Suppression of Cytochrome P450 3A Protein Levels by Proteasome Inhibitors

Zangar, Richard C.; Kocarek, Thomas A.; Shen, Shang Yue; Bollinger, Nikki; Dahn, Michael S.; Lee, Donna W.

doi:10.1124/jpet.102.044628

Cited by 17 publications

(11 citation statements)

References 16 publications

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“…10C). 4 Together, our findings account for the profound CYP3A suppression by MG132 at 200 M concentrations detected by immunoblotting analyses (Zangar et al, 2003(Zangar et al, , 2008. They also rationalize the differential CYP3A4 suppression observed over 6 h between treatment with the NFB activation inhibitor 6-amino-4-(4-phenoxyphenyl-ethylamino)quinazoline (40%) and that with MG132 plus cycloheximide (75%) (Zangar et al, 2008).…”

Section: Discussionsupporting

confidence: 56%

RETRACTION: Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational Shutoff

2009

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Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH) 2 (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-B activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0 -300 M) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 M with marked suppression at Ͼ100 M. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2␣ (eIF2␣) kinase. Indeed, we found a marked (Ϸ4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2␣-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2␣ kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2␣ kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes.Hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored integral proteins responsible for the metabolism of various endobiotics as well as xenobiotics, including drugs, toxins, and carcinogens. Of these, human liver CYP3A4 and its mammalian orthologs are noteworthy not only because they biotransform a host of clinically relevant drugs, but also because they are inducible by their substrates via enhanced transcriptional/translational activity, or protein stabilization. Such substrate-mediated regulation of hepatic CYP3A 1 content can result in clinically relevant drugdrug interactions (DDIs), respectively prototyped by the DDIs encountered between rifampin-ethinylestradiol cotherapy on one hand, and grapefruit juice furanocoumarins and felodipine cointake, on the other (Yang et al., 2008). The 1 CYP3A or P450s 3A refer to rat liver P450s 3A2, 3A23, 3A18, and 3A9 and/or human liver CYP3A4/CYP3A5.

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Section: Discussionsupporting

confidence: 56%

RETRACTION: Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational Shutoff

2009

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“…It is interesting to note that the CYP4A1/3 activities and protein expression levels in our study were unchanged, whereas Roberts (1997) showed that proteasome inhibitors could enhance CYP4A degradation in rat hepatocyte cultures. Studies by Zangar et al (2003) also showed that rat hepatocytes treated with proteasome inhibitors resulted in a decrease of CYP3A protein and mRNA levels. However, this is contrary to the CYP3A-ubiqutin-proteasome pathway, thus suggesting that CYP3A may be degraded by multiple pathways or proteasomes could be responsible for the degradation of proteins that suppress CYP3A expression.…”

Section: Discussionmentioning

confidence: 99%

Investigation of Drug-Drug Interaction Potential of Bortezomib in Vivo in Female Sprague-Dawley Rats and in Vitro in Human Liver Microsomes

