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The Metabolism of Pyrazoloacridine (NSC 366140) by Cytochromes P450 and Flavin Monooxygenase in Human Liver Microsomes

¹ Department of Oncology and ² Mass Spectrometry Facility, Mayo Clinic, Rochester, Minnesota, and ³ Wayne State University School of Medicine, Detroit, Michigan

	ABSTRACT

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Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary electrophoresis coupled with electrospray ionization mass spectrometry, we have identified three oxidative PZA metabolites, 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. The cytochrome P450 (CYP) isoforms involved in PZA metabolism were characterized by studies with CYP chemical inhibitors, correlation of marker activities for selected CYPs with formation of the metabolites using a human liver panel, and PZA metabolism by cDNA-expressed CYPs. 9-Desmethyl-PZA formation was catalyzed by CYP1A2, whereas N-demethyl-PZA formation was catalyzed by CYP3A4. PZA N-oxide formation was catalyzed by flavin monooxygenase (FMO) rather than CYP, as determined by studies with chemical inhibitors of FMO and metabolism by cDNA-expressed human flavin monooxygenase. After administration of [10b-¹⁴C]PZA to mice, six urinary metabolites were detected by high-performance liquid chromatography UV and radiochromatograms including 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. Trace concentrations of 9-desmethyl-PZA and PZA N-oxide were detected in mouse plasma. PZA N-oxide and N-demethyl-PZA were detected in urine from patients after PZA administration. PZA, 9-desmethyl-PZA, and PZA N-oxide inhibited growth of A375 human melanoma cells. IC₅₀ values were 0.17, 0.11, and 7.0 µM, respectively, for the three molecules.

	INTRODUCTION

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Pyrazoloacridine [PZA (NSC 366140)] is an acridine analog presently under investigation in Phase II clinical trials. In preclinical studies, PZA had greater cytotoxicity against solid tumor cell lines than against leukemia cell lines and selective cytotoxicity against hypoxic cells compared with nonhypoxic cells (1 , 2) . In vitro activity was schedule independent against early- and advanced-stage colon 38 and pancreatic carcinoma 03 tumor models. PZA was only weakly cross-resistant against multidrug-resistant tumor cells (3) . In a recent study, PZA was active in multidrug-resistant neuroblastoma cell lines. That activity was substantially increased after prolonged exposure (4) . PZA was equally effective against noncycling and exponentially growing Chinese hamster ovary cells (1) . Consistent with this observation, PZA inhibited RNA synthesis more effectively than DNA synthesis (1) . PZA inhibited topoisomerases I and II by a mechanism that is distinct from other dual topoisomerase I/II inhibitors (5) , possibly due to direct enzyme inhibition (5 , 6) . PZA also induced apoptosis in p53-deficient Hep3B human hepatoma cells (7) .

In preclinical murine studies, in vivo activity was schedule independent against early- and advanced-stage colon 38 and pancreatic carcinoma 03 tumor models (2) . Central nervous system toxicity, attributed to high PZA peak plasma concentrations, was dose-limiting after bolus administration but was not observed after 4–6-h infusions. Hematological toxicity was dose-limiting when the drug was administered by 4–6-h infusion (2) .

In Phase I studies, neurological toxicity was dose-limiting after a 1-h infusion, whereas hematological toxicity was dose-limiting after a 3-h (8 , 9) or 24-h infusion (10) . Hematological toxicity was also dose-limiting in pediatric patients given either a 1-h or 24-h infusion of PZA (11) . In contrast to adults, mild neurological toxicity was observed in only 1 of 22 pediatric patients given a 1-h infusion (11) . Pharmacokinetic data from the adult trials revealed rapid clearance and possibly enterohepatic recirculation of drug after i.v. PZA administration (8 , 9) . Substantial interpatient variation was observed for PZA pharmacokinetics in adults and children (8, 9, 10, 11) . PZA metabolism was not investigated in those Phase I studies.

