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References and notes
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Figure 2. Effect of 20a on skin-allograft survival: Full thickness skins of BALB/c
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Table 5
Non-GLP in vitro toxicity in a rat hepatoma (H4IIE) cell line
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a
Compound
TC50
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CsA
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48
300
75
300
42
300
42
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were over 300 lM, whereas CsA showed much lower TC50 values
than 20a, supporting lower in vivo toxicity of 20a than CsA.
In summary, we have synthesized 39 colchicine-derived ana-
logues, which were evaluated by MLR, lymphoproliferation assay,
inhibitory effects on inflammatory genes and in vivo skin-allograft
model. The analog 9b possessing nitrate substituent as an exoge-
nous NO donor turned out to be equipotent to CsA. In addition,
optimization of 9b afforded more potent analogue 20a, which
was comparable to CsA on skin-allograft model and less toxic than
CsA in in vitro toxicity. Considering low molecular weight of 20a,
its equipotent immunosuppressive activity implies that 20a would
be usable as orally available immunosuppressive agent. The struc-
ture–activity relationship of colchicine for immunosuppressive
activity was also established. Currently, the immunosuppressant
efficacy evaluation on 20a for in vivo heart transplantation animal
model is in progress and the successful results will be reported in
due course.
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22. Because the parent compound (9b) of these analogues showed non-selective
activity for B and T cells, we just performed lymphoproliferation assay on T-
lymphocyte-activated cells as a general thing.
23. The TC50 values are the exposure concentrations (empirically estimated from a
log dose versus response curve) that produce a half (50%) maximal response
relative to controls. If a 50% response could not be determined, the TC50 value
was expressed as greater than the highest exposure concentration. Each dose
response curve is composed of a vehicle (DMSO) group and seven treatment
groups spanning an exposure range of 0.1–300
represents the mean of 4–8 wells in a plate. The
l
a
M. Each value in the curve
-GST and MTT assays were
run from one plate as were the GSH/8-isoprostane assays, while the cell
proliferation and ATP assays were run from separate plates.
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Acknowledgment
This work was supported by grant from KT&G and in part by the
Research Institute of Pharmaceutical Science, Seoul National
University.