CYP7A1 Gene Induction via SHP-Dependent or Independent Mechanisms can Increase the Risk of Drug-Induced Liver Injury Independently or in Synergy with BSEP Inhibition.

Drug-induced liver injury (DILI) is a frequent cause of clinical trial failures during drug development. While inhibiting bile salt export pump (BSEP) is a well-documented DILI mechanism, interference with genes related to bile acid (BA) metabolism and transport can further complicate DILI development. Here, the effects of twenty-eight compounds on genes associated with BA metabolism and transport were evaluated, including those with discontinued development or use, boxed warnings, and clean labels for DILI. The study also included rifampicin and omeprazole, pregnane X receptor and aryl hydrocarbon receptor ligands, and four mitogen-activated protein kinase kinase (MEK1/2) inhibitors. BSEP inhibitors with more severe DILI, notably pazopanib and CP-724714, significantly upregulated the expression of 7 alpha-hydroxylase (CYP7A1), independent of small heterodimer partner (SHP) expression. CYP7A1 expression was marginally induced by omeprazole. In contrast, its expression was suppressed by mometasone (10-fold), vinblastine (18-fold), hexachlorophene (2-fold), bosentan (2.1-fold), and rifampin (2-fold). All four MEK1/2 inhibitors that show clinical DILI were not potent BSEP inhibitors but significantly induced CYP7A1 expression, accompanied by a significant SHP gene suppression. Sulfotransferase 2A1 and BSEP were marginally upregulated, but no other genes were altered by the drugs tested. Protein levels of CYP7A1 were increased with the treatment of CYP7A1 inducers and decreased with obeticholic acid, an farnesoid X receptor ligand. CYP7A1 inducers significantly increased bile acid (BA) production in hepatocytes, indicating the overall regulatory effects of BA metabolism. This study demonstrates that CYP7A1 induction via various mechanisms can pose a risk for DILI, independently or in synergy with BSEP inhibition, and it should be evaluated early in drug discovery. SIGNIFICANCE STATEMENT: Kinase inhibitors, pazopanib and CP-724714, inhibit BSEP and induce CYP7A1 expression independent of small heterodimer partner (SHP) expression, leading to increased bile acid (BA) production and demonstrating clinically elevated drug-induced liver toxicity. MEK1/2 inhibitors that show BSEP-independent drug-induced liver injury (DILI) induced the CYP7A1 gene accompanied by SHP suppression. CYP7A1 induction via SHP-dependent or independent mechanisms can pose a risk for DILI, independently or in synergy with BSEP inhibition. Monitoring BA production in hepatocytes can reliably detect the total effects of BA-related gene regulation for de-risking.

Carbon-carbon Bond Cleavage Catalyzed by Human Cytochrome P450 Enzymes: α-ketol as the Key Intermediate Metabolite in Sequential Metabolism of Olanexidine.

BACKGROUND: Carbon-carbon bond cleavage of a saturated aliphatic moiety is rarely seen in xenobiotic metabolism. Olanexidine (Olanedine®), containing an n-octyl (C8) side chain, was mainly metabolized to various shortened side chain (C4 to C6) acid-containing metabolites in vivo in preclinical species. In liver microsomes and S9, the major metabolites of olanexidine were from multi-oxidation on its n-octyl (C8) side chain. However, the carbon-carbon bond cleavage mechanism of n-octyl (C8) side chain, and enzyme(s) responsible for its metabolism in human remained unknown. METHODS: A pair of regioisomers of α-ketol-containing C8 side chain olanexidine analogs (3,2-ketol olanexidine and 2,3-ketol olanexidine) were synthesized, followed by incubation in human liver microsomes, recombinant human cytochrome P450 enzymes or human hepatocytes, and subsequent metabolite identification using LC/UV/MS. RESULTS: Multiple shortened side chain (C4 to C6) metabolites were identified, including C4, C5 and C6- acid and C6-hydroxyl metabolites. Among 19 cytochrome P450 enzymes tested, CYP2D6, CYP3A4 and CYP3A5 were identified to catalyze carbon-carbon bond cleavage. CONCLUSION: 3,2-ketol olanexidine and 2,3-ketol olanexidine were confirmed as the key intermediates in carbon-carbon bond cleavage. Its mechanism is proposed that a nucleophilic addition of iron-peroxo species, generated by CYP2D6 and CYP3A4/5, to the carbonyl group caused the carbon-carbon bond cleavage between the adjacent hydroxyl and ketone groups. As results, 2,3-ketol olanexidine formed a C6 side chain acid metabolite. While, 3,2-ketol olanexidine formed a C6 side chain aldehyde intermediate, which was either oxidized to a C6 side chain acid metabolite or reduced to a C6 side chain hydroxyl metabolite.

