Idiosyncratic hepatotoxicity has been associated with the oral tyrosine kinase inhibitor lapatinib, which is used in metastatic breast cancer therapy. Lapatinib is extensively metabolized by ...cytochrome P450 3A4/5 to yield an O-debenzylated metabolite, which can undergo further oxidation to a reactive quinone imine. A recent clinical study reported that concomitant use of lapatinib with dexamethasone increased the incidence of hepatotoxicity in metastatic breast cancer patients treated with lapatinib, and so we hypothesized that induction of CYP3A enhances the bioactivation of lapatinib to reactive intermediates that contribute to hepatotoxicity. Therefore, we examined the effect of CYP3A4 induction on the cytotoxicity and metabolism of lapatinib in the HepaRG human hepatic cell line. Differentiated HepaRG cells were pretreated with dexamethasone (100 μM) or the prototypical CYP3A4 inducer rifampicin (4 μM) for 72 hours, followed by incubation with lapatinib (0-100 μM) for 24 hours. Cell viability was monitored using WST-1 assays, and metabolites were quantified by liquid chromatography coupled to tandem mass spectrometry. Induction of CYP3A4 by dexamethasone or rifampicin enhanced lapatinib-induced cytotoxicity, compared with treatment with lapatinib alone. A direct comparison of the cytotoxicity of lapatinib versus O-debenzylated lapatinib demonstrated that the O-debenzylated metabolite was significantly more cytotoxic than lapatinib itself. Furthermore, pretreatment with 25 μM l-buthionine sulfoximine to deplete intracellular glutathione markedly enhanced lapatinib cytotoxicity. Cytotoxicity was correlated with increased formation of O-debenzylated lapatinib and cysteine adducts of the putative quinone imine intermediate. Collectively, these data suggest that CYP3A4 induction potentiates lapatinib-induced hepatotoxicity via increased reactive metabolite formation.
Abstract
Mechanism-based inactivation (MBI) of CYP450 enzymes is a unique form of inhibition in which the enzymatic machinery of the victim is responsible for generation of the reactive metabolite. ...This precondition sets up a time-dependency for the inactivation process, a hallmark feature that characterizes all MBI. Yet, MBI itself is a complex biochemical phenomenon that operates in different modes, namely, covalent binding to apoprotein, covalent binding of the porphyrin group and also complexation of the catalytic iron. Using lapatinib as a recent example of toxicological interest, we present an example of a mixed-function MBI that can confound clinical drug-drug interactions manifestation. Lapatinib exhibits both covalent binding to the apoprotein and formation of a metabolite-intermediate complex in an enzyme-selective manner (CYP3A4 versus CYP3A5), each with different reactive metabolites. The clinical implication of this effect is also contingent upon genetic polymorphisms of the enzyme involved as well as the co-administration of other substrates, inhibitors or inducers, culminating in drug-drug interactions. This understanding recapitulates the importance of applying isoform-specific mechanistic investigations to develop customized strategies to manage such outcomes.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Reactive oxygen species (ROS) and oxidative stress have been considered in a variety of disease models, and cytochrome P450 (P450) enzymes have been suggested to be a source of ROS. Induction of ...P450s by phenobarbital (PB), β-naphthoflavone (βNF), or clofibrate in a mouse model increased ROS parameters in the isolated liver microsomes, but isoniazid treatment did not. However, when F2-isoprostanes (F2-IsoPs) were measured in tissues and urine, PB showed the strongest effect and βNF had a measurable but weaker effect. The same trend was seen when an Nfr2-based transgene reporter sensitive to ROS was analyzed in the mice. This pattern had been seen earlier with F2-IsoPs both in vitro and in vivo with rats (Dostalek, M., Brooks, J. D., Hardy, K. D., Milne, G. L., Moore, M. M., Sharma, S., Morrow, J. D., and Guengerich, F. P. (2007) Mol. Pharmacol. 72, 1419–1424). One possibility for the general in vitro-in vivo discrepancy in oxidative stress found in both mice and rats is that PB treatment might attenuate protective systems. One potential candidate suggested by an mRNA microarray was nicotinamide N-methyltransferase. PB was found to elevate nicotinamide N-methyltransferase activity 3- to 4-fold in mice and rats and to attenuate levels of NAD+, NADP+, NADH, and NADPH in both species (20–40%), due to the enhanced excretion of (N-methyl)nicotinamide. PB also down-regulated glutathione peroxidase and glutathione reductase, which together constitute a key enzymatic system that uses NADPH in protecting against oxidative stress. These multiple effects on the protective systems are proposed to be more important than P450 induction in oxidative stress and emphasize the importance of studying in vivo models.
Previously published studies have shown that cytochrome P450 (P450) enzyme systems can produce reactive oxygen species and
suggest roles of P450s in oxidative stress. However, most of the studies ...have been done in vitro, and the potential link between
P450 induction and in vivo oxidative damage has not been rigorously explored with validated biomarkers. Male Sprague-Dawley
rats were pretreated with typical P450 inducers (β-naphthoflavone, phenobarbital (PB), Aroclor 1254, isoniazid, pregnenolone
16α-carbonitrile, and clofibrate) or the general P450 inhibitor 1-aminobenztriazole; induction of P4501A, -2B, -2E, -3A, and
-4A subfamily enzymes was confirmed by immunoblotting and the suppression of P450 by 1-aminobenztriazole using spectral analysis.
