Chemistry Reference
In-Depth Information
were exceptionally good substrates for Cat A, they were also unstable when
incubated with hepatocytes. This was not unexpected since hepatocytes also
contain Cat A, in addition to many other esterases, leading to the conclusion
that the optimal prodrug would not necessarily be the best Cat A substrate.
Rather, the best prodrug would finely balance lability toward Cat A (and other
esterases) to improve intestinal and hepatic stability whilst also allowing e-
cient PBMC loading. The role of other esterases was suspected because some
prodrugs, such as 30i, were very poor Cat A substrates, yet hepatic stability was
the same as the much superior Cat A substrate 30f. Overall, it was found that
the monoamidate alanine prodrugs, especially n-propyl ester 30f and ethyl ester
30g, demonstrated the appropriate balance of Cat A lability in combination
with hepatic and intestinal stability. The generally improved hepatic stability
for the alanine prodrugs over the phenylalanine-based prodrugs was inter-
preted to be due to their lower lipophilicity. Therefore, a focused effort toward
modifying the alanine ester groups was the next logical step in the optimization
process, with the aim of maintaining low lipopholicity. A systematic explora-
tion of hindered, proximally branched esters, such as sec-butyl, isopropyl, and
small cycloalkyl rings, was moderately successful and resulted in the identifi-
cation of alanine cyclobutyl ester 30h, which demonstrated both greater hepatic
stablility and promising Cat A substrate properties. Indeed, compound 30h
provided a balance of hepatic stability and Cat A lability very close to that of
the targeted profile exemplified by compound 11.
10.3.3 In vivo Evaluation of Prodrugs
An evaluation method using intravenous (iv) administration in beagle dogs was
developed to identify the best prodrugs for oral studies and also reduced the
burden for large quantities of prodrugs during the discovery process.
29
The
method involved 30min iv infusion of prodrug, followed by measuring the
plasma prodrug over 24 h, and measuring the PBMC levels of 22 and its
metabolites over the same time period. The ratio of 22 in PBMCs ([AUC] over
24 h) compared to plasma prodrug ([AUC] over 24 h) exposure was defined as
the loading eciency of the prodrug. Clearly, high levels of 22 inside PBMCs
from relatively low exposures of prodrug would indicate an ecient prodrug
for loading the PBMCs. Using this method, amidate 11 demonstrated a loading
eciency of 0.58 and a moderate clearance rate, as shown in Table 10.5.
Both bisamidate 29d and monoamidate 30d had high clearance rates, which
was expected given their lower hepatic stabilities, but resulted in markedly
different loading eciencies! The difference can be attributed to the Cat A
cleavage rates, which in the case of 30d was suciently high to load PBMCs
despite the rapid clearance. Thus, the ability to effectively load the PBMCs by
iv was reflected by the balance between the rate of Cat A cleavage and clearance
of prodrug. Unfortunately upon oral administration, 30d had only 2.6% F due
to a significant first-pass effect, and minimal intracellular PBMC levels of 22
and diphosphate 22-DP were observed. The alanine prodrugs proved to be
more effective due to their improved hepatocyte stabilities. Cyclobutyl prodrug
Search WWH ::
Custom Search