PZA demonstrated modest antitumor activity against cisplatin and Taxol-resistant ovarian cancer in one of the adult Phase I trials (8 , 9) . In subsequent Phase II trials, one complete response was observed in a study with hormone-refractory prostate cancer patients (12) , and modest activity was noted in patients with platinum-resistant (13) or recurrent platinum-sensitive ovarian cancer (14) . Several other studies reported insignificant activity (15, 16, 17, 18) . PZA is presently under investigation in two Phase II trials for glioblastoma and one Phase I trial for neuroblastoma. PZA metabolism was not characterized during preclinical and Phase I development, primarily due to the difficulty in isolation and identification of putative metabolites. We previously developed a capillary electrophoresis (CE) method that effectively separated PZA and several metabolites (19) . Recent technical developments allow coupling of CE with electrospray ionization (ESI)-mass spectrometry (MS) providing a configuration ideal for rapid on-line metabolite characterization (20 , 21) . Using this methodology, we identified the oxidative PZA metabolites from mouse and human hepatic microsomes and characterized the reaction phenotype for those metabolites with combined data from studies with selective chemical inhibitors, correlation analysis, and metabolism by recombinant P450s and flavin monooxygenase (FMO3). We also characterized the growth inhibition of these metabolites against human tumor cell lines for comparison with parent drug cytotoxicity data.

	MATERIALS AND METHODS

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Chemicals and Reagents
PZA methanesulfonate and [10b-¹⁴C]PZA methanesulfonate hydrate (specific activity, 18 mCi/mmol) were provided by the Pharmaceutical Resources Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). 9-Desmethyl-PZA was generously provided by Warner-Lambert (Ann Arbor, MI). Metyrapone, -naphthoflavone, coumarin, tolbutamide, quinidine, disulfiram, erythromycin, quercitin, chlorozoxanzone, phenacetin, sulfaphenazole, caffeine, methimazole, and N-octylamine were obtained from Sigma Chemical Co. (St. Louis, MO). All solvents were high-performance liquid chromatography (HPLC) grade.

PZA N-oxide was synthesized from PZA-free base according to the procedure described by Elslager (22) as follows: PZA-free base was isolated by treating a solution of the methanesulfonate salt with excess sodium bicarbonate and extracting the basic solution with ethyl acetate. The proton NMR spectrum obtained after drying and evaporating the organic solvent was consistent with PZA-free base. PZA-free base was added to acetone and 30% hydrogen peroxide and allowed to stand at ambient temperature for 24 h. Additional hydrogen peroxide was added. After 1 week, an orange crystalline solid precipitated from the aqueous solution. The proton NMR spectrum of the filtered product was consistent with the N-oxide of the parent molecule.

PZA and PZA N-oxide stock solutions (1 mg/ml) were prepared in distilled water and stored frozen at -20°C. 9-Desmethyl-PZA (1 mg/ml) stock solutions were prepared in DMSO. Stock solutions of each molecule were diluted in HPLC mobile phase for chromatographic analysis and in methanol for CE-ESI-MS analysis.

In Vitro Metabolism by Human Liver Microsomes
Human Liver Microsomes.
Human liver microsomes used to characterize PZA oxidative metabolism (HL8) were provided by Jerry M. Collins (Center for Drug Evaluation and Research, United States Food and Drug Administration, Rockville, MD). Human liver samples, medically unsuitable for liver transplantation, were acquired under the auspices of the Washington Regional Transplant Consortium (Washington, D.C.). Preparation and characterization of the liver microsomal fractions have been described previously (23) .

