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gastrointestinal tract, blood (or circulation), and the liver (Figure 11.5). Our
desire was to identify a prodrug that was stable in simulated gastric fluid,
simulated intestinal fluid, and blood, but that would decompose readily to the
monophosphate derivative when exposed to liver S9 fractions. At the same
time, we evaluated the safety profile of our compounds by assessing cytotoxi-
city against a panel of cell lines and by assessing mitochondrial toxicity.
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This
initial assessment phase led to a selection of a set of compounds that we pro-
gressed into PK evaluation in rats, where we looked not only for circulating
levels of prodrug and key metabolites, but also at liver levels of prodrug and
key metabolites (particularly the pharmacologically active PSI-6202 triphos-
phate metabolite).
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Simultaneously, we evaluated the potential for these
prodrugs to generate the active PSI-6206 triphosphate metabolite in isolated
primary rat, human, dog, and monkey hepatocytes.
After the initial screening phase, a selected set of compounds met the criteria
of improved potency over PSI-6130 in the HCV replicon assay, lack of cyto-
toxicity in a panel of different cell lines, lack of mitochondrial toxicity, adequate
gastrointestinal stability, and a half-life when incubated with a liver S9 fraction
that supported rapid conversion in the liver by first-pass metabolism (see Figure
11.6). Rats were dosed orally with each of these compounds to compare their
metabolic fate and to assess if they generated appropriate liver levels of the
desired triphosphate-active metabolite. In fact, each compound demonstrated
high levels of the triphosphate in rat livers, thus supporting the concept that a
phosphoramidate prodrug of PSI-6206 could be delivered effectively to the liver
Phosphoramidate
Prodrugs
HCV Replicon Activity + r-RNA Cytotoxicity
Cytotoxicity Evaluation: Cell Line Panel
Stability: SGF, SIF, Plasma,
Liver S9
Mitochondrial Tox,
Bone Marrow Tox
Cellular TP Levels
Rat, Dog, PK
Rat Tox
Select Compound for further preclinical development
Figure 11.5
To select a preclinical development candidate, the phosphoramidate
prodrugs of PSI-6206 were progressed through a screening process
assessing potency, in vitro and in vivo toxicity, and PK characteristics.
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