrhetinic acid (Sigma) to give a final con-centration of 5 mM. Control experiments were performed in thepresence of only DMSO. At 24 h after plating, the medium wasreplaced with the buffer containing both CCl and an appropriate drug. The monolayers were further cultured for 24 h, and culturemedium was collected and centrifuged at 24,000g for 20 min. GOTand lactate dehydrogenase (LDH) were assayed as described above.
One unit was defined as the amount of activity catalyzing formationof 1 ␮mol of product/1 min.
Pharmacokinetic Analysis in Normal Rats. Under ether an-
Fig. 1. Chemical structures of JBP923 and JBP485.
esthesia, JBP923 (3.13 or 25 mg/kg) dissolved in saline was admin- Although both JBP923 and JBP485 have simple chemical istered through the penis vein, through the portal vein, or into the structures with dipeptide backbone (Fig. 1), here we report stomach with a gastric sonde. This ether anesthesia was sufficient to their potent antihepatotoxic activity in rats. It is notable that allow portal vein injection, which was performed over a period of 10 these compounds are active in vivo after oral administration.
min using an infusion pump. Plasma was collected from the externaljugular vein at the indicated times, and the JBP923 concentration in To support our hypothesis that these dipeptides are orally plasma was determined by HPLC as described below. The plasma absorbed and directly interact with hepatocytes to restore concentration (Cp)-time profiles of JBP923 after i.v. and oral admin- their functions, we investigated the gastrointestinal absorp- istration were fitted to the following equations, respectively: tion in vivo and antihepatitis activity in primary culturedhepatocytes. Our findings demonstrate that these dipeptides Cp ⫽ A exp(⫺␣t) ⫹ B exp(⫺␤t) may be applicable as oral drugs for the treatment of liver 兲t兲兲/共␣ ⫺ k 兲 aFexp(⫺kat)共A共1 ⫺ exp(⫺共␣ ⫺ ka ⫹ B共1 ⫺ exp(⫺共␤ ⫺ k 兲 a t兲)/共␤ ⫺ ka ) where k and F were absorption rate constant and bioavailability, Animals and Materials. Male Wistar rats weighing 250 and
respectively. The plasma clearance (CL ) was calculated by: 150 g (Nisseizai, Tokyo, Japan) for in vivo and in vitro studies, respectively, were used throughout the experiments. All animals were treated humanely. The studies reported in this article have is area under the plasma concentration-time profile been carried out in accordance with the Guide for the Care and Use after i.v. injection.
of Laboratory Animals as adopted and promulgated by the NationalInstitutes of Health. JBP923 and JBP485 were synthesized by Wa- tanabe Chemical Industries Company (Hiroshima, Japan). Acetoni- The hepatic availability (F ) was calculated as: trile, tetrahydrofuran, trifluoroacetic acid (TFA), dioxane, and dis- tilled water, all of HPLC grade, were purchased from Wako Pure Chemical Industries (Osaka, Japan). 4-Fluoro-7-nitro-2,1,3-benzox-adiazole was from Tokyo Kasei Co. (Tokyo, Japan).
was AUC after portal vein injection. The AUC during Antihepatotoxic Activities In Vivo. ANIT (Sigma, St. Louis,
the 10-min portal vein infusion was calculated by trapezoidal rule.
MO) dissolved in olive oil was injected i.p. at a dose of 50 mg/kg body The AUC after the end of infusion was obtained by eq. 4, where the wt. For i.v. administration, JBP923 and JBP485 (25 mg/ml), dis- plasma concentration-time profile was fitted also to eq. 1. The input solved in saline, or glycyrrhizin injection (Minophagen Pharmaceu- data for all the fitting were weighted as the reciprocal of the square tical Co., Tokyo, Japan) were administered through the penis vein.
of the observed values, and the algorithm used for the fitting was the For oral administration, JBP923 and JBP485 dissolved in saline or damping Gauss-Newton method.