Incubation Conditions.
Incubations of human microsomal suspensions were performed in amber glass vials maintained at 37°C in a shaker bath. Each 100-µl incubation mixture contained human liver microsomes (0.1 mg of protein), NADP+ (0.4 mM), glucose-6-phosphate (25 mM), glucose-6-phosphate dehydrogenase (0.7 unit/ml), magnesium chloride (5 mM), and potassium phosphate (100 mM) buffer adjusted to pH 7.4. Control incubation mixtures contained boiled microsomes or active microsomes with a nitrogen or CO/O₂ atmosphere. The incubation mixtures were preincubated for 2 min before the initiation of the reaction upon the addition of PZA. At the end of the incubation period, reactions were terminated by the addition of a 20-µl aliquot to 40 µl of ice-cold methanol. The aqueous methanol supernatants obtained after centrifugation (10,000 x g for 2 min) were diluted 1:1 with HPLC mobile phase for analysis. In selected experiments, P450-selective chemical inhibitors [caffeine metyrapone and SKF525A dissolved in water; coumarin, quinidine, erythromycin, quercitin, chlorzoxazone, phenacetin, tolutamide, methimazole, paclitaxel, and sulfaphenazole dissolved in methanol; and -naphthoflavone dissolved in DMSO (20 or 200 µM)] were incubated with PZA (100 µM) in reaction buffer for 30 min as described above. Appropriate controls containing methanol or DMSO only were performed in parallel incubations.

Correlation of PZA Metabolism with Marker Activities of Selected Cytochrome P450 (CYP) Forms.
A panel of microsomal fractions from 10 individual human livers with defined catalytic activities for several CYP-selective substrates was obtained from Human Biologics, Inc. (Phoenix, AZ). Microsomal suspensions were prepared in the reaction buffer to achieve a final protein concentration of 1 mg/ml and incubated with PZA (100 µM) for a 30-min reaction period as described above.

Metabolite Formation by cDNA-Expressed Human P450 Enzymes.
Microsomal suspensions from the B-lymphoblastoid cell line AHH-1 TK+/- expressing cDNA constructs for CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2E1, and CYP3A4 were obtained from Gentest Corporation (Woburn, MA). After dilution with the reaction buffer to achieve a final protein concentration of 1 mg/ml, microsomal suspensions were incubated with PZA (100 µM) for reaction periods of 30 and 120 min. Control reactions included microsomal preparations from cells that contained the expression vector alone.

	In Vitro Metabolism by Mouse Liver Microsomes

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Mouse Liver Microsomes.
Mouse liver homogenates were prepared in 0.15 M KCl or 0.25 M sucrose after excision from mice sacrificed by cervical dislocation. S-9 supernatant fractions were obtained by centrifugation (9000 x g, 10 min) of liver homogenates prepared in 0.15 M KCl. Hepatic microsomes were obtained by differential centrifugation of liver homogenates prepared in 0.25 M sucrose (24) . The pellet obtained after ultracentrifugation was resuspended in 0.15 M KCl. Protein content was determined by the Lowry method (25) .

Incubation Conditions.
PZA or p-nitrobenzoic acid (0.5 mM in 3 ml) was incubated with mouse liver microsomes (3 mg protein/ml) in the presence of a NADP-generating system containing NADP+ (0.4 mM), glucose-6-phosphate (23.6 mM), glucose-6-phosphate dehydrogenase (0.47 units/ml), magnesium chloride (5 mM) and Tris buffer (50 mM, pH 7.4). Where appropriate, nicotinamide (16.7 mM) and/or flavin-mononucleotide (0.5 mM) was added to incubation mixtures.

In some experiments, the above mixtures were incubated under reductive conditions. Air was replaced by nitrogen in incubation flasks by alternating vacuum (2 min x 3 times) with nitrogen (2 min x 3 times) via a needle through the septum of the sealed flask. After a 2-min preincubation period, drug was added to the sealed flask, and aliquots were removed at appropriate times with needle and syringe and frozen immediately for later HPLC analysis.