Aminopyridinecarboxamide-based inhaled IKK-2 inhibitors for asthma and COPD: Structure-activity relationship.

Installation of sites for metabolism in the lead compound PHA-767408 was the key focus of the IKK-2 inhaled program. This paper reports our efforts to identify a novel series of aminopyridinecarboxamide-based IKK-2 inhibitors, which display low nanomolar potency against IKK-2 with long duration of action (DOA), and metabolically labile to phase I and/or phase II metabolizing enzymes with potential capability for multiple routes of clearance. Several compounds have demonstrated their potential usefulness in the treatment of asthma and chronic obstructive pulmonary disease (COPD).

Two branched polar groups and polar linker moieties of thiophene amide derivatives are essential for MRP2/ABCC2 recognition.

Previously we demonstrated that the torsion angle between two biphenyl rings forming a three-dimensional conformation is the determinant factor for multi-drug resistance protein 2 (Mrp2/Abcc2) interaction [1]. More recently, we reported a heterocyclic compound, 1-(1-(4-bromophenyl)-3-carbamoyl-1H-pyrazol-4-yl) urea that shares the polar head groups with the biphenyl-substituted heterocycles, is highly secreted from bile by Mrp2/Abcc2, [2]. Collectively we hypothesized that the two branched polar groups and linkers might be essential with proposed Mrp2/Abcc2 recognition fitting in two primarily positive regions deep in the binding site. To test the hypothesis, a discovery lead compound (Compound 1) was examined to confirm the Mrp2/Abcc2 involvement resulting in hepatobiliary secretion in rats. The structural requirement of Mrp2/Abcc2 recognition was further explored in a series of thiophene amides derivatives divided into eight structural classes, with structural changes focused on the amide linker orientation or substitution from amide and sulfonamide to alkene, alkane, or alkyne linkers. In Caco-2 cell bidirectional transport assays and Mrp2/Abcc2 membrane vesicle uptake assays, the involvement of Mrp2/Abcc2 mediated transport was confirmed in structural classes 1 - 5, which contains polar amide or sulfonamide linker, but not in classes 6 - 8 with non-polar aliphatic linker. The Mrp2/Abcc2 recognition showed strong correlation with structural descriptors in predictive Bayesian model, as well as with polar surface area and lipophilicity (LogP). The result provided valuable information for predicting transporter recognition in silico, for improved predictions of transporter involved ADME in early drug discovery.

Evaluating the suitability of using rat models for preclinical efficacy and side effects with inhaled corticosteroids nanosuspension formulations.

Inhaled corticosteroids (ICS) are often prescribed as first-line therapy for patients with asthma Despite their efficacy and improved safety profile compared with oral corticosteroids, the potential for systemic side effects continues to cause concern. In order to reduce the potential for systemic side effects, the pharmaceutical industry has begun efforts to generate new drugs with pulmonary-targeted topical efficacy. One of the major challenges of this approach is to differentiate both efficacy and side effects (pulmonary vs. systemic) in a preclinical animal model. In this study, fluticasone and ciclesonide were used as tool compounds to explore the possibility of demonstrating both efficacy and side effects in a rat model using pulmonary delivery via intratracheal (IT) instillation with nanosuspension formulations. The inhibition of neutrophil infiltration into bronchoalveolar lavage fluid (BALF) and cytokine (TNFα) production were utilized to assess pulmonary efficacy, while adrenal and thymus involution as well as plasma corticosterone suppression was measured to assess systemic side effects. Based on neutrophil infiltration and cytokine production data, the ED50s for ciclesonide and fluticasone were calculated to be 0.1 and 0.03 mg, respectively. At the ED50, the average adrenal involution was 7.6 ± 5.3% for ciclesonide versus 16.6 ± 5.1% for fluticasone, while the average thymus involution was 41.0 ± 4.3% for ciclesonide versus 59.5 ± 5.8% for fluticasone. However, the differentiation became less significant when the dose was pushed to the EDmax (0.3 mg for ciclesonide, 0.1 mg for fluticasone). Overall, the efficacy and side effect profiles of the two compounds exhibited differentiation at low to mid doses (0.03-0.1 mg ciclesonide, 0.01-0.03 mg fluticasone), while this differentiation diminished at the maximum efficacious dose (0.3 mg ciclesonide, 0.1 mg fluticasone), likely due to overdosing in this model. We conclude that the rat LPS model using IT administration of nanosuspensions of ICS is a useful tool to demonstrate pulmonary-targeted efficacy and to differentiate the side effects. However, it is only suitable at sub-maximum efficacious levels.