glycyrrhizin tablets (Minophagen Pharmaceutical Co.) dissolved in Determination of JBP923 in Plasma by HPLC. To 12.5 ␮l of
5% glucose solution were administered via esophagus with a gastric plasma, 500 ␮l of methanol was added, and the mixture was centri- sonde. Administrations of these drugs were performed at 30 min fuged at 600g for 5 min for deproteinization. The supernatant was before and 8, 22, 32, and 46 h after ANIT treatment. All the admin- collected and dried under reduced pressure using a centrifugal evap- istrations were performed under ether anesthesia. Serum was col- orator. For the derivatization of an imino group in JBP923, 20 ␮l of lected 48 h after ANIT treatment. For serum collection, rats were 50 mM borate buffer (pH ⫽ 8.0) and 30 ␮l of 20 mM 4-fluoro-7-nitro- anesthetized with ether, and approximately 10 ml of blood was 2,1,3-benzoxadiazole dissolved in acetonitrile were added to the sampled from the aorta abdominalis. Blood was then left on ice for 20 dried sample (Fukushima et al., 1995). The reaction mixture was min and centrifuged at 1000g for 5 min to obtain the supernatant as heated at 60°C for 5 min, and 450 ␮l of 1% TFA in water was added serum. The total bilirubin concentration (BIL) and activity of liver- to the mixture to stop the reaction. Twenty microliters of the result- specific cytosolic enzymes, such as GPT, LAP, ALP, glutamic-oxaloa- ant solution was subjected to HPLC analysis. The HPLC system cetic transaminase (GOT) and ␥-GTP, in the rat serum were deter- consisted of a model L-6320 Intelligent pump (Hitachi, Tokyo, Japan) mined using the appropriate assay kits (Wako Pure Chemical and a model F-1050 fluorescence spectrophotometer (Hitachi). An ODS-COSMOSIL (4.6 ⫻ 150 mm, i.d.) column (Nacalai Tesque, To- Determination of Biochemical Marker Leakage from Pri-
kyo, Japan) was used. The excitation and emission wavelengths were mary Cultured Rat Hepatocytes. Parenchymal hepatocytes were
fixed at 470 and 540 nm, respectively. A gradient HPLC system was plated at a density of 1.25 ⫻ 105 cells/1.88 cm2 and cultured for 24 h adopted. Eluent A, water/acetonitrile (95.5:4.5, v/v) containing 4.5% Liu et al.
dioxane, 1% tetrahydrofuran, and 0.05% TFA, and eluent B (aceto- represent the transport velocity in the presence nitrile) were used. The elution program was as follows: eluent A, 100 and absence of inhibitor, respectively; I is the inhibitor concentra- to 0% from 0 to 18 min; eluent B, 100 to 0% from 18.1 to 35 min; tion. Equation 6 is based on the assumption of competitive inhibition eluent A, 100% from 35.1 to 45 min. The flow rate was 1 ml/min.
in a case when the Michaelis constant (K ) is much higher than the substrate concentration. In a preliminary study, we found that the (BBMVs). BBMVs were prepared by the method of Kessler (Kessler
K for Gly-Sar uptake was 15.5 mM in rabbit BBMVs, and therefore et al., 1978). Briefly, the approximately 50-cm proximal portion of the substrate concentration chosen for this experiment was 0.668 the jejunum was isolated from male rabbits (2.0 –2.5 kg; Nisseizai).