Nitro-reductase activity in microsomal or S-9 preparations was monitored by measuring the amount of p-aminobenzoic acid formed from p-nitrobenzoic acid by the Bratton and Marshall method (26) as modified by Fouts and Brodie (27) . Microsomal or S-9 incubation aliquots were added to screw-capped glass vials that contained H₂O (3 ml) and 15% trichloroacetic acid (0.8 ml) to stop the reaction and precipitate proteins. Vials were allowed to stand on ice for 5 min. Standard curve samples were prepared by serial dilution of stock p-aminobenzoic acid (1 mg/ml) such that the final concentration of p-aminobenzoic acid in the acid extracts was 0.1–1 µg/ml. After the addition of 0.1% sodium nitrate (0.4 ml) to 4 ml of the acid extract, the mixture was allowed to stand at room temperature for 3 min. After a 2-min reaction with 0.5% ammonium sulfamate (0.4 ml), 0.1% N-(1-naphthyl)ethylene diamine dihydrochloride (0.4 ml) was added to the reaction mixtures, and the absorbance of the samples was monitored spectrophotometrically at 540 nm. The unknown sample was compared with an appropriate standard curve that was treated as described above.

	Urinary Excretion of PZA

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Non-tumor-bearing male CD2F₁ mice (20–30 g), supplied by the National Cancer Institute, were housed 5 animals/cage on commercially obtained pure wood shaving bedding in an on-site facility with light provided from 6 a.m. to 8 p.m. Food (Purina Rodent Chow) and tap water were provided ad libitum.

PZA was prepared in sterile water or normal saline, and doses of 37.5 mg/kg were administered slowly (3–5 min) via the tail vein to male CD2F₁ mice. In selected experiments, mice were given [10b-¹⁴C]PZA (1 µCi/dose). Urine was collected from mice (4 mice/cage) placed in glass metabolism cages after drug administration. At the end of each 24-h collection period, the urine volume was recorded, and the samples were stored frozen (-20°C) until analysis.

Human subjects were three patients with advanced cancer participating in a Phase I trial of PZA at Wayne State University (Detroit, MI). PZA (600 mg/m²) was given by a 1-h i.v. infusion. Urine was collected for a 24-h period beginning at the start of the infusion.

	Metabolite Purification

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Urine was extracted with ethyl acetate/isopropanol (9:1, v/v), and the organic layer was removed, dried over magnesium sulfate, and concentrated under reduced pressure. Microsomal incubation mixtures were deproteinated by the addition of methanol (2:1). After centrifugation (1000 x g, 5 min), the supernatant was transferred to a conical centrifuge tube and evaporated to dryness under a gentle stream of nitrogen. Urine and microsomal extracts were reconstituted in HPLC mobile phase, and peak fractions with retention times corresponding to putative metabolites (determined from the elution pattern of radioactivity in mice that had received radiolabeled PZA) were pooled and concentrated to dryness. Individual peak fraction concentrates were extracted with ethyl acetate:isopropanol (9:1, v/v) and chromatographed by HPLC until the final organic extract exhibited a single peak by HPLC (usually 3 organic extraction/HPLC cycles).

	Analytical Methods

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Reverse-phase HPLC separations were achieved on a Lichrosorb C2 or C8 (E.M. Science) analytical column (250 x 4.0-mm inner diameter, 10-µm particles) fitted with a Brownlee RP2 (Chromtech) guard column (15 x 3.2-mm inner diameter, 7-µm particles). The mobile phase consisted of acetonitrile:tetrahydrofuran:100 mM potassium phosphate (20:5:80) adjusted to an apparent pH 4.0 with 10% phosphoric acid after mixing the solvents and was delivered at a flow rate of 1.0 ml/min. UV absorbance of the column effluent was monitored at 450 nm.

Mouse urine (0.2 ml) was incubated with 0.4 ml of 500 units/ml type VII ß-glucuronidase (Sigma Chemical Co.) in 75 mM potassium phosphate (pH 6.8) overnight at 37°C to investigate the presence of glucuronide conjugates. Active ß-glucuronidase in the incubation mixtures was ascertained in parallel incubations of phenolphthalein glucuronide in urine from untreated mice.

