Herein, the in vitro metabolism of tyrosine kinase inhibitor cabozantinib, the drug used for the treatment of metastatic medullary thyroid cancer and advanced renal cell carcinoma, was studied using hepatic microsomal samples of different human donors, human recombinant cytochromes P450 (CYPs), flavin-containing mono-oxygenases (FMOs) and aldehyde oxidase. After incubation with human microsomes, three metabolites, namely cabozantinib N-oxide, desmethyl cabozantinib and monohydroxy cabozantinib, were detected. Significant correlations were found between CYP3A4 activity and generation of all metabolites. The privileged role of CYP3A4 was further confirmed by examining the effect of CYP inhibitors and by human recombinant enzymes. Only four of all tested human recombinant cytochrome P450 were able to oxidize cabozantinib, and CYP3A4 exhibited the most efficient activity. Importantly, cytochrome b5 (cyt b5) stimulates the CYP3A4-catalyzed formation of cabozantinib metabolites. In addition, cyt b5 also stimulates the activity of CYP3A5, whereas two other enzymes, CYP1A1 and 1B1, were not affected by cyt b5. Since CYP3A4 exhibits high expression in the human liver and was found to be the most efficient enzyme in cabozantinib oxidation, we examined the kinetics of this oxidation. The present study provides substantial insights into the metabolism of cabozantinib and brings novel findings related to cabozantinib pharmacokinetics towards possible utilization in personalized medicine.

• Keywords
• cabozantinib, cytochrome P450, cytochrome b5, tyrosine kinase inhibitor,
• Publication type
• Journal Article MeSH

The metabolism of vandetanib, a tyrosine kinase inhibitor used for treatment of symptomatic/progressive medullary thyroid cancer, was studied using human hepatic microsomes, recombinant cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs). The role of CYPs and FMOs in the microsomal metabolism of vandetanib to N-desmethylvandetanib and vandetanib-N-oxide was investigated by examining the effects of CYP/FMO inhibitors and by correlating CYP-/FMO-catalytic activities in each microsomal sample with the amounts of N-desmethylvandetanib/vandetanib-N-oxide formed by these samples. CYP3A4/FMO-activities significantly correlated with the formation of N-desmethylvandetanib/ vandetanib-N-oxide. Based on these studies, most of the vandetanib metabolism was attributed to N-desmethylvandetanib/vandetanib-N-oxide to CYP3A4/FMO3. Recombinant CYP3A4 was most efficient to form N-desmethylvandetanib, while FMO1/FMO3 generated N-oxide. Cytochrome b5 stimulated the CYP3A4-catalyzed formation of N-desmethylvandetanib, which is of great importance because CYP3A4 is not only most efficient in generating N-desmethylvandetanib, but also most significant due to its high expression in human liver. Molecular modeling indicated that binding of more than one molecule of vandetanib into the CYP3A4-active center can be responsible for the high efficiency of CYP3A4 N-demethylating vandetanib. Indeed, the CYP3A4-mediated reaction exhibits kinetics of positive cooperativity and this corresponded to the in silico model, where two vandetanib molecules were found in CYP3A4-active center.

• Keywords
• cytochromes P450, flavin-containing monoxygenases, metabolism, tyrosine kinase inhibitor, vandetanib,
• MeSH
• Quinazolines chemistry   pharmacology   MeSH
• Cytochrome P-450 CYP3A chemistry   metabolism   MeSH
• Enzymes chemistry   metabolism   MeSH
• Protein Kinase Inhibitors chemistry   pharmacology   MeSH
• Microsomes, Liver metabolism   MeSH
• Rabbits MeSH
• Rats MeSH
• Humans MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Mice MeSH
• Oxidation-Reduction * MeSH
• Piperidines chemistry   pharmacology   MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Recombinant Proteins MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Quinazolines MeSH
• Cytochrome P-450 CYP3A MeSH
• Enzymes MeSH
• Protein Kinase Inhibitors MeSH
• Piperidines MeSH
• Antineoplastic Agents MeSH
• Recombinant Proteins MeSH
• vandetanib MeSH Browser

The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.