PB and Aroclor 1254 significantly enhanced malondialdehyde and H 2 O 2 generation and NADPH oxidation in vitro and significantly enhanced formation in vivo, in both liver and plasma. Some of the
other treatments changed in vitro parameters but none did in vivo. The PB-mediated increases in liver and plasma F 2 -isoprostanes could be ablated by 1-aminobenztriazole, implicating the PB-induced P450(s) in the F 2 -isoprostane elevation. The markers of in vivo oxidative stress were influenced mainly by PB and Aroclor 1254, indicative
of an oxidative damage response only to barbiturate-type induction and probably related to 2B subfamily enzymes. These studies
define the contribution of P450s to oxidative stress in vivo, in that the phenomenon is relatively restricted and most P450s
do not contribute substantially.
15-Deoxy-Δ¹²,¹⁴-prostaglandin J₂ (15-d-PGJ₂) is a reactive cyclopentenone eicosanoid generated from the dehydration of cyclooxygenase-derived prostaglandin D₂ (PGD₂). This compound possesses an ...α,β-unsaturated carbonyl moiety that can readily adduct thiol-containing biomolecules such as glutathione and cysteine residues of proteins via the Michael addition. Due to its reactivity, 15-d-PGJ₂ is thought to modulate inflammatory and apoptotic processes and is believed to be an endogenous ligand for peroxisome proliferator-activated receptor-γ. However, the extent to which 15-d-PGJ₂ is formed in vivo and the mechanisms that regulate its formation are unknown. Previously, we have reported the formation of PGD₂ and PGJ₂-like compounds, termed D₂/J₂-isoprostanes (D₂/J₂-IsoPs), produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid (AA). Based on these findings, we investigated whether 15-d-PGJ₂-like compounds are also formed via this nonenzymatic pathway. Here we report the generation of novel 15-d-PGJ₂-like compounds, termed deoxy-J₂-isoprostanes (deoxy-J₂-IsoPs), in vivo, via the nonenzymatic peroxidation of AA. Levels of deoxy-J₂-IsoPs increased 12-fold (6.4 ± 1.1 ng/g liver) in rats after oxidant insult by CCl₄ treatment, compared with basal levels (0.55 ± 0.21 ng/g liver). These compounds may have important bioactivities in vivo under conditions associated with oxidant stress.
15-Deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2) is a reactive cyclopentenone eicosanoid generated from the dehydration of cyclooxygenase-derived prostaglandin D2 (PGD2). This compound possesses an ...α,β-unsaturated carbonyl moiety that can readily adduct thiol-containing biomolecules such as glutathione and cysteine residues of proteins via the Michael addition. Due to its reactivity, 15-d-PGJ2 is thought to modulate inflammatory and apoptotic processes and is believed to be an endogenous ligand for peroxisome proliferator-activated receptor-γ. However, the extent to which 15-d-PGJ2 is formed in vivo and the mechanisms that regulate its formation are unknown. Previously, we have reported the formation of PGD2 and PGJ2-like compounds, termed D2/J2-isoprostanes (D2/J2-IsoPs), produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid (AA). Based on these findings, we investigated whether 15-d-PGJ2-like compounds are also formed via this nonenzymatic pathway. Here we report the generation of novel 15-d-PGJ2-like compounds, termed deoxy-J2-isoprostanes (deoxy-J2-IsoPs), in vivo, via the nonenzymatic peroxidation of AA. Levels of deoxy-J2-IsoPs increased 12-fold (6.4 ± 1.1 ng/g liver) in rats after oxidant insult by CCl4 treatment, compared with basal levels (0.55 ± 0.21 ng/g liver). These compounds may have important bioactivities in vivo under conditions associated with oxidant stress.
Reactive oxygen species (ROS) and oxidative stress have been considered in
a variety of disease models, and cytochrome P450 (P450) enzymes have been
suggested to be a source of ROS. Induction of ...P450s by phenobarbital (PB),
β-naphthoflavone (βNF), or clofibrate in a mouse model increased ROS
parameters in the isolated liver microsomes, but isoniazid treatment did not.
However, when F
2
-isoprostanes (F
2
-IsoPs) were measured
in tissues and urine, PB showed the strongest effect and βNF had a
measurable but weaker effect. The same trend was seen when an Nfr2-based
transgene reporter sensitive to ROS was analyzed in the mice. This pattern had
been seen earlier with F
2
-IsoPs both
in vitro
and
in
vivo
with rats (Dostalek, M., Brooks, J. D., Hardy, K. D., Milne, G. L.,
Moore, M. M., Sharma, S., Morrow, J. D., and Guengerich, F. P. (2007)
Mol.
Pharmacol.
72, 1419–1424). One possibility for the general
in
vitro
-
in vivo
discrepancy in oxidative stress found in both mice
and rats is that PB treatment might attenuate protective systems. One
potential candidate suggested by an mRNA microarray was nicotinamide
N
-methyltransferase. PB was found to elevate nicotinamide
N
-methyltransferase activity 3- to 4-fold in mice and rats and to
attenuate levels of NAD
+
, NADP
+
, NADH, and NADPH in both
species (20–40%), due to the enhanced excretion of
(
N
-methyl)nicotinamide. PB also down-regulated glutathione peroxidase
and glutathione reductase, which together constitute a key enzymatic system
that uses NADPH in protecting against oxidative stress. These multiple effects
on the protective systems are proposed to be more important than P450
induction in oxidative stress and emphasize the importance of studying
in
vivo
models.
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