Gallegos²,

Li³

et al. 2006

Drug Metab Dispos

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ABSTRACT:Bortezomib (Velcade, PS-341), a dipeptidyl boronic acid, is a firstin-class proteasome inhibitor approved in 2003 for the treatment of multiple myeloma. In a preclinical toxicology study, bortezomibtreated rats resulted in liver enlargement (35%). Ex vivo analyses of the liver samples showed an 18% decrease in cytochrome P450 (P450) content, a 60% increase in palmitoyl coenzyme A ␤-oxidation activity, and a 41 and 23% decrease in CYP3A protein expression and activity, respectively. Furthermore, liver samples of bortezomib-treated rats had little change in CYP2B and CYP4A protein levels and activities. To address the likelihood of clinical drug-drug interactions, the P450 inhibition potential of bortezomib and its major deboronated metabolites M1 and M2 and their dealkylated metabolites M3 and M4 was evaluated in human liver microsomes for the major P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4/5. Bortezomib, M1, and M2 were found to be mild inhibitors of CYP2C19 (IC 50 ϳ 18.0, 10.0, and 13.2 M, respectively), and M1 was also a mild inhibitor of CYP2C9 (IC 50 ϳ 11.5 M). However, bortezomib, M1, M2, M3, and M4 did not inhibit other P450s (IC 50 values > 30 M). There also was no time-dependent inhibition of CYP3A4/5 by bortezomib or its major metabolites. Based on these results, no major P450-mediated clinical drug-drug interactions are anticipated for bortezomib or its major metabolites. To our knowledge, this is the first report on P450-mediated drug-drug interaction potential of proteasome inhibitors or boronic acid containing therapeutics.The approval of bortezomib (Velcade, PS-341) by the U.S. Food and Drug Administration for treatment of multiple myeloma made it the first drug in a new class of medicines called proteasome inhibitors. The proteasome is an enzyme complex found in the nucleus and cytoplasm of all cells in the body. It degrades intracellular proteins, such as IB and p53, through the ubiquitin proteasome pathway (Ciechanover, 1994) and regulates cell growth, apoptosis, and cell adhesion. In tumor cells, the blockage of degradation of IB by proteasome inhibitors makes the inflammatory nuclear factor B remain in an inactive form, thus enhancing tumor cell apoptosis (Palombella et al., 1998;Berenson et al., 2001;Garg and Aggarwal, 2002). In addition, proteasome inhibitors block the degradation of the tumor suppressor protein p53. When cells undergo radiation or chemotherapy, the p53 expressed in normal cells allows the arrest of cell proliferation and permits the repair of damaged DNA. In contrast, tumor cells express mutated forms of p53, which hinders cell cycle arrest and the ability to repair damaged DNA (Kuerbitz et al.,1992). Thus proteasome inhibitors help normal cells to recover from DNA damage while allowing tumor cells to undergo apoptosis (Adams, 2001;Chauhan et al., 2005).Bortezomib, a dipeptidyl boronic acid ( Fig. 1; Wu et al., 2000), is a potent, selective, and reversible inhibitor of the proteasome in mammalian cells (Adams, 2001;Richardson et al., 2003;Chauhan et al., 2005). Bo...

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“…The ability of proteasome inhibitors to reduce P450 expression in vitro has been described previously (Zangar et al, 2003(Zangar et al, , 2008Anwar-Mohamed et al, 2008;Acharya et al, 2009), but the mechanism of this effect remains unclear.…”

Section: Discussionmentioning

confidence: 99%

Clinical Pharmacokinetics, Metabolism, and Drug-Drug Interaction of Carfilzomib

Wang¹,

Jiao²,

Kirk³

et al. 2012

Drug Metab Dispos

125

128

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Carfilzomib, an irreversible proteasome inhibitor, has a favorable safety profile and significant antitumor activity in patients with relapsed and refractory multiple myeloma (MM). Here we summarize the clinical pharmacokinetics (PK), metabolism, and drug-drug interaction (DDI) profile of carfilzomib. The PK of carfilzomib, infused over 2-10 minutes, was evaluated in patients with solid tumors or MM. Metabolites of carfilzomib were characterized in patient plasma and urine samples. In vitro drug metabolism and DDI studies were conducted in human liver microsomes and hepatocytes. A clinical DDI study was conducted in patients with solid tumors to evaluate the effect of carfilzomib on CYP3A activity. Plasma concentrations of carfilzomib declined rapidly and in a biphasic manner after intravenous administration. The systemic half-life was short and the systemic clearance rate was higher than hepatic blood flow. Carfilzomib was cleared largely extrahepatically via peptidase cleavage and epoxide hydrolysis. Cytochrome P450-mediated metabolism played a minor role, suggesting that coadministration of P450 inhibitors or inducers is unlikely to change its PK profile. Carfilzomib showed direct and time-dependent inhibition of CYP3A in human liver microsome preparations and exposure to carfilzomib resulted in reductions in CYP3A and 1A2 gene expression in cultured human hepatocytes. However, administration of carfilzomib did not affect the PK of midazolam in patients with solid tumors, and there were no safety signals indicative of potential drug interactions. We conclude that the rapid systemic clearance and short half-life of carfilzomib limit clinically significant DDI.

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Suppression of Cytochrome P450 3A Protein Levels by Proteasome Inhibitors

Cited by 17 publications

References 16 publications

Investigation of Drug-Drug Interaction Potential of Bortezomib in Vivo in Female Sprague-Dawley Rats and in Vitro in Human Liver Microsomes

Clinical Pharmacokinetics, Metabolism, and Drug-Drug Interaction of Carfilzomib

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