Novel metabolic bioactivation mechanism for a series of anti-inflammatory agents (2,5-diaminothiophene derivatives) mediated by cytochrome p450 enzymes.

The thiophene moiety is considered a structural alert in molecular design in drug discovery, largely because several thiophene-containing drugs, including tienilic acid and suprofen, have been withdrawn from the market because of toxicities. Reactive thiophene intermediates, activated via sulfur oxidation or ring epoxidation, are possible culprits for these adverse side effects. In this work, the metabolic activation of an anti-inflammatory agent, 1-(3-carbamoyl-5-(2,3,5-trichlorobenzamido)thiophen-2-yl)urea), containing a 2,5-diaminothiophene structure, was studied in liver microsomes in the presence of glutathione or N-acetylcysteine as trapping agents. In addition, the glutathione conjugate was detected in bile from a bile duct-cannulated rat study. The structure of the glutathione conjugate was identified by mass spectrometry and (1)H NMR. The glutathione molecule was attached to the thiophene ring, replacing the existing proton. Metabolic phenotyping experiments, using chemical inhibitors or recombinant cytochromes P450 (P450), demonstrated that CYP3A4 was the major P450 enzyme responsible for the metabolic activation, followed by CYP1A2, 2Cs, and 2D6. A novel metabolic activation mechanism is proposed whereby the 2,5-diaminothiophene moiety undergoes oxidation to a 2,5-diimine thiophene reactive intermediate. This mechanism was used to support efforts to eliminate reactive metabolite generation via structural modification of ring substituents using structure-activity relationships. The disruption of formation of the 2,5-diimine reactive intermediate resulted in the elimination of glutathione conjugate formation both in vitro and in vivo and provided a rational approach to mitigating potential safety risks associated with this class of thiophenes in drug research and development.

Euthanasia method for mice in rapid time-course pulmonary pharmacokinetic studies.

To develop a means of euthanasia to support rapid time-course pharmacokinetic studies in mice, we compared retroorbital and intravenous lateral tail vein injection of ketamine-xylazine with regard to preparation time, utility, tissue distribution, and time to onset of euthanasia. Tissue distribution and time to onset of euthanasia did not differ between administration methods. However, retroorbital injection could be performed more rapidly than intravenous injection and was considered to be a technically simple and superior alternative for mouse euthanasia. Retroorbital ketamine-xylazine, CO(2) gas, and intraperitoneal pentobarbital then were compared as euthanasia agents in a rapid time-point pharmacokinetic study. Retroorbital ketamine-xylazine was the most efficient and consistent of the 3 methods, with an average time to death of approximately 5 s after injection. In addition, euthanasia by retroorbital ketamine-xylazine enabled accurate sample collection at closely spaced time points and satisfied established criteria for acceptable euthanasia technique.

Advancement of structure-activity relationship of multidrug resistance-associated protein 2 interactions.

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is mainly expressed in the apical phase of barrier membranes. It functions as a critical efflux pump in the biliary excretion of endogenous substances, such as conjugated bilirubin and bile salts, as well as many structurally diverse xenobiotics and their metabolites. Due to its important role in defining ADME/Tox properties, efforts have emerged to build the structure-activity relationship (SAR) for MRP2/ABCC2 at early stages of drug discovery process. MRP2/ABCC2 is a member of the integral membrane protein family whose high-resolution crystal structure has not been described. To overcome the obstacle of lacking detailed structural depiction, various molecular modeling approaches have been applied to derive the structural requirements for binding interactions with MRP2/ABCC2 protein, including two-dimensional (2D) and three-dimensional (3D) quantitative SAR (QSAR) analysis, pharmacophore models, and homology modeling of the transporter. Here we summarize recent progresses in understanding the SAR of MRP2/ABCC2 recognition of substrates and/or inhibitors, and describe some of the useful in vitro tools for characterizing the interactions with the transporter.

