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(Figure 11.3).
19
The existence of the uridine metabolite and the discovery of the
metabolic pathway for the formation of the uridine triphosphate (an active
inhibitor of the HCV polymerase) added a unique dimension to the under-
standing of how PSI-6130 functioned as an HCV inhibitor. However, it also
raised an issue regarding how PSI-6130 would be metabolized in humans and
how the extent of formation of either the inactive unphosphorylated uridine
metabolite (PSI-6206) or the active triphosphate uridine metabolite (PSI-6206-
TP) was going to influence the pharmacokinetics (PK) and e
cacy. As
described later, the extent of PSI-6206 metabolite formation would become an
issue in the clinic.
11.2.3 Resistance Profile of PSI-6130
Because nucleoside analogs that possess the 2
0
-C modification (1 and 4) were
known to select for the S282T mutation (which resides in the active site of the
HCV RdRp), PSI-6130 was tested for cross resistance using a HCV genotype
1b replicon that contained this mutation.
12
In general, this mutation caused a
20- to 100-fold reduction in sensitivity to 2
0
-C-methyl-substituted compounds
such as NM107 (1), and 2
0
-C-methyladenosine (4).
20-22
In contrast, only a 2.4-
to 6-fold reduction in activity was observed for PSI-6130 with the S282T
mutant replicon.
12,23
Enzyme assays were performed using a RdRp that con-
tained the S282T mutation to assess the ability of PSI-6130-TP and PSI-6206-
TP to inhibit this mutant enzyme.
19,23
The presence of the S282T mutation in
the RdRp resulted in a 7.5-fold and 23.7-fold reduction in the ability of PSI-
6130-TP and PSI-6206-TP to inhibit the enzyme, respectively.
19
Further in vitro
selection studies with PSI-6130 performed by Ali and colleagues demonstrated
that long-term culturing of replicon cells in the presence of compound resulted
in the emergence of the S282T mutation in a complex pattern with other amino
acid changes.
23
However, the S282T mutation appeared to be the only muta-
tion that provoked reduced sensitivity to PSI-6130. The presence of the S282T
mutation led to a 15% decrease in the replication fitness of the replicon. Pair-
wise combinations with S282T resulted in a further decrease in replication fit-
ness; however, certain multiple mutations combined with S282T resulted in
enhanced replication fitness.
The modest impact of the S282T mutation on PSI-6130 activity and the
challenge observed in obtaining this mutation was somewhat surprising.
Previous mutation studies with 2
0
-methylcytidine (1, NM107) and 2
0
-methyl-
adenosine (4) nucleosides showed that the S282T mutation arose relatively
quickly, and that these 2
0
-C-Me nucleosides were quite sensitive to the
mutation. Based on these results, it was speculated that the 2
0
-methyl group
was responsible for the mutational sensitivity; therefore, one would have
expected PSI-6130 to show similar sensitivity.
20
The relatively low fold-change
in sensitivity to the S282T mutation again demonstrates that the 2
0
-F-2
0
-C-
methyl combination imparts differentiating characteristics relative to the
unfluorinated analog.
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