• Keywords
• Cytochrome P450, Cytochrome b(5), DNA Adducts, Metabolism, Mouse models,
• MeSH
• DNA Adducts metabolism   MeSH
• Aryl Hydrocarbon Hydroxylases metabolism   MeSH
• Cytochrome P-450 CYP3A MeSH
• Cytochrome-B(5) Reductase deficiency   genetics   MeSH
• Cytochromes b5 deficiency   genetics   MeSH
• Ellipticines metabolism   pharmacology   MeSH
• Phenotype MeSH
• Genotype MeSH
• Hepatocytes enzymology   MeSH
• Microsomes, Liver enzymology   MeSH
• Liver enzymology   MeSH
• Activation, Metabolic MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• NADPH-Ferrihemoprotein Reductase metabolism   MeSH
• Antineoplastic Agents metabolism   pharmacology   MeSH
• Cytochrome P-450 Enzyme System metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Aryl Hydrocarbon Hydroxylases MeSH
• CYP3A protein, mouse MeSH Browser
• Cytochrome P-450 CYP3A MeSH
• Cytochrome-B(5) Reductase MeSH
• Cytochromes b5 MeSH
• Ellipticines MeSH
• ellipticine MeSH Browser
• NADPH-Ferrihemoprotein Reductase MeSH
• Antineoplastic Agents MeSH
• Cytochrome P-450 Enzyme System MeSH

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) can induce cytochrome P450 1A1 (CYP1A1) via a p53-dependent mechanism. The effect of different p53-activating chemotherapeutic drugs on CYP1A1 expression, and the resultant effect on BaP metabolism, was investigated in a panel of isogenic human colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/-) or TP53(-/-) were treated for up to 48 h with 60 μM cisplatin, 50 μM etoposide or 5 μM ellipticine, each of which caused high p53 induction at moderate cytotoxicity (60-80% cell viability). We found that etoposide and ellipticine induced CYP1A1 in TP53(+/+) cells but not in TP53(-/-) cells, demonstrating that the mechanism of CYP1A1 induction is p53-dependent; cisplatin had no such effect. Co-incubation experiments with the drugs and 2.5 μM BaP showed that: (i) etoposide increased CYP1A1 expression in TP53(+/+) cells, and to a lesser extent in TP53(-/-) cells, compared to cells treated with BaP alone; (ii) ellipticine decreased CYP1A1 expression in TP53(+/+) cells in BaP co-incubations; and (iii) cisplatin did not affect BaP-mediated CYP1A1 expression. Further, whereas cisplatin and etoposide had virtually no influence on CYP1A1-catalysed BaP metabolism, ellipticine treatment strongly inhibited BaP bioactivation. Our results indicate that the underlying mechanisms whereby etoposide and ellipticine regulate CYP1A1 expression must be different and may not be linked to p53 activation alone. These results could be relevant for smokers, who are exposed to increased levels of BaP, when prescribing chemotherapeutic drugs. Beside gene-environment interactions, more considerations should be given to potential drug-environment interactions during chemotherapy.

• Keywords
• Benzo[a]pyrene, Cisplatin, Cytochrome P450, Ellipticine, Etoposide, Tumour suppressor p53,
• MeSH
• DNA Adducts metabolism   MeSH
• Benzo(a)pyrene pharmacokinetics   pharmacology   MeSH
• Cisplatin pharmacology   MeSH
• Cytochrome P-450 CYP1A1 biosynthesis   metabolism   MeSH
• Cytochrome P-450 CYP3A biosynthesis   metabolism   MeSH
• Ellipticines pharmacokinetics   pharmacology   MeSH
• Enzyme Induction drug effects   MeSH
• Etoposide pharmacology   MeSH
• Genes, p53 MeSH
• HCT116 Cells MeSH
• Carcinogens pharmacokinetics   pharmacology   MeSH
• Colorectal Neoplasms drug therapy   genetics   metabolism   pathology   MeSH
• Humans MeSH
• Activation, Metabolic MeSH
• Tumor Suppressor Protein p53 deficiency   genetics   metabolism   MeSH
• DNA Damage MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Benzo(a)pyrene MeSH
• Cisplatin MeSH
• CYP1A1 protein, human MeSH Browser
• CYP3A4 protein, human MeSH Browser
• Cytochrome P-450 CYP1A1 MeSH
• Cytochrome P-450 CYP3A MeSH
• Ellipticines MeSH
• ellipticine MeSH Browser
• Etoposide MeSH
• Carcinogens MeSH
• Tumor Suppressor Protein p53 MeSH
• TP53 protein, human MeSH Browser

