Osamu Cynshi, Kunio Tamura, and Etsuo Niki
3.4. Evaluation of PK Profiles and
Design, Synthesis, and Action of Antiatherogenic Antioxidants 103
Table 6.2
Inhibitory effects of BO-653 and BO-313 on AAPH- and lipoxygenase-catalyzed LDL oxidationa
Fluorescence (% inhibition)
Fluorescence (% inhibition)
Conc. AAPHb LO Conc. AAPH LO
Control 892± 38 308± 5 Control 221± 15 245± 2
BO-313 Low 972 (+9) 324 (+5) BO-653 Low 239 (+8) 258 (+6)
High 700 (–22) 356 (+16) High 148 (–33) 34 (–91)
Probucol Low 798 (–11) 306 (–1) Probucol Low 179 (–19) 248 (+1)
High 371 (–88) 63 (–84) High 111 (–50) 61 (–79)
aRabbit LDL at a concentration of 200g/mL was incubated with AAPH or SLO at 37◦C for 24 h in the presence or absence of test compound. Fluorescence (Ex: 360 nm, Em: 430 nm) was integrated in whole LDL fraction determined by gel permeation chromatography (GPC). Low and high concentrations of test compound were 10 and 100M for AAPH-catalyzed oxidation, respectively, and 0.5 and 5 M for LO-catalyzed oxidation, respectively. The control contained 1% DMSO as vehicle.
bThe fluorophotometric detector in the GPC system was replaced with another one in this assay.
types of LDL oxidation due to the design of the drug that is responsible for the inhibition of the propagation process (steps) of LDL oxidation.
3.4. Evaluation of PK
104 Cynshi, Tamura, and Niki
methanol and the solution was then analyzed on an ECD-HPLC system with methanol/tert-butyl alcohol (90/10, v/v) contain-ing 50 mM NaClO4as the eluents at a flow rate of 0.8 mL/min.
Figure 6.5 shows that both compounds were absorbed and emerged in plasma more easily than probucol. Following frac-tionating the plasma into very-low-density lipoprotein (VLDL), LDL, and HDL fractions at 24 h after the final administration of the test drug, we confirmed the affinity of the test compound to LDL. The results indicate that the oral administration of both compounds distributed in the VLDL and LDL fractions rather than in the HDL fraction (data not shown) (13),
0 20 40 60 80 100
0 2 4 6 8 10
Days
Plasma Concentration
Probucol BO-313 BO-653
0 2 4 6 8 10
0 2 4 6 8 10
Days
Fig. 6.5. Plasma concentration of BO-653 and BO-313 in WHHL rabbits. Rabbits (n=2) were orally administered once a day with the test compound (250 mg/kg) or probucol (500 mg/kg) for 9 days and were evaluated at 24 h after each daily administration. The graph on theright shows a magnification of the plasma concentration from 0 to 10 (g/mL).
3.4.2. Evaluation of Atherosclerotic Lesions of the
Antioxidant-Treated WHHL Rabbits
Male WHHL rabbits were maintained for 6 or 8 months on a normal diet (CR3) without additional antioxidants or with 0.5%
BO-653 or BO-313. A diet with 1% probucol was used as the reference for an antiatherogenic compound. The atherosclerotic lesion area of the WHHL rabbits was determined as the area of fatty region on the aortic inner surface using SigmaScan Pro software (Jandel Scientific Software) with the photos of the aor-tic inner surface (24). To evaluate the inhibitory potency against atherosclerosis, we conducted an experiment for each compound;
that is, experiment 1 is with BO-313 and experiment 2 is with BO-653.
Although both compounds inhibited the Cu2+-catalyzed LDL oxidation, BO-313 and BO-653 also showed contrary results as shown in Fig. 6.6. BO-653 ameliorated atherosclerosis in WHHL rabbits, whereas BO-313 deteriorated it. This result suggests that the mechanism of an antioxidant is essential for exerting its antiatherogenic effect in vivo and that the inhibition of a common process in various types of LDL oxidation, e.g., the propagation process in LDL oxidation, is more critical for design-ing an effective antiatherogenic antioxidant.
Design, Synthesis, and Action of Antiatherogenic Antioxidants 105
Control BO-313 Control BO-653
Experiment 1
0 20 40 60 80 100
Probucol
Lesion Area (%)
Total Abdominal
Experiment 2
0 20 40 60 80 100
Probucol
Lesion Area (%)
Total Abdominal
Control BO-313 Control BO-653
(A) (B)
Fig. 6.6. Inhibitory effect of BO-653 and BO-313 on atherosclerosis in WHHL rabbits. Male rabbits (n=5) were maintained for 6 or 8 months on a diet containing 0.5, 0.5, or 1% concentration of BO-313, BO-653, or probucol, respectively. (a) Representative photographs of the aortic inner surface of WHHL rabbits. (b) Effects of BO-313 or BO-653 on intimal surface area with atherosclerotic lesions of the whole aorta and abdominal aorta in two experiments. All results are shown as mean± SD.
4. Notes
1. In GPC analysis, the LDL solution, in particular its oxidation form, easily adheres to the gel permeation column. There-fore, a column prepared specifically for lipoprotein analysis should be used. An eluent specially used for lipoprotein anal-ysis is currently available and provided by TOSOH Corpo-ration which offers a stable GPC analysis.
2. For PK analysis, the test compound was administered orally as a suspension in 3% gum Arabic solution. If the gum Arabic content were lower, a uniform suspension would be difficult to prepare due to separation of the test compound from the gum Arabic in solution. As most of the test compounds in this study were oils, a uniform suspension was prepared with an electric homogenizer and had to be used within 1 h of its preparation. If a stock solution of the suspension is used, the solution would need to be resuspended with the electric homogenizer.
Acknowledgments
The authors thank Drs. T. Kodama at Tokyo University and T. Kita at Kyoto University for providing excellent advice on atherosclerosis in animal models. We also thank Drs. Y. Kawabe, Y. Kato, T. Suzuki, Y. Takashima, M. Takeda, H. Kaise, M. Kim, J. Aono, and Y. Ohba for their collaboration on this project in Chugai Pharmaceutical Co., Ltd. and Ms Ford Frances for her editorial assistance.
106 Cynshi, Tamura, and Niki
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