The mucosa was scraped off and homogenized in a volume of ice-cold Statistical Analysis. Statistical analysis was performed by Stu-
buffer A (2 mM Tris/HEPES buffer containing 50 mM D-mannitol, dent's t test to identify significant differences between various treat- pH ⫽ 7.1). The homogenization was carried out with a Waring ment groups.
blender for 2 min at a speed of 18,000 rpm. Solid CaCl was added to the homogenate to give a final concentration of 10 mM, and the mixture was stirred in an ice bath for 15 min. It was then centrifugedat 500g for 15 min, and the supernatant was centrifuged at 1500g for Antihepatotoxic Effect of JBP923 and JBP485 in
30 min. The pellet was homogenized with buffer A in a glass/Teflon ANIT-Intoxicated Rats. To examine whether JBP923 and
Potter homogenizer at a speed of 1000 rpm. The mixture then was JBP485 promote the repair of injured liver function in ANIT- centrifuged at 750g for 30 min. The pellet was homogenized in a intoxicated rats, we determined the change in BIL and ac- glass/Teflon Potter homogenizer again with the same buffer and the tivities of liver cytosolic enzymes in serum of ANIT-intoxi- speed mentioned above. The supernatant was centrifuged at 48,000gfor 30 min, and then the pellet was suspended with a 23-gauge cated rats after administration of JBP923 and JBP485 (Table needle. The centrifugation was performed again with the same speed 1). The increase in BIL and liver cytosolic enzyme activities and time, and then the pellet was suspended with a 27-gauge needle.
caused by ANIT intoxification were countered by i.v. and oral Finally, protein concentration in the suspension was determined administration of JBP923 and JBP485 (Table 1). The reduc- using a Bio-Rad protein assay kit with BSA as a standard; the tion in all the marker values were significant at i.v. and oral concentration was 25 mg/ml with transport buffer (10 mM Tris/ doses of more than 1.36 and 25 mg/kg, respectively, both for HEPES buffer with 270 mM D-mannitol, pH ⫽ 7.5).
JBP923 and JBP485 (Table 1). When the i.v. and oral doses Uptake of [14C]glycylsarcosine (Gly-Sar) by BBMVs was measured were increased up to 6.25 and 25 mg/kg, respectively, the by the rapid filtration method described by Hopfer (Hopfer et al., bilirubin, LAP, and ALP levels were almost comparable with 1973). The uptake was started by adding 4 ␮l of BBMVs (100 ␮g) to those values in normal rats (Table 1).
16 ␮l of transport buffer (20 mM Tris-citrate buffer, pH ⫽ 5.5)containing JBP923, JBP485, and [14C]Gly-Sar at 37°C. The final Comparison of the Antihepatotoxic Effect of JBP923
substrate concentration was 68 and 600 ␮M [14C]Gly-Sar (2.96 GBq/ and JBP485 with Glycyrrhizin. Because glycyrrhizin is
mmol) and unlabeled Gly-Sar, respectively. The reaction was one of the most frequently administered drugs in chronic stopped at the desired time by adding 1 ml of ice-cold stop buffer liver-injured patients, the antihepatitis activity was com- (pH ⫽ 7.5), which contained 20 mM Tris, 20 mM HEPES, and 300 pared among JBP923, JBP485, and glycyrrhizin (Fig. 2).
mM mannitol. Then, 0.9 ml of the diluted sample was applied im- Intravenous or oral administration of JBP923 caused the mediately on a Millipore filter (HAWP, 0.45-␮m pore size) and reduction of GPT at almost the same doses (Fig. 2A). The washed rapidly twice with 5 ml of ice-cold stop buffer. The uptake of reduction in GPT activity was also observed after i.v. or oral [14C]Gly-Sar by BBMVs trapped on the Millipore filter was mea- administration of JBP485, although oral administration ex- sured in a liquid scintillation spectrometer. The inhibition constant hibited weaker antihepatitis activity (Fig. 2B). Minimal re- (K ) was obtained by fitting the data to the following equation: duction was found after oral administration of glycyrrhizin, 1/共1 ⫹ I/Ki whereas its i.v. administration decreased GPT level (Fig. 2C).
TABLE 1Change in BIL and activity of liver cytosolic enzymes in serum in ANIT-intoxicated rats treated with JBP923 and JBP485Each value represents the mean ⫾ S.E. of three animals.
(ANIT-treated rat) (ANIT-treated rat) (ANIT-treated rat) (ANIT-treated rat) * P ⬍ .05, ** P ⬍ .01, significantly different from ANIT-treated rats.