Novel tight-binding inhibitory factor-kappaB kinase (IKK-2) inhibitors demonstrate target-specific anti-inflammatory activities in cellular assays and following oral and local delivery in an in vivo model of airway inflammation.

Nuclear factor-kappaB (NF-kappaB) is one of the major families of transcription factors activated during the inflammatory response in asthma and chronic obstructive pulmonary disease. Inhibitory factor-kappaB kinase 2 (IKK-2) has been shown to play a pivotal role in cytokine-induced NF-kappaB activation in airway epithelium and in disease-relevant cells. Nevertheless, the potential toxicity of specific IKK-2 inhibitors may be unacceptable for oral delivery in chronic obstructive pulmonary disease. Therefore, local delivery to the lungs is an attractive alternative that warrants further exploration. Here, we describe potent and selective small-molecule IKK-2 inhibitors [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (PHA-408) and 8-(2-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-5-chloroisonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (PF-184)] that are competitive for ATP have slow off-rates from IKK-2 and display broad in vitro anti-inflammatory activities resulting from NF-kappaB pathway inhibition. Notably, PF-184 has been designed to have high systemic clearance, which limits systemic exposure and maximizes the effects locally in the airways. We used an inhaled lipopolysaccharide-induced rat model of neutrophilia to address whether inhibiting NF-kappaB activation locally within the airways would show anti-inflammatory effects in the absence of systemic exposure. PHA-408, a low-clearance compound previously shown to be efficacious orally in a rodent model of arthritis, dose-dependently attenuated inhaled lipopolysaccharide-induced cell infiltration and cytokine production. Interestingly, PF-184 produced comparable dose-dependent anti-inflammatory activity by intratracheal administration and was as efficacious as intratracheally administered fluticasone propionate (fluticasone). Together, these results support the potential therapeutic utility of IKK-2 inhibition in inflammatory pulmonary diseases and demonstrate anti-inflammatory efficacy of an inhaled IKK-2 inhibitor in a rat airway model of neutrophilia.

Simultaneous determination of LogD, LogP, and pK(a) of drugs by using a reverse phase HPLC coupled with a 96-well plate auto injector.

For years, the physicochemical properties of drug candidates have been used to predict their in vivo pharmacokinetic behaviors. Several theories and empirical correlations have been established by various researchers with the overall goal of expediting the drug candidate selection process, with greater confidence and faster turnaround. This study describes a 96-well reverse phase HPLC method, simultaneously determining LogD, LogP, and pK(a) values of drugs in a throughput mode. The LogD and LogP values of each compound were determined, based on the octanol-aqueous partitioning behavior of the charged and non-charged species under different pH values. The pK(a) value was determined by using the Polynomial fit between LogP and LogD and the equation LogD (pK(a)) approximately LogP-0.301. The advantages of this method are: low sample consumption, suitability for low solubility compounds, less restriction on compound purity, potential for higher throughput, precise data, and multiple determinations in one assay.

Pharmacokinetic and pharmacodynamic evaluation of the suitability of using fluticasone and an acute rat lung inflammation model to differentiate lung versus systemic efficacy.

Inhaled corticosteroids (ICSs) are often prescribed as the first line therapy for pulmonary diseases such as asthma. The biggest concern of using steroid therapy is the systemic side effects at high dose. To reduce the side effects, the pharmaceutical industry has been putting effort to generate new drugs with maximized topical efficacy. One of the key challenges is to differentiate efficacy from local versus systemic contribution in preclinical animal models. Fluticasone with various formulations was used as a model compound to explore the possibilities to demonstrate lung targeted efficacy by intratracheally instillation in the lipopolysaccharide induced inflammation rat model. Fluticasone formulations contained various surfactant concentrations and particle sizes to achieve lung retention and lower systemic exposure. Neutrophil infiltration in broncoalveolar lavage fluid and cytokine production in whole blood were measured to assess pulmonary efficacy versus systemic efficacy. PK/PD characterization of fluticasone with various formulations in the rat inflammation model provided an integrated approach in preclinical to evaluate lung targeted efficacy for ICS. Our study concluded that the combination of the rat LPS model and fluticasone is not suitable to use for establishing potency and dose requirement for new drug candidate designed for topical only efficacy.