CE was performed on a Beckman P/ACE 2100 instrument through an uncoated fused silica capillary (65-cm x 50-µm inner diameter). The separation buffer consisted of methanol containing 20 mM ammonium acetate with 1% acetic acid, and the separation voltage was 30 kV. The CE capillary was maintained at ambient temperature (25°C). Capillary flow to the electrospray ion source was supplemented with a coaxillay delivered liquid sheath of 60:40:1 (v/v/v) isopropanol-water-acetic acid at a flow rate of 2 µl/min through the ESI needle and served as a ground for the CE capillary as described previously (20 , 21) . The ESI voltage was 3400 V. The CE-MS scan range was 80–600 Da (exponential magnet scan from low to high mass) at a rate of 2 s/decade, and the instrument resolution was 1000. MS was performed on a Finnigan MAT 900 mass spectrometer (Bremen, Germany) of EB configuration (where E and B are electric and magnetic sectors, respectively). A position- and time-resolved ion counter PATRIC focal plane detector was used with an 8% mass window. A modified Analytica electrospray ion source (Branford, CT) operated in a positive ion mode with the needle assembly at ground potential was used throughout. The sample needle of the ESI source was replaced by the CE capillary, from which 2–3 mm of the polyamide coating at the MS end had been removed by treatment with hydrofluoric acid.

	Growth Inhibition Assay

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Exponentially growing A375 (human melanoma) tumor cells in DMEM (100 µl, without phenol red) with 10% FCS and 1% penicillin-streptomycin were added to each well of 96-well cell culture plates at a cell density of 1000 cells/well and placed in an incubator (37°C, 5% CO₂/95% air, 100% relative humidity) for a 24-h incubation period. Fresh medium (100 µl) containing appropriate concentrations of drugs or PZA metabolites was added to each well. After a 72-h incubation period, 50 µl of 2 mg/ml 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide in PBS were added to each well. After 4 h, medium was removed carefully from the wells, leaving 50 µl in each well, which was mixed with 150 µl of DMSO by repeated pipeting. After 5 min, absorbance at 570 nm was determined using a microtiter plate reader.

Each experiment included 6 wells/drug concentration and 6 control wells and was repeated two to three times. IC₅₀ values were calculated as the concentration of drug required to cause a 50% decrease in absorbance by drug-treated cells compared with untreated cells.

	RESULTS

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Identification of PZA Oxidative Metabolites.
Three oxidative metabolites were detected by HPLC analysis after incubation of PZA with NADPH-fortified human liver microsomal incubations (Fig. 1) . Efforts to characterize these metabolites by HPLC/MS analysis were unsuccessful due to poor ionization under ESI and atmospheric pressure chemical ionization conditions. The CE-ESI-MS method was developed to overcome this limitation by using on-line isotachophoresis preconcentration before separation and MS (19) Accordingly, these metabolites (also among those detected in mouse and human urine after an i.v. dose of PZA; see below) were identified by CE-ESI-MS analysis of the corresponding HPLC peak fractions. The "soft" ionization of each product as it was analyzed by on-line CE-ESI-MS yielded prominent pseudo-molecular ion (MH⁺) responses. In addition, the bond linking the substituted alkyl nitrogen moiety to the ring was rather weak, causing fragmentation during ionization processes. Hence, for each product, there was facile loss of the substituted alkyl nitrogen moiety. These fragment ions were used in conjunction with MH⁺ responses to identify the metabolic products of PZA (Fig. 2) . 9-Desmethyl-PZA was identified by m/z 354 and m/z 309 ions representing MH⁺ and loss of the dimethylamino [-N(CH₃)₂] moiety, respectively (Fig. 2C) . N-Demethyl-PZA was identified by m/z 354 and m/z 323 ions representing MH⁺ and the loss of monomethylamino [-NH(CH₃)] moiety, respectively (Fig. 2B) . PZA N-oxide was identified by m/z 384 and m/z 323 ions representing MH⁺ and loss of the -N(CH₃)₂ moiety, respectively (Fig. 2D) .

