3.1 In vitro experiments
Mean (SD) intrinsic hepatocyte clearance (n=2) of SPL026 was 19.4 (0.8)
µL/min/million cells, with a half-life of 98.9 (3.9) min. Separately,
MAO inhibition had a minimal impact on the intrinsic hepatocyte
clearance rate of SPL026, 13.8 µL/min/million cells (half-life, 92.4
min) with MAO inhibitors vs 13.24 µL/min/million cells (half-life, 96.1
min) in the absence of MAO inhibition.
The contribution of MAO in the metabolism of SPL026 was further assessed
in human liver mitochondrial fractions. The intrinsic clearance of
SPL026 was reduced by approximately 11-fold with MAO-A inhibition
compared to vehicle control (<3.9 vs 42.9 µL/min/mg protein),
with an accompanying increase in half-life (>373.7 vs 33.7
min); however, there was no influence of MAO-B inhibition (intrinsic
clearance, 42.7 µL/min/mg protein; half-life, 32.5 min). Blood:plasma
ratio was 1.53 for SPL026 (Table 2). Around 70% of SPL026 was not bound
to plasma proteins, suggesting greater efficiency for diffusion within
the blood plasma, with a large proportion of the free drug available and
subject to both circulation and metabolism. In addition, a mean
logD7.4 value of 0.15 indicated that the lipophilicity
of SPL026 was very low.
CYP phenotyping using 8 human enzymes showed that the intrinsic
clearance of SPL026 was <29 µL/min/nmol CYP. No turnover of
SPL026 with CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP3A4 and CYP3A5 was
detected for the duration of the study indicating that SPL026 is not a
substrate for these isozymes. SPL026 intrinsic clearance was 801
µL/min/nmol CYP (half-life, 9 min) for CYP2D6 and 37 µL/min/nmol CYP
(half-life, 189 min) for CYP2C19. This data corresponds to an estimated
clearance rate of 9.5 and 0.3 µL/min/mg microsomal protein with CYP2D6
and CYP2C19, respectively, indicating a role for CYP2D6 and minor role
of CYP2C19 in DMT metabolism in human liver microsomes.