The document investigates how cis/trans isomerization impacts the in vivo distribution and clearance of two pairs of ozonide antimalarial drug isomers. In vitro experiments showed the trans isomers degraded more rapidly than the cis isomers in rat blood and liver microsomes, despite higher protein binding of the trans isomers. In vivo pharmacokinetic studies in rats demonstrated the trans isomers had approximately two-fold shorter half-lives and five to nine-fold higher unbound plasma clearance than the corresponding cis isomers. The results emphasize the importance of conformation in determining the in vivo properties of this class of antimalarial drugs.

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Cis/trans Isomerism is an Important Determinant of
Clearance Processes for Ozonides Antimalarials
Ozonides are an important new class of
synthetic peroxide antimalarials [1,2] and are
thought to act by alkylation of parasite targets
following reductive cleavage of the peroxide by
iron(II) species [1]. An important determinant of
peroxide reactivity is the degree of steric
crowding associated with conformers of the
cyclohexyl group, which in turn is influenced by
cis/trans isomerisation at the 8’-carbon [3].
Previous studies have shown that Fe(II)
reactivity is influenced by conformation
differences [3]. Since the in vivo clearance
occurs in part by blood-mediated degradation,
most likely catalysed by Fe(II) in the labile iron
pool [2], conformation is also likely to impact in
vivo clearance processes.
Objectives
To investigate the impact of 8’-cis/trans
isomerisation on the determinants of in vivo
distribution and clearance of two pairs of
ozonide isomers.
Conclusions
Cis/trans isomerisation has a substantial
impact on the blood- and CYP-mediated
degradation of the ozonide pairs. In each in
vitro system, the trans isomers degraded more
rapidly than the cis isomers, in spite of being
more highly bound to proteins. This translated
to more rapid in vivo clearance for the trans
isomers. This work emphasizes the importance
of conformation in dictating in vivo disposition
of this important new class of antimalarials.
Acknowledgements
We acknowledge Prof. J Vennerstom (University of
Nebraska) for providing the ozonides and Dr K Katneni
for assistance with the binding experiments.
References
[1] J.L. Vennerstrom et al. (2004) Nature 430:900-904
[2] S.A. Charman et al. (2011) PNAS 108:4400-4405
[3] D.J. Creek et al. (2008) J Molec Graph Model 27:394-400; (2007) J Pharm Sci 96:2945-2956
[4] L. Zhou et al. (2008) Bioorg Med Chem Lett 18:1555-1558
Methods
• Stability was assessed in vitro in rat blood
and plasma and rat liver microsomes (RLM)
with and without NADPH supplementation.
Blood-to-plasma partitioning ratios (B/P)
were determined in fresh rat blood.
• Compound binding to plasma and
microsomal proteins was assessed by
ultracentrifugation.
• The IV pharmacokinetics were defined in
male Sprague Dawley rats. All samples were
analysed by LC/MS. PK parameters were
determined by using WinNonLin and
unbound clearance and volume of
distribution values were calculated using
standard equations.
• The half-life of each trans isomer was ~2-fold lower than for the corresponding cis isomer and unbound
plasma clearance values of the trans isomers were 5 to 9-fold higher than those of cis isomers.
• The higher unbound volumes of distribution for the trans isomers compared to the cis suggests that the
differences in half-life can be ascribed to the differences in clearance, which are consistent with the trends
observed in vitro in blood and RLM where the trans isomers were more rapidly degraded.
OZ209
8’-cis
OZ269
8’-trans
OZ277
8’-cis
OZ395
8’-trans
fu,microsomes 0.074 ± 0.007 0.047 ± 0.005 0.104 ± 0.009 0.071 ± 0.007
fu,plasma 0.052 ± 0.007 0.010 ± 0.002 0.077 ± 0.004 0.035 ± 0.004
B/P 2.5 ± 0.2 2.2 ± 0.2 2.1 ± 0.1 1.8 ± 0.1
CLint,u* 519 ± 102 2655 ± 78 358 ± 69 2140 ± 80
Introduction Results and Discussion
Both pairs of cis/trans
isomers had comparable
B/P ratios, however plasma
and microsomal protein
binding were higher for the
trans than for the cis
isomers.
Janne Mannila, Julia Morizzi, Anne Mannila, Eileen Ryan, Francis C.K. Chiu, David M. Shackleford, Susan A.
Charman and Darren J. Creek
Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia
OZ209
8’-cis
OZ269
8’-trans
OZ277
8’-cis
OZ395
8’-trans
t1/2 (h) 1.3 ± 0.2 0.6 ± 0.0 1.3 ± 0.1 0.6 ± 0.0
Plasma CL (mL/min/kg) 183 ± 8 309 ± 49 141 ± 12 298 ± 30
Plasma Vss (L/kg) 13.6 ± 2.3 8.2 ± 1.3 7.4 ± 1.7 5.8 ± 0.9
Binding-corrected PK parameters
Plasma CLu (mL/min/kg) 3511 ± 152 30342 ± 4841 1840 ± 158 8393 ± 835
Plasma Vss, u (L/kg) 261 ± 43 804 ± 124 97± 22 163 ± 25
The cis isomers were more stable
in RLM than the trans isomers even
though the unbound fraction was
higher for the cis isomers. There
was no evidence of peroxide
cleavage. Metabolism most likely
occurs through hydroxylation on
the adamantane ring [4].
The cis isomers were more stable
in blood than the trans, in spite of
there being a higher unbound
fraction for the cis isomers.
Results are consistent with
previous data demonstrating that
the cis isomers are more stable to
Fe(II) mediated degradation than
the trans isomers [3].
RatWholeBloodRatLiverMicrosomes
8’-cis isomer 8’-trans isomer
OZ277 OZ395
OZ209 OZ269
8’
8’ 8’
8’
*µL/min/mg protein
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