A novel, highly selective, tight binding IkappaB kinase-2 (IKK-2) inhibitor: a tool to correlate IKK-2 activity to the fate and functions of the components of the nuclear factor-kappaB pathway in arthritis-relevant cells and animal models.

Nuclear factor (NF)-kappaB activation has been clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The central role that IkappaB kinase-2 (IKK-2) plays in regulating NF-kappaB signaling in response to inflammatory stimuli has made this enzyme an attractive target for therapeutic intervention. Although diverse chemical classes of IKK-2 inhibitors have been identified, the binding kinetics of these inhibitors has limited the scope of their applications. In addition, safety assessments of IKK-2 inhibitors based on a comprehensive understanding of the pharmacokinetic/pharmacodynamic relationships have yet to be reported. Here, we describe a novel, potent, and highly selective IKK-2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide]. PHA-408 is an ATP-competitive inhibitor, which binds IKK-2 tightly with a relatively slow off rate. In arthritis-relevant cells and animal models, PHA-408 suppresses inflammation-induced cellular events, including IkappaBalpha phosphorylation and degradation, p65 phosphorylation and DNA binding activity, the expression of inflammatory mediators, and joint pathology. PHA-408 was efficacious in a chronic model of arthritis with no adverse effects at maximally efficacious doses. Stemming from its ability to bind tightly to IKK-2, as a novelty, we demonstrated that PHA-408-mediated inhibition of IKK-2 activity correlated very well with its ability to modulate the fate of IKK-2 substrates and downstream transcriptional events. We ultimately directly linked IKK-2 activity ex vivo and in vivo to markers of inflammation with the inhibitor plasma concentrations. Thus, PHA-408 represents a powerful tool to further gain insight into the mechanisms by which IKK-2 regulates NF-kappaB signaling and validates IKK-2 as a therapeutic target.

Saturation of multidrug-resistant protein 2 (mrp2/abcc2)-mediated hepatobiliary secretion: nonlinear pharmacokinetics of a heterocyclic compound in rats after intravenous bolus administration.

Multidrug-resistant protein 2 (MRP2/ABCC2), expressed on the canalicular membrane of hepatocytes, mediates the secretion of conjugated or nonconjugated compounds into bile and plays an important role in physiology and drug elimination. A heterocyclic compound, BPCPU [1-(1-(4-bromophenyl)-3-carbamoyl-1H-pyrazol-4-yl) urea], which was metabolically stable in vitro in rat liver microsomes and freshly isolated rat hepatocytes, demonstrated a saturable nonlinear pharmacokinetic profile in the rat. Polarized efflux was observed for this compound in Caco-2 cells, with a low K(m) = 1.06 +/- 0.06 microM. The Caco-2 efflux was dose-dependent and saturable. Coadministration of 25 microM MK571 ([3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]), an MRP inhibitor, blocked the polarized efflux in Caco-2 cells. In contrast, this compound did not inhibit calcein efflux in MRP2 gene-transfected Madin-Darby canine kidney cells, suggesting that it is a substrate, not an inhibitor, of the MRP2/ABCC2 transporter. To investigate the mechanism for the nonlinear pharmacokinetics, bile duct-cannulated rats were used to obtain time profiles of plasma concentration, biliary, and urinary excretion after intravenous administration at various doses. The plasma clearance increased remarkably with decreased dose, from 1.5 ml/min/kg at 5 mg/kg to 14.9 ml/min/kg at 0.05 mg/kg. A dose-dependent biliary excretion also was observed. The results revealed that saturation of hepatobiliary secretion played a role in the dose-dependent changes in total body clearance and biliary clearance. Saturating concentrations of the Mrp2/Abcc2 substrate, BPCPU, causing decreased hepatobiliary clearance could be the major cause for the nonlinear pharmacokinetics observed in rats.

Evaluation of Aerosol Delivery of Nanosuspension for Pre-clinical Pulmonary Drug Delivery.

