Chad D. Moore

Metabolic Pathways of Inhaled Glucocorticoids by the CYP3A Enzymes

Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and ∆6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of ∆6-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography–mass spectrometry and NMR analysis.

Dehydrogenation of the Indoline-Containing Drug 4-Chloro-N-(2-methyl-1-indolinyl)-3-sulfamoylbenzamide (Indapamide) by CYP3A4: Correlation with in Silico Predictions

4-Chloro-N-(2-methyl-1-indolinyl)-3-sulfamoylbenzamide (indapamide), an indoline-containing diuretic drug, has recently been evaluated in a large Phase III clinical trial (ADVANCE) with a fixed-dose combination of an angiotensin-converting enzyme inhibitor, perindopril, and shown to significantly reduce the risks of major vascular toxicities in people with type 2 diabetes. The original metabolic studies of indapamide reported that the indoline functional group was aromatized to indole through a dehydrogenation pathway by cytochromes P450. However, the enzymatic efficiency of indapamide dehydrogenation was not elucidated. A consequence of indoline aromatization is that the product indoles might have dramatically different therapeutic potencies. Thus, studies that characterize dehydrogenation of the functional indoline of indapamide were needed. Here we identified several indapamide metabolic pathways in vitro with human liver microsomes and recombinant CYP3A4 that include the dehydrogenation of indapamide to its corresponding indole form, and also hydroxylation and epoxidation metabolites, as characterized by liquid chromatography/mass spectrometry. Indapamide dehydrogenation efficiency (Vmax/Km = 204 min/mM) by CYP3A4 was approximately 10-fold greater than that of indoline dehydrogenation. In silico molecular docking of indapamide into two CYP3A4 crystal structures, to evaluate the active site parameters that control dehydrogenation, produced conflicting results about the interactions of Arg212 with indapamide in the active site. These conflicting theories were addressed by functional studies with a CYP3A4R212A mutant enzyme, which showed that Arg212 does not seem to facilitate positioning of indapamide for dehydrogenation. However, the metabolites of indapamide were precisely consistent with in silico predictions of binding orientations using three diverse computer methods to predict drug metabolism pathways.

IMPROVED CYTOCHROME P450 3A4 MOLECULAR MODELS ACCURATELY PREDICT THE PHE215 REQUIREMENT FOR RALOXIFENE DEHYDROGENATION SELECTIVITY

The use of molecular modeling in conjunction with site-directed mutagenesis has been extensively used to study substrate orientation within cytochrome P450 active sites and to identify potential residues involved in the positioning and catalytic mechanisms of these substrates. However, because docking studies utilize static models to simulate dynamic P450 enzymes, the effectiveness of these studies is strongly dependent on accurate enzyme models. This study employed a cytochrome P450 3A4 (CYP3A4) crystal structure (Protein Data Bank entry 1W0E) to predict the sites of metabolism of the known CYP3A4 substrate raloxifene. In addition, partial charges were incorporated into the P450 heme moiety to investigate the effect of the modified CYP3A4 model on metabolite prediction with the ligand docking program Autodock. Dehydrogenation of raloxifene to an electrophilic diquinone methide intermediate has been linked to the potent inactivation of CYP3A4. Active site residues involved in the positioning and/or catalysis of raloxifene supporting dehydrogenation were identified with the two models, and site-directed mutagenesis studies were conducted to validate the models. The addition of partial charges to the heme moiety improved the accuracy of the docking studies, increasing the number of conformations predicting dehydrogenation and facilitating the identification of substrate-active site residue interactions. On the basis of the improved model, the Phe215 residue was hypothesized to play an important role in orienting raloxifene for dehydrogenation through a combination of electrostatic and steric interactions. Substitution of this residue with glycine or glutamine significantly decreased dehydrogenation rates without concurrent changes in the rates of raloxifene oxygenation. Thus, the improved structural model predicted novel enzyme-substrate interactions that control the selective dehydrogenation of raloxifene to its protein-binding intermediate.