Neuroblastoma (NBL) originates from undifferentiated cells of the sympathetic nervous system. Chemotherapy is judged to be suitable for successful treatment of this disease. Here, the influence of histone deacetylase (HDAC) inhibitor valproate (VPA) combined with DNA-damaging chemotherapeutic, ellipticine, on UKF-NB-4 and SH-SY5Y neuroblastoma cells was investigated. Treatment of these cells with ellipticine in combination with VPA led to the synergism of their anticancer efficacy. The effect is more pronounced in the UKF-NB-4 cell line, the line with N-myc amplification, than in SH-SY5Y cells. This was associated with caspase-3-dependent induction of apoptosis in UKF-NB-4 cells. The increase in cytotoxicity of ellipticine in UKF-NB-4 by VPA is dictated by the sequence of drug administration; the increased cytotoxicity was seen only after either simultaneous exposure to these drugs or after pretreatment of cells with ellipticine before their treatment with VPA. The synergism of treatment of cells with VPA and ellipticine seems to be connected with increased acetylation of histones H3 and H4. Further, co-treatment of cells with ellipticine and VPA increased the formation of ellipticine-derived DNA adducts, which indicates an easier accessibility of ellipticine to DNA in cells by its co-treatment with VPA and also resulted in higher ellipticine cytotoxicity. The results are promising for in vivo studies and perhaps later for clinical studies of combined treatment of children suffering from high-risk NBL.

• Keywords
• DNA damage, acetylation of histones, apoptosis, ellipticine, neuroblastoma, valproate,
• MeSH
• Apoptosis MeSH
• Ellipticines toxicity   MeSH
• Histone Deacetylase Inhibitors toxicity   MeSH
• Valproic Acid toxicity   MeSH
• Humans MeSH
• Mutagens toxicity   MeSH
• Cell Line, Tumor MeSH
• Neuroblastoma metabolism   MeSH
• Neurons drug effects   metabolism   MeSH
• Drug Synergism MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ellipticines MeSH
• ellipticine MeSH Browser
• Histone Deacetylase Inhibitors MeSH
• Valproic Acid MeSH
• Mutagens MeSH

ABSTRACT: Cytochrome P450 (CYP) 1A1 is the most important enzyme activating and detoxifying the human carcinogen benzo[a]pyrene (BaP). In the previous studies, we had shown that not only the canonic NADPH:CYP oxidoreductase (POR) can act as electron donor but also cytochrome b5 and its reductase, NADH:cytochrome b5 reductase. Here, we studied the role of the expression system used on the metabolites generated and the levels of DNA adducts formed by activated BaP. We used an eukaryotic and a prokaryotic cellular system (Supersomes, microsomes isolated from insect cells, and Bactosomes, a membrane fraction of Escherichia coli, each transfected with cDNA of human CYP1A1 and POR). These were reconstituted with cytochrome b5 with and without NADH:cytochrome b5 reductase. We evaluated the effectiveness of each cofactor, NADPH and NADH, to mediate BaP metabolism. We found that both systems differ in catalysing the reactions activating and detoxifying BaP. Two BaP-derived DNA adducts were generated by the CYP1A1-Supersomes, both in the presence of NADPH and NADH, whereas NADPH but not NADH was able to support this reaction in the CYP1A1-Bactosomes. Seven BaP metabolites were found in Supersomes with NADPH or NADH, whereas NADPH but not NADH was able to generate five BaP metabolites in Bactosomes. Our study demonstrates different catalytic efficiencies of CYP1A1 expressed in prokaryotic and eukaryotic cells in BaP bioactivation indicating some limitations in the use of E. coli cells for such studies.