Asthma and chronic obstructive pulmonary disease (COPD) are pulmonary diseases that are characterized by inflammatory cell infiltration, cytokine production, and airway hyper-reactivity. Most of the effector cells responsible for these pathologies reside in the lungs. One of the most direct ways to deliver drugs to the target cells is via the trachea. In a pre-clinical setting, this can be achieved via intratracheal (IT), intranasal (IN), or aerosol delivery in the desired animal model. In this study, we pioneered the aerosol delivery of a nanosuspension formulation in a rodent model. The efficiency of different dosing techniques and formulations to target the lungs were compared, and fluticasone was used as the model compound. For the aerosol particle size determination, a ten-stage cascade impactor was used. The mass median aerodynamic diameter (MMAD) was calculated based on the percent cumulative accumulation at each stage. Formulations with different particle size of fluticasone were made for evaluation. The compatibility of regular fluticasone suspension and nanosuspension for aerosol delivery was also investigated. The in vivo studies were conducted on mice with optimized setting. It was found that the aerosol delivery of fluticasone with nanosuspension was as efficient as intranasal (IN) dosing, and was able to achieve dose dependent lung deposition.

Structure-activity relationships for interaction with multidrug resistance protein 2 (ABCC2/MRP2): the role of torsion angle for a series of biphenyl-substituted heterocycles.

Multidrug resistance protein 2 (ABCC2/MRP2) is an ATP-binding cassette transporter involved in the absorption, distribution, and excretion of drugs and xenobiotics. Identifying compounds that are ABCC2/MRP2 substrates and/or inhibitors and understanding their structure-activity relationships (SARs) are important considerations in the selection and optimization of drug candidates. In the present study, the interactions between ABCC2/MRP2 and a series of biphenyl-substituted heterocycles were evaluated using Caco-2 cells and human ABCC2/MRP2 gene-transfected Madin-Darby canine kidney cells. It was observed that ABCC2/MRP2 transport and/or inhibition profile, both in nature and in magnitude, depends strongly on the substitution patterns of the biphenyl system. In particular, different ortho-substitutions cause various degrees of twisting between the two-phenyl rings, resulting in changing interactions between the ligands and ABCC2/MRP2. The compounds with small ortho functions (hydrogen, fluorine, and oxygen) and, thus, the ones displaying the smallest torsion angles of biphenyl (37-45 degrees) are neither substrates nor inhibitors of human ABCC2/MRP2. The transporter interactions increase as the steric bulkiness of the ortho-substitutions increase. When the tested compounds are 2-methyl substituted biphenyls, they exhibit moderate torsion angles (54-65 degrees) and behave as ABCC2/MRP2 substrates as well as mild inhibitors [10-40% compared with 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethyl-sulfanyl)methylsulfanyl] propionic acid (MK571)]. For the 2,2'-dimethyl substituted biphenyls, the torsions are enhanced (78-87 degrees) and so is the inhibition of ABCC2/MRP2. This class of compounds behaves as strong inhibitors of ABCC2/MRP2. These results can be used to define the three-dimensional structural requirements of ABCC2/MRP2 interaction with their substrates and inhibitors, as well as to provide SAR guidance to support drug discovery.

Further studies on the biosynthesis of the manumycin-type antibiotic, asukamycin, and the chemical synthesis of protoasukamycin.

Asukamycin (2), a metabolite of Streptomyces nodosus ssp. asukaensis ATCC 29757 and a member of the manumycin family of antibiotics, is assembled from three components, an "upper" polyketide chain initiated by cyclohexanecarboxylic acid, a "lower" polyketide chain initiated by the novel starter unit, 3-amino-4-hydroxybenzoic acid (3,4-AHBA), and a cyclized 5-aminolevulinic acid moiety, 2-amino-3-hydroxycyclopent-2-enone (C(5)N unit). To shed light on the order in which these components are assembled, we synthesized in labeled form various potential intermediates and evaluated their incorporation into 2. The assembly of the molecular framework of 2 from 3,4-AHBA and cyclohexanecarboxylic acid apparently does not involve free, unactivated intermediates. However, protoasukamycin (12), the total synthesis of which is reported, was efficiently converted into 2, demonstrating that the modification of the aromatic ring to the epoxyquinol structure is the terminal step in the biosynthesis. The results suggest that the two polyketide chains are synthesized separately and that the "upper" chain must be connected to the "lower" polyketide chain before the C(5)N unit.

Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450s): major differences in selective enantiomer formation by various P450 isoforms.