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A high-performance liquid chromatography chromatogram of an aliquot of a human liver microsomal suspension after 120 min of incubation with 0.1 mM pyrazoloacridine in the presence of a NADPH-generating system.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Ion electropherograms of pyrazoloacridine (PZA) and into oxidative metabolites after purification of urine from humans (PZA, N-demethyl-PZA, and PZA N-oxide) and mice (9-desmethyl-PZA). A, the PZA pseudo-molecular ion was monitored at m/z 368. B, the N-demethyl-PZA pseudo-molecular ion was monitored at m/z 354. C, the 9-desmethyl-PZA pseudo-molecular ion was monitored at m/z 354. D, the PZA N-oxide pseudo-molecular ion was monitored at m/z 384. The inset structures illustrate the major ions observed by electrospray ionization mass spectrometry.

The kinetics of PZA N-oxide, N-demethyl-PZA, and 9-desmethyl-PZA formation by human liver microsomes are summarized in Table 1 . The K_m values for formation of each metabolite were similar. The V_max value for PZA N-oxide formation was 15-fold greater than the value for 9-desmethyl-PZA formation, whereas the V_max for N-demethyl-PZA could not be determined due to absence of an authentic standard.

Inhibition of PZA Metabolism by Form-Selective Chemical Inhibitors.
To characterize the involvement of CYP and FMO in PZA metabolism by human liver microsomes, studies were conducted with selective inhibitors of the two enzyme systems. Results of microsomal PZA N- and O-demethylation and N-oxidation studies are summarized in Table 2 . Chemicals with the greatest inhibitory effect on PZA metabolism were known substrates or inhibitors of CYP1A [-naphthoflavone (29) , quercitin (30) ], CYP3A [-naphthoflavone (29) , erythromycin (31) , quercitin (32) , quinidine (33) , and paclitaxel (23) ], and FMO [methimazole (34) ]. Specifically, 9-desmethyl-PZA formation was reduced by -naphthoflavone and quercitin, but not by erythromycin, quinidine, paclitaxel, or methimazole. N-Demethyl-PZA formation was reduced to the greatest extent by erythromycin, quercitin, and methimazole; reduced to a lesser extent by -naphthoflavone, quinidine, and paclitaxel, but not reduced by the selective CYP1A inhibitors phenacitin or caffeine. PZA N-oxide formation was reduced substantially by methimazole and -naphthoflavone.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. High-performance liquid chromatography chromatograms of aliquots from 120-min incubations of 0.1 mM pyrazoloacridine with recombinant human cytochrome P450 1A2, cytochrome P450 3A4, and flavin monooxygenase in the presence of a NADPH-generating system.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. A, high-performance liquid chromatography (HPLC) chromatogram and radiochromatogram (inset) of urine from mice treated with 37.5 mg/kg [10b-14C]pyrazoloacridine (PZA). Peaks labeled A-G are not present in control urine. B, representative HPLC chromatogram of urine from one of three cancer patients treated with 600 mg/m2 PZA. C, HPLC chromatogram of a mouse plasma specimen obtained 5 min after i.v. administration of 37.5 mg/kg PZA. Peaks: A, B, D, and G, unknown metabolites; C, 9-desmethyl PZA; E, PZA; F, PZA N-oxide.

	DISCUSSION

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CYP1A2 was the predominant enzyme responsible for 9-desmethyl-PZA formation, based on strong inhibition by the CYP1A-selective inhibitor -naphthoflavone, high correlations between 9-desmethyl-PZA formation and two CYP1A marker activities (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), and the observation that CYP1A2 was the only recombinant CYP to catalyze formation of 9-desmethyl-PZA.

The predominant enzyme responsible for N-demethyl-PZA formation was CYP3A4, based on selective inhibition by the CYP3A-selective inhibitors erythromycin, quinidine, and quercitin; a high correlation with the CYP3A4 marker activity testosterone 6ß-hydroxylation; and the observation that CYP3A4 was the only human cDNA-expressed CYP to catalyze N-demethyl-PZA formation. Enzymes in the CYP2C subfamily may have a modest role in N-demethyl-PZA formation because quinidine, quercitin, and paclitaxel inhibit CYP2C isoforms and there was a correlation with tolbutamide methyl hydroxylation (a marker for CYP2C activity).

