RT Journal Article
SR Electronic
T1 Time- and NADPH-Dependent Inhibition of Cytochrome P450 3A4 by the Cyclopentapeptide Cilengitide: Significance of the Guanidine Group and Accompanying Spectral Changes
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1438
OP 1446
DO 10.1124/dmd.114.059295
VO 42
IS 9
A1 Mirza Bojić
A1 Luca Barbero
A1 Hugues Dolgos
A1 Achim Freisleben
A1 Dieter Gallemann
A1 Simona Riva
A1 F. Peter Guengerich
YR 2014
UL http://dmd.aspetjournals.org/content/42/9/1438.abstract
AB Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed unexpected inhibition of cytochrome P450 (P450) 3A4 at high concentrations, that is, a 15-mM concentration caused attenuation of P450 3A4 activity (depending on the probe substrate): 15–19% direct inhibition, 10–23% time-dependent inhibition (30-minute preincubation), and 54–60% metabolism-dependent inhibition (30-minute preincubation). The inactivation efficiency determined with human liver microsomes was 0.003 ± 0.001 min−1 mM−1 and was 0.04 ± 0.01 min−1 mM−1 with baculovirus-based microsomes containing recombinant P450 3A4. Neither heme loss nor covalent binding to apoprotein could explain the observed reductions in residual activity. Slowly forming type II difference spectra were observed, with maximum spectral changes after 2 hours. Binding to both reduced and oxidized P450 3A4 was observed, with apparent Kd values of 0.66 μM and 6 μM. The significance of the guanidine group in inhibition was demonstrated using ligand binding spectral changes and inactivation assays with guanidine analogs (debrisoquine, N-acetylarginine-O-methyl ester) and the acetylated ornithine derivative of cilengitide. The observed inhibition could be explained by direct inhibition, plus by formation of stable complexes with both ferric and ferrous forms of heme iron and to some extent by the formation of reactive species capable to react to the protein or heme. Formation of the complex required time and NADPH and is attributed to the guanidino group. Thus, the NADPH-dependent inhibition is considered to be mainly due to the formation of a stable complex rather than the formation of reactive species.