Methoxychlor, a currently used pesticide that in mammals elicits proestrogenic/estrogenic activity and reproductive toxicity, has been classified as a prototype endocrine disruptor. Methoxychlor is prochiral, and its metabolites 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M); 1,1,1-trichloro- 2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M); and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M) are chiral; whereas 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M) is achiral. These metabolites are formed during methoxychlor incubation with liver microsomes or recombinant cytochrome p450s (rp450s). Since methoxychlor-metabolite enantiomers may have different estrogenic/antiestrogenic/antiandrogenic activities than corresponding racemates, the possibility that p450s preferentially generate or use R or S enantiomers, was examined. Indeed, rCYP1A2 and r2A6 mono-demethylated methoxychlor primarily into (R)-mono-OH-M at 91 and 75%, respectively, whereas rCYP1A1, 2B6, 2C8, 2C9, 2C19, and 2D6 formed the (S)-enantiomer at 69, 66, 75, 95, 96, and 80%, respectively. However, rCYP3A4, 3A5, and 2B1(rat) weakly demethylated methoxychlor without enantioselectivity. Human liver microsomes generated (S)-mono-OH-M (77-87%), suggesting that CYP1A2 and 2A6 display only minor catalytic contribution. P450 inhibitors demonstrated that CYP2C9 and possibly 2C19 are major hepatic catalysts forming (S)-mono-OH-M, and CYP1A2 is primarily involved in forming the (R)-mono-OH-M. Demethylation rate of (S)-mono-OH-M versus (R)-mono-OH-M forming achiral bis-OH-M by rCYP1A2 was 97/3, compared with 15/85 and 17/83 for rCYP2C9 and 2C19, respectively, indicating opposite substrate enantioselectivity of rCYP1A2 versus 2C9 and 2C19. Also, rCYP1A2 preferentially O-demethylated (R)-catechol-M into (R)-tris-OH-M (at 80%), contrasting r2C9 and r2C19 that yielded (S)-tris-OH-M at 80 and 77%, respectively. Ortho-hydroxylation of mono-OH-M into catechol-M and bis-OH-M into tris-OH-M was primarily by 3A4 and was not enantioselective. In conclusion, enantiomeric abundance of methoxychlor metabolites depends on the relative catalytic activity of the hepatic p450 isoforms.

Metabolism of the endocrine disruptor pesticide-methoxychlor by human P450s: pathways involving a novel catechol metabolite.

The metabolism of methoxychlor, a proestrogenic pesticide (endocrine disruptor), was investigated with cDNA expressed human cytochrome P450s and liver microsomes (HLM). In addition to 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M), 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M), and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M), a new metabolite was identified as 1,1,1-trichloro-2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M; previously assumed to be ring-OH-M) and as a key metabolic intermediate. A novel metabolic route was proposed involving methoxychlor O-demethylation to mono-OH-M, followed by bifurcation of the pathway, both leading to the same final product tris-OH-M: pathway a, mono-OH-M is demethylated to bis-OH-M, followed by ortho-hydroxylation forming tris-OH-M and pathway b, mono-OH-M is ortho-hydroxylated forming catechol-M that is O-demethylated forming tris-OH-M. Among the human cDNA-expressed P450s examined, CYP1A2, 2A6, 2C8, 2C9, 2C19, and 2D6 exhibited mainly O-demethylation, with CYP2C19 being the most catalytically competent. CYP3A4, 3A5, and rat 2B1 catalyzed primarily ortho-hydroxylation of mono-OH-M (CYP3A4 being catalytically the most active) but were weak in O-demethylation. CYP1A1, 1B1, 2E1, and 4A11 demonstrated little or no catalytic activity. CYP2B6 appeared unique, catalyzing effectively both O-demethylation and ortho-hydroxylation. Thus, CYP2B6 demethylated methoxychlor to mono-OH-M and ortho-hydroxylated the mono-OH-M forming catechol-M; however, 2B6 did not appreciably demethylate mono-OH-M or ortho-hydroxylate bis-OH-M, suggesting a narrow substrate specificity. CYP2C19-catalyzed demethylation of methoxychlor, mono-OH-M and catechol-M, demonstrating relatively good substrate affinity (K(m) = 0.23 - 0.41 microM). However, the 3A4 ortho-hydroxylation of mono-OH-M and bis-OH-M exhibited lower affinity, K(m) = 12 and 25 microM, respectively. Thus, a phenolic group seems essential for efficient ortho-hydroxylation, forming catechol-M and tris-OH-M. Inhibition studies with HLM and P450s indicate that CYP2C9 and likely 2C19 are catalysts of methoxychlor-mono-demethylation.