Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305.

1. 5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors. 2. It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy. 3. The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4. 4. Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, KI = 10.8 ± 2.14 µM) and testosterone 6ß-hydroxylation (CYP3A4, KI = 9.1 ± 2.82 µM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively. 5. In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug-drug interactions in potent multidrug therapy.

Metabolic transformation of antitumor acridinone C-1305 but not C-1311 via selective cellular expression of UGT1A10 increases cytotoxic response: implications for clinical use.

The acridinone derivates 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) are promising antitumor agents with high activity against several experimental cellular and tumor models and are under evaluation in preclinical and early phase clinical trials. Recent evidence from our laboratories has indicated that both compounds were conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. The present studies were designed to test the ability and selectivity of UGT1A10 in the glucuronidation of acridinone antitumor agents in a cellular context. We show that in KB-3 cells, a HeLa subline lacking expression of any UGT isoforms, both C-1305 and C-1311 undergo metabolic transformation to the glucuronidated forms on overexpression of UGT1A10. Furthermore, UGT1A10 overexpression significantly increased the cytotoxicity of C-1305, but not C-1311, suggesting that the glucuronide was more potent than the C-1305 parent compound. These responses were selective for UGT1A10 because documented overexpression of UGT2B4 failed to produce glucuronide products and failed to alter the cytotoxicity for both compounds. These findings contribute to our understanding of the mechanisms of action of these agents and are of particular significance because data for C-1305 contradict the dogma that glucuronidation typically plays a role in detoxification or deactivation. In summary, these studies suggest that extrahepatic UGT1A10 plays an important role in the metabolism and the bioactivation of C-1305 and constitutes the basis for further mechanistic studies on the mode of action of this drug, as well as translational studies on the role of this enzyme in regulation of C-1305 toxicity in cancer.

Role of human UDP-glucuronosyltransferases in the biotransformation of the triazoloacridinone and imidazoacridinone antitumor agents C-1305 and C-1311: highly selective substrates for UGT1A10.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311 (NSC-645809), is an antitumor agent shown to be effective against breast cancer in phase II clinical trials. A similar compound, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, shows high activity against experimental tumors and is expected to have even more beneficial pharmacological properties than C-1311. Previously published studies showed that these compounds are not substrates for cytochrome P450s; however, they do contain functional groups that are common targets for glucuronidation. Therefore, the aim of this work was to identify the human UDP-glucuronosyltransferases (UGTs) able to glucuronidate these two compounds. High-performance liquid chromatography analysis was used to examine the activities of human recombinant UGT1A and UGT2B isoforms and microsomes from human liver [human liver microsomes (HLM)], whole human intestinal mucosa [human intestinal microsomes (HIM)], and seven isolated segments of human gastrointestinal tract. Recombinant extrahepatic UGT1A10 glucuronidated 8-hydroxyl groups with the highest catalytic efficiency compared with other recombinant UGTs, V(max)/K(m) = 27.2 and 8.8 µl · min⁻¹ · mg protein⁻¹, for C-1305 and C-1311, respectively. In human hepatic and intestinal microsomes (HLM and HIM, respectively), high variability in UGT activities was observed among donors and for different regions of intestinal tract. However, both compounds underwent UGT-mediated metabolism to 8-O-glucuronides by microsomes from both sources with comparable efficiency; V(max)/K(m) values were from 4.0 to 5.5 µl · min⁻¹ · mg protein⁻¹. In summary, these studies suggest that imid azoacridinone and triazoloacridinone drugs are glucuronidated in human liver and intestine in vivo and may form the basis for future translational studies of the potential role of UGTs in resistance to these drugs.

Flavin monooxygenases, FMO1 and FMO3, not cytochrome P450 isoenzymes, contribute to metabolism of anti-tumour triazoloacridinone, C-1305, in liver microsomes and HepG2 cells.

5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, being the close structural analogue of the clinically tested imidazoacridinone anti-tumour agent, C-1311, expressed high activity against experimental tumours and is expected to have more advantageous pharmacological properties than C-1311. The aim of this study was to elucidate the role of selected liver enzymes in the metabolism of C-1305. We demonstrated that the studied triazoloacridinone was transformed with rat and human liver microsomes, HepG2 hepatoma cells and with human recombinant flavin-containing monooxygenases FMO1, FMO3 but not with CYPs. Furthermore, this compound was an effective inhibitor of CYP1A2 and CYP3A4. The product of FMO catalysed metabolism was shown to be identical to the main metabolite from liver microsomes and HepG2 cells. It was identified as an N-oxide derivative and, under hypoxia, it underwent retroreduction back to C-1305, what was extremely effective with participation of CYP3A4. In summary, this work revealed that the involvement of the P450 enzymatic system in microsomal and cellular metabolism of C-1305 was negligible, whereas this agent was an inhibitor of CYP1A2 and CYP3A4. In contrast, FMO1 and FMO3 were crucial for metabolism of C-1305 by liver microsomes and in HepG2 cells, which makes C-1305 an attractive potent anti-tumour agent.

Imidazoacridinone antitumor agent C-1311 as a selective mechanism-based inactivator of human cytochrome P450 1A2 and 3A4 isoenzymes.

BACKGROUND: 5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311), a promising antitumor agent that is also active against autoimmune diseases, was determined to be a selective inhibitor of the cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1311 might modulate the effectiveness of other drugs used in multidrug therapy. The present work aimed to identify the mechanism of the observed C-1311-mediated inactivation of CYP1A2 and CYP3A4. METHODS: The inactivation experiments were performed in vitro using the human recombinant CYP1A2 and CYP3A4 (Bactosomes). CYP isoenzyme activities were determined using the CYP-specific reactions, 7-ethoxycoumarin O-deethylation (CYP1A2) and testosterone 6ß-hydroxylation (CYP3A4). The concentrations of CYP-specific substrates and their metabolites formed by CYP isoenzymes were measured by RP-HPLC with UV-Vis detection. RESULTS: The inhibition of CYPs by C-1311 was time-, concentration- and NADPH-dependent, which suggested a mechanism-based mode of action. Using a 10-fold dilution method and potassium ferricyanide we demonstrated the irreversible nature of the inhibition. In addition, the inhibition was attenuated by the presence of alternate substrates (alternative active site ligands) but not by a nucleophilic trapping agent (glutathione) or a reactive oxygen scavenger (catalase), which further supported a mechanism-based action. Substrate depletion partition ratios of 299 and 985 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively. CONCLUSIONS: Our results indicated that C-1311 is a potent mechanism-based inactivator of CYP1A2 and CYP3A4. This finding provided new insights into the mechanism of C-1311 antitumor action, particularly in relation to potential pharmacokinetic drug-drug interactions between C-1311 and/or its derivatives and the substrates of CYP isoforms.