Dipeptides Orally Exhibiting Antihepatitis Activities
Fig. 2. Comparison of antihepatitis effect on ANIT-intoxicated rats among JBP923 (A), JBP485 (B), and glycyrrhizin (C). JBP923, JBP485, or
glycyrrhizin was administered through the penis vein (F) or orally (E) 30 min before and 8, 22, 32, and 46 h after ANIT treatment. Serum was collectedat 48 h, and the activity of GPT was determined. The values are expressed as means ⫾ S.E. of three rats. *P ⬍ .05; **P ⬍ .01, significantly differentfrom saline alone.
Thus, these dipeptides exhibit antihepatotoxic effect after pass elimination of JBP923, its plasma concentration-time oral administration, but glycyrrhizin does not.
profiles in rats were determined after i.v., oral, and portal Antihepatotoxic Effect on Primary Cultured Hepa-
vein administrations (Fig. 4). The plasma concentration of tocytes. The decrease in leakage of liver cytosolic enzyme by
JBP923 was gradually decreased after i.v. administration these compounds was also examined in vitro in primary with a terminal phase half-life of 21 to 24 min (Fig. 4). The cultured hepatocytes intoxicated with CCl (Fig. 3). The GOT gastrointestinal absorption of JBP923 was rapid with a k of activity in the medium was decreased by addition of JBP923 0.01 to 0.04 min⫺1 and maximum plasma concentration ob- and JBP485 in a concentration-dependent manner, the GOT served within 30 min (Fig. 4 and Table 2). The AUC after oral activity at the highest concentration being almost compara- administration was almost comparable with that after i.v.
ble with that of hepatocytes without CCl intoxication (Fig.
administration both at 3.13 and 25 mg/kg (Table 2), suggest- 3A). Both glycyrrhizin and 18␤-glycyrrhetinic acid also de- ing almost complete oral absorption. The AUC after portal creased the GOT activity, although such effect at concentra- vein administration was also comparable with that after i.v.
tions greater than 50 ␮M was smaller than JBP923 and administration at 3.13 mg/kg (Table 2), suggesting that he- JBP485 (Fig. 3A). The reduction in LDH activity was found patic first-pass elimination is not so remarkable.
in the presence of any compounds examined (Fig. 3B). Such Effect of JBP923 and JBP485 on Uptake of [14C]Gly-
reduction in the presence of JBP923 or JBP485 was found at Sar in Intestinal BBMVs. To examine the interaction of
a lower concentration than that found in the presence of these dipeptides with oligopeptide-specific transporters ex- glycyrrhizin or 18␤-glycyrrhetinic acid.
pressed in small intestines, their inhibitory effect on uptake Pharmacokinetics of JBP923 in Normal Rats. To ex-
of Gly-Sar, a typical substrate of peptide transporter PEPT1, amine the gastrointestinal absorption as well as hepatic first- by rabbit intestinal BBMVs was investigated (Fig. 5). The Fig. 3. The recovery of the GOT and LDH leakage by JBP923, JBP485, glycyrrhizin, or 18␤-glycyrrhetinic acid in the medium of primary cultured rat
hepatocytes exposed to CCl . Parenchymal hepatocytes were cultured for 24 h, then the cultured medium was changed to the fresh medium containing
5 mM CCl (control) or 5 mM CCl with various concentrations of the indicated compounds. Hepatocytes were further cultured for 24 h, and the activities of GOT and LDH in the medium were determined. CCl (⫺), medium without treatment of CCl ; CCl (⫹), medium with CCl in 0.45% of DMSO; DMSO, medium containing DMSO alone. The values are expressed as means ⫾ S.E. of three rats.
Liu et al.
Fig. 4. Plasma concentration-time profiles of JBP923 after i.v., portal vein, and oral administrations in rats. JBP923 at 3.13 (A) or 25 (B) mg/kg was
administered through the penis vein (F), portal vein (Œ), and orally (E) to rats. Plasma was collected from the external jugular vein at the indicated
time, and the JBP923 concentration in plasma was determined by HPLC. Points are expressed as means ⫾ S.E. of three to five rats.
