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Fig. 2. Effects of PFD and Its Metabolites on TGF-1 Stimulated Increases in Hydroxyproline in WI-38 Cells
PFD and its metabolites with TGF-1 (10 ng/mL) were applied to WI-38 cells, followed by incubation for 24 h at 37C with 5% CO2. After incubation, the intracellular
hydroxyproline amount was determined; the data are shown as the ratio of intracellular hydroxyproline to amount of DNA. Each point represents the meanS.D. (n=4).
* p<0.05 and ** p<0.01: signicantly different from TGF-1 alone.
nated time point, rats were anesthetized using intraperitoneal shown in Fig. 2. PFD at 100 M (18.5 g/mL), 300 M (55.6 g/
injections of pentobarbital sodium at a dose of 40 mg/kg and mL), and 1000 M (185 g/mL), and PFD-OH at 300 M
blood was collected from the jugular vein. The concentration (60.4 g/mL) and 1000 M (201 g/mL), and PFD-COOH at
of PFD and its metabolites was measured in each sample by 300 M (64.6 g/mL) and 1000 M (215 g/mL) signicantly
HPLC as described below. The pharmacokinetic analysis was decreased the TGF-1-induced hydroxyproline content in
performed using the non-compartment analytical method.10) WI-38 cells without cellular toxicity. PFD and its metabolites
Determination of Hydroxyproline by HPLC The con- were stable at 37C in medium for 24 h (data not shown).
centrations of hydroxyproline in samples were measured by These results indicate that PFD-OH and PFD-COOH have
HPLC following fluorescent derivatization, using the method antibrotic activities, which inhibit collagen synthesis in lung
of Hutson et al.11) The derivative in samples was subjected broblasts. In addition, after intravenous administration of
to HPLC using a system (Jasco Corporation, Tokyo, Japan) PFD to rats, the PFD-COOH concentration in plasma was
involving an InertSustain C18 (3.0250 mm internal diameter; comparable to that of PFD (Fig. 3). The calculated terminal
GL Sciences, Inc., Torrance, CA, U.S.A.). The mobile phase elimination half-life (T1/2) of PFD, PFD-OH, and PFD-COOH
was 85 m M acetic buffer (pH 4.3)acetonitrile (68 : 32). Separa- were 0.740.12, 0.790.26, and 0.840.26 h, respectively. The
tion was performed at a flow rate of 0.4 mL/min at 45C, and areas under the concentrationtime curves (AUC) for PFD,
the eluate from the column was monitored by fluorescence PFD-OH, and PFD-COOH were 30.5, 3.9, and 24.2 g*h/mL,
detection (excitation wavelength of 250 nm and emission respectively. These results indicate that the antibrotic effect
wavelength of 310 nm). because of the presence of PFD-COOH in plasma cannot be
Determination of PFD and Its Metabolites by HPLC neglected. The plasma concentrations of both PFD and PFD-
The concentrations of PFD, PFD-OH, and PFD-COOH COOH were lower than effective concentrations of those in
in plasma were measured by HPLC, using the method by vitro. We reasoned that PFD can possibly exert antibrotic
Wang et al.12) The prepared samples were subjected to HPLC effect in synergy with PFD-COOH in vivo. Recently, Huang
using a system involving a Mightysil RP-18GPII column et al. have reported that PFD-COOH concentration in plasma
(3.0250 mm, Kanto Chemical Co., Tokyo, Japan). The mobile was approximately 60% of PFD concentration in plasma after
phase was 0.2% acetic acid/methanol (74 : 26). The separation oral administration to human.9) Thus, it is thought that PFD-
was performed at a flow rate of 0.4 mL/min at 45C and the COOH may participate in the antibrotic effects of PFD in
column was monitored by UV absorbance detection (absor- the treatment of IPF. On the other hands, PFD-OH pharmaco-
bance wavelength of 310 nm). kinetics in human are not well understood. The involvement
Statistics Statistical analysis was performed using the of PFD-OH in the antibrotic action of a therapy for IPF in
Dunnetts t-test and SPSS software version 21 (IBM Inc., Ar- human is thought to need examination in both pharmacody-
monk, NY, U.S.A.). namics and pharmacokinetics in human.
PFD has many mechanisms of pharmacological action.
RESULTS AND DISCUSSION PFD has been shown to reduce collagen I expression,13) block
the proliferative effects of platelet-derived growth factor
The present study evaluated the antibrotic effects of the (PDGF),14) and inhibit the expression of heat shock protein
PFD metabolites PFD-OH and PFD-COOH. The effects of (HSP) 47 on lung broblasts.13) In this study, the inhibition of
different concentrations of PFD, PFD-OH, and PFD-COOH collagen synthesis by PFD-OH and PFD-COOH were weaker
on hydroxyproline content, a major component of the protein than that of the parent compound (Fig. 3). Since many mecha-
collagen, in WI-38 cells, a human lung broblast cell line, are nisms contribute to brosis,1315) the pharmacological effects
October 20131527