Neither inhibitor, correlation, nor cDNA studies clearly defined a P450 catalytic activity responsible for PZA N-oxide formation. PZA N-oxide formation was inhibited by -naphthoflavone and quercitin, but not other CYP1A, CYP3A, and CYP2C subfamily-selective inhibitors. Moreover, PZA N-oxide formation was increased during incubation with the classical P450 inhibitor SKF525A correlated with coumarin 7-hydroxylation and S-mephenytoin 4'-hydroxylation, markers for CYP2A6 and CYP2C19, respectively, and PZA N-oxide was absent after incubation with recombinant CYP3A4.

Given the conflicting findings, we investigated the catalytic activity of FMO, a NADPH-dependent enzyme that catalyzes N-oxidation. PZA-N-oxide formation was inhibited by mild heating and methimazole, both of which inhibit FMO (34) . Most importantly, PZA N-oxide, but not 9-desmethyl-PZA or N-demethyl-PZA, was detected in incubations with recombinant human FMO3. These observations are consistent with FMO catalytic activity for PZA N-oxidation.

Whereas the in vitro metabolism data are consistent with CYP1A2, CYP3A4, and FMO3 in the oxidative metabolism of PZA, the urinary excretion data clearly implicate additional metabolic pathways for PZA metabolism. Palomino et al. (35) detected a PZA reduction product, 5-aminopyrazoloacridine, in mouse urine. In our studies, PZA was not metabolized under reductive conditions. Because p-aminobenzoic acid formed p-nitrobenzoic acid in parallel reactions, it would appear that PZA is not metabolized by mouse hepatic nitroreductase. These observations suggest that intestinal microflora catalyze the reduction of the PZA nitro moiety because other investigators found that nitroreductase activity present in isolated cecal contents was much greater than that present in the liver (36 , 37) . We were also unable to identify PZA conjugates in mouse urine based on enzyme hydrolysis studies.

Interindividual variation in drug metabolism and differences in biological activity of the resulting metabolites may contribute to differences among patients in efficacy and/or toxicity of anticancer agents (38) . In this regard, the equivalent cytotoxicity of PZA and 9-desmethyl-PZA suggests that PZA activity is probably maintained after metabolism by CYP1A2 to the 9-desmethyl metabolite. In contrast, the markedly reduced cytotoxicity of PZA N-oxide compared with the parent molecule suggests that oxidation by FMO3 may contribute to deactivation of PZA. Furthermore, FMO3 exhibits high levels of expression in brain, and enzyme variants with low catalytic activity have been identified (39) . Because primate studies found that PZA crosses the blood-brain barrier (40) , individuals that have low FMO3 activity variants may be more susceptible to PZA-related neurological toxicity.

In conclusion, we characterized the in vitro metabolism of PZA by mouse and human liver microsomes. PZA N-oxide and N-demethyl-PZA were the major products of NADPH-dependent human liver microsomal metabolism of PZA. Formation of these metabolites was catalyzed by FMO3 and CYP3A4, respectively. 9-Desmethyl-PZA was a minor metabolite, and formation was catalyzed by CYP1A2. Whereas N-oxidation is a deactivation pathway, and PZA antiproliferative activity is maintained after O-demethylation, the role of N-demethylation in the activity of PZA is presently unknown. Based on these data, we have incorporated pharmacokinetic studies in two clinical trials of PZA. A Phase I/II trial of the combination of PZA and carboplatin for patients with recurrent glioma, initiated based on in vitro synergy between the two drugs against T98G human glioblastoma cells (41) , incorporated pharmacokinetic studies to investigate the effect of enzyme-inducing anticonvulsants on the disposition of PZA and its metabolites. A Phase I trial of PZA and stem cell or bone marrow transplantation to treat children with recurrent or refractory neuroblastoma includes pharmacokinetic studies to monitor PZA peak concentrations and explore PZA metabolism in children. PZA is also under investigation in a Phase I/II trial sponsored by New Approaches to Brain Tumor Treatment consortium to determine the maximum tolerated dose and activity of PZA combined with radiation treatment for patients with supratentorial glioblastoma multiforme.

	ACKNOWLEDGMENTS