TABLE 2Pharmacokinetic parameters of JBP923 in rats F or Fh ␮g min/ml 1.11 ⫾ 0.13a 0.040 ⫾ 0.006c 1.12 ⫾ 0.12a 1.05 ⫾ 0.12b pv, portal vein.
a Bioavailability (F) obtained from eq. 2.
b Hepatic availability (Fh) obtained from eq. 5.
c Significantly different (P ⬍ .05).
Fig. 5. Inhibitory effects of JBP923 and JBP485 on [14C]Gly-Sar uptake by rabbit intestine BBMVs. Membrane vesicles were preloaded with transport
buffer (pH ⫽ 7.5). Uptake of [14C]Gly-Sar (68 ␮M) with cold Gly-Sar (600 ␮M) in 20 mM Tris-citrate transport buffer (pH ⫽ 5.5) was measured at 37°C
for 2 min as a control. After incubating the membrane vesicles containing various concentrations of JBP923 and JBP485 at 37°C for 2 min, the uptake
of [14C]Gly-Sar (68 ␮M) with cold Gly-Sar (600 ␮M) was measured. Points are expressed as means ⫾ S.E. of three independent experiments.
uptake of [14C]Gly-Sar exhibited proton dependence because The antihepatitis activity of JBP923 was observed both after the uptake was higher in medium at pH 5.5 than that in its i.v. and oral administration. This was compatible with our medium at pH 7.5 (data not shown). Both JBP923 and finding that gastrointestinal absorption of JBP923 is almost JBP485 inhibited the uptake of [14C]Gly-Sar in a concentra- complete (Table 2). JBP923 decreased both GOT and LDH tion-dependent manner (Fig. 5) with K values of 13 and 31 activity in the medium of in vitro primary cultured hepato- mM for JBP923 and JBP485, respectively.
cytes (Fig. 3). Such direct effect on hepatocytes was found atthe concentrations above ⬃50 ␮M. On the other hand, the plasma JBP923 concentration after its oral administration at In this study, we found two dipeptide compounds, JBP923 25 mg/kg was higher than 50 ␮M (10 ␮g/ml) until at least 2 h and JBP485, that decrease BIL and hepatic cytosolic en- (Fig. 4B). This means that effective JBP923 concentration zymes activities in serum of ANIT-intoxicated rats (Table 1).
was maintained after oral administration. Thus, it seems to Dipeptides Orally Exhibiting Antihepatitis Activities
be that JBP923 showed pharmacological activity after its these activities are actually related to its protective effect on gastrointestinal absorption and subsequent interaction with liver function (Yi et al., 1996; Liu et al., 1998; Shaikh et al., hepatocytes, although this finding does not deny the possi- 1999). Therefore, the mechanism of antihepatitis activity of bility of the existence of its active metabolites.
these two dipeptides should also be clarified to further dem- Although glycyrrhizin also decreased the liver function onstrate their applicability to clinical stages.
marker enzyme both in vivo and in vitro (Figs. 2 and 3), itspharmacological effect was minimal after oral administra- tion (Fig. 3C). This is compatible with the fact that glycyr- Arase Y, Ikeda K, Murashima N, Chayama K, Tsubota A and Koida I (1997) The long rhizin was usually administered i.v. for the treatment of term efficacy of glycyrrhizin in chronic hepatitis C patients. Cancer 79:1494 –1500.
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glycyrrhizin has been reported to exert many types of biolog-ical activity, including antioxidant effect, anti-apoptosis ac- Send reprint requests to: Professor Yuichi Sugiyama, Ph.D., Graduate
tion, and enhancement of nitric oxide production from acti- School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, unkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama@seizai.f.u-tokyo.ac.jp vated macrophages, although it is still unknown which of

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