3-Methylindole is Mutagenic and a Possible Pulmonary Carcinogen

Previous work has shown that bioactivation of the cigarette smoke pneumotoxicant 3-methylindole (3MI) by pulmonary cytochrome P450 enzymes is directly associated with formation of DNA adducts. Here, we present evidence that normal human lung epithelial cells, exposed to low micromolar concentrations of 3MI, showed extensive DNA damage, as measured by the comet assay, with similar potency to the prototypical genotoxic agents, doxorubicin and irinotecan. The DNA damage caused by 3MI was predominantly caused by single-strand breaks. Furthermore, we show that this damage decreased with time, given a subtoxic concentration, with detectable DNA fragmentation peaking 4 h after exposure and diminishing to untreated levels within 24 h. Pretreatment with an inhibitor of poly(ADP-ribose) polymerase 1 (PARP1), NU1025, nearly doubled the DNA damage produced by 5 microM 3MI, implying that PARP1, which among other activities, functions to repair single-strand breaks in DNA, repaired at least some of the 3MI-induced DNA fragmentation. A key cellular response to DNA damage, phosphorylation, and nuclear localization of p53 was seen at subtoxic levels of 3MI exposure. 3MI was highly mutagenic, with essentially the same potency as the prototype carcinogen, benzo[a]pyrene, only when a lung-expressed CYP2F3 enzyme was used to dehydrogenate 3MI to its putative DNA-alkylating intermediate. Conversely, a rat liver S9 metabolic system did not bioactivate 3MI to its mutagenic intermediate(s). Concentrations higher than 25 microM caused apoptosis, which became extensive at 100 microM, similar to the response seen with 10 microM doxorubicin. Our findings indicate that there is a low concentration window in which 3MI can cause extensive DNA damage and mutation, without triggering apoptotic defenses, reinforcing the hypothesis that inhaled 3MI from cigarette smoke may be a potent lung-selective carcinogen.

Metabolism of Beclomethasone Dipropionate by Cytochrome P450 3A Enzymes

Inhaled glucocorticoids, such as beclomethasone dipropionate (BDP), are the mainstay treatment of asthma. However, ∼30% of patients exhibit little to no benefit from treatment. It has been postulated that glucocorticoid resistance, or insensitivity, is attributable to individual differences in glucocorticoid receptor-mediated processes. It is possible that variations in cytochrome P450 3A enzyme-mediated metabolism of BDP may contribute to this phenomenon. This hypothesis was explored by evaluating the contributions of CYP3A4, 3A5, 3A7, and esterase enzymes in the metabolism of BDP in vitro and relating metabolism to changes in CYP3A enzyme mRNA expression via the glucocorticoid receptor in lung and liver cells. CYP3A4 and CYP3A5 metabolized BDP via hydroxylation ([M4] and [M6]) and dehydrogenation ([M5]) at similar rates; CYP3A7 did not metabolize BDP. A new metabolite [M6], formed by the combined action of esterases and CYP3A4 hydroxylation, was also characterized. To validate the results observed using microsomes and recombinant enzymes, studies were also conducted using A549 lung and DPX2 liver cells. Both liver and lung cells produced esterase-dependent metabolites [M1–M3], with [M1] correlating with CYP3A5 mRNA induction in A549 cells. Liver cells produced both hydroxylated and dehydrogenated metabolites [M4, M5, and M6], but lung cells produced only the dehydrogenated metabolite [M5]. These studies show that CYP3A4 and CYP3A5 metabolize BDP to inactive metabolites and suggest that differences in the expression or function of these enzymes in the lung and/or liver could influence BDP disposition in humans.

Respective Roles of CYP2A5 and CYP2F2 in the Bioactivation of 3-Methylindole in Mouse Olfactory Mucosa and Lung: Studies Using Cyp2a5-Null and Cyp2f2-Null Mouse Models

The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice. In lung reactions, GS-A1, GS-A2, and HMI were detected as major products, and I-3-C and MOI, as minor metabolites. In OM reactions, all five metabolites were detected in ample amounts. The loss of CYP2F2 affected formation of all 3MI metabolites in the lung and formation of HMI, GS-A1, and GS-A2 in the OM. In contrast, loss of CYP2A5 did not affect formation of 3MI metabolites in the lung but caused substantial decreases in I-3-C and MOI formation in the OM. Thus, whereas CYP2F2 plays a critical role in the 3MI metabolism in the lung, both CYP2A5 and CYP2F2 play important roles in 3MI metabolism in the OM. Furthermore, the fate of the reactive metabolites produced by the two enzymes through common dehydrogenation and epoxidation pathways seemed to differ with CYP2A5 supporting direct conversion to stable metabolites and CYP2F2 supporting further formation of reactive iminium ions. These results provide the basis for understanding the respective roles of CYP2A5 and CYP2F2 in 3MIs toxicity in the respiratory tract.

Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli

Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E. coli at yields greater than 250 nmol/L culture. We hypothesized that a limited number of sequence differences between P450s 2F1 and 2F3 could limit P450 2F1 expression in E. coli and that problematic P450 2F1 sequence elements could be identified by directed evolution. A library of P450 2F1/2F3 mutants was created by DNA family shuffling and screened for expression in E. coli. Three generations of DNA shuffling revealed a mutant (named JH_2F_F3_1_007) with 96.5% nucleotide sequence identity to P450 2F1 and which expressed 119 ± 40 pmol (n = 3, mean ± SD) hemoprotein in 1 mL microaerobic cultures. Across all three generations, two regions were observed where P450 2F3-derived sequence was consistently substituted for P450 2F1 sequence in expressing mutants, encoding nine amino acid differences between P450s 2F1 and 2F3: nucleotides 191-278 (amino acids 65-92) and 794-924 (amino acids 265-305). Chimeras constructed to specifically test the importance of these two regions confirmed that P450 2F3 sequence is essential in both regions for expression in E. coli but that other non-P450 2F1 sequence elements outside of these regions also improved the expression of mutant JH_2F_F3_1_007. Mutant JH_2F_F3_1_007 catalyzed the dehydrogenation of 3MI to 3-MEI as indicated by the observation of glutathione adducts after incubation in the presence of glutathione. The JH_2F_F3_1_007 protein differs from P450 2F1 at only 20 amino acids and should facilitate further studies of the structure-activity relationships of P450s of the 2F subfamily.

Regulation of CYP3A genes by glucocorticoids in human lung cells.

Inhaled glucocorticoids are the first-line treatment for patients with persistent asthma. However, approximately thirty percent of patients exhibit glucocorticoid insensitivity, which may involve excess metabolic clearance of the glucocorticoids by CYP3A enzymes in the lung. CYP3A4, 3A5, and 3A7 enzymes metabolize glucocorticoids, which in turn induce CYP3A genes. However, the mechanism of CYP3A5 mRNA regulation by glucocorticoids in lung cells has not been determined. In hepatocytes, glucocorticoids bind to the glucocorticoid receptor (GR), which induces the expression of the constitutive androstane receptor or pregnane X receptor; both of which bind to the retinoid X receptor alpha, leading to the induction of CYP3A4, 3A5, and 3A7. There is also evidence to suggest a direct induction of CYP3A5 by GR activation in liver cells. In this study, these pathways were evaluated as the mechanism for CYP3A5 mRNA induction by glucocorticoids in freshly isolated primary tracheal epithelial, adenocarcinomic human alveolar basal epithelial (A549), immortalized bronchial epithelial (BEAS-2B), primary normal human bronchial/tracheal epithelial (NHBE), primary small airway epithelial (SAEC), and primary lobar epithelial lung cells. In A549 cells, beclomethasone 17-monopropionate ([M1]) induced CYP3A5 mRNA through the glucocorticoid receptor. CYP3A5 mRNA induction by five different glucocorticoids was attenuated by inhibiting the glucocorticoid receptor using ketoconazole, and for beclomethasone dipropionate, using siRNA-mediated knock-down of the glucocorticoid receptor. The constitutive androstane receptor was not expressed in lung cells. SAEC cells, a primary lung cell line, expressed CYP3A5, but CYP3A5 mRNA was not induced by glucocorticoid treatment despite evaluating a multitude of cell culture conditions. None of the other lung cells expressed CYP3A4, 3A5 or 3A7 mRNA. These studies demonstrate that CYP3A5 mRNA is induced by glucocorticoids in A549 cells via the glucocorticoid receptor, but that additional undefined regulatory processes exist in primary lung cells.