• Keywords
• Coenzymes, DNA, Enzymes, Membranes, Proteins,
• Publication type
• Journal Article MeSH

ABSTRACT: Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase and/or cytochrome b5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b5 can act as an allosteric modifier of the CYP3A4 oxygenase.

• Keywords
• Coenzymes, DNA, Enzymes, High pressure liquid chromatography,
• Publication type
• Journal Article MeSH

Cytochrome P450 1A2 (P450 1A2, CYP1A2) is a membrane-bound enzyme that oxidizes a broad range of hydrophobic substrates. The structure and dynamics of both the catalytic and trans-membrane (TM) domains of this enzyme in the membrane/water environment were investigated using a multiscale computational approach, including coarse-grained and all-atom molecular dynamics. Starting from the spontaneous self-assembly of the system containing the TM or soluble domain immersed in randomized dilauroyl phosphatidylcholine (DLPC)/water mixture into their respective membrane-bound forms, we reconstituted the membrane-bound structure of the full-length P450 1A2. This structure includes a TM helix that spans the membrane, while being connected to the catalytic domain by a short flexible loop. Furthermore, in this model, the upper part of the TM helix interacts directly with a conserved and highly hydrophobic N-terminal proline-rich segment of the catalytic domain; this segment and the FG loop are immersed in the membrane, whereas the remaining portion of the catalytic domain remains exposed to aqueous solution. The shallow membrane immersion of the catalytic domain induces a depression in the opposite intact layer of the phospholipids. This structural effect may help in stabilizing the position of the TM helix directly beneath the catalytic domain. The partial immersion of the catalytic domain also allows for the enzyme substrates to enter the active site from either aqueous solution or phospholipid environment via several solvent- and membrane-facing tunnels in the full-length P450 1A2. The calculated tunnel dynamics indicated that the opening probability of the membrane-facing tunnels is significantly enhanced when a DLPC molecule spontaneously penetrates into the membrane-facing tunnel 2d. The energetics of the lipid penetration process were assessed by the linear interaction energy (LIE) approximation, and found to be thermodynamically feasible.

• MeSH
• Cytochrome P-450 CYP1A2 chemistry   MeSH
• Phosphatidylcholines MeSH
• Phospholipids chemistry   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Humans MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1,2-dilauroylphosphatidylcholine MeSH Browser
• Cytochrome P-450 CYP1A2 MeSH
• Phosphatidylcholines MeSH
• Phospholipids MeSH

Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by (32)P-postlabeling was characterized as 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP.

• MeSH
• DNA Adducts metabolism   MeSH
• Benzo(a)pyrene toxicity   MeSH
• Cytochrome-B(5) Reductase metabolism   MeSH
• Humans MeSH
• Oxidation-Reduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Benzo(a)pyrene MeSH
• Cytochrome-B(5) Reductase MeSH

Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5 , to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP-7,8-dihydrodiol and BaP-9-ol, which are intermediates in BaP-derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP-3-ol, a metabolite that is a 'detoxified' product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP-7,8-dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP-9-ol. BaP-3-ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active.

• Keywords
• benzo[a]pyrene, cytochrome P450, human liver, metabolism,
• MeSH
• DNA Adducts metabolism   MeSH
• Benzo(a)pyrene metabolism   pharmacokinetics   MeSH
• Microsomes, Liver metabolism   MeSH
• Liver enzymology   metabolism   MeSH
• Rabbits MeSH
• Humans MeSH
• Inactivation, Metabolic MeSH
• Oxidation-Reduction MeSH
• Cytochrome P-450 Enzyme System genetics   metabolism   MeSH
• Chromatography, High Pressure Liquid MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• benzo(a)pyrene-DNA adduct MeSH Browser
• Benzo(a)pyrene MeSH
• Cytochrome P-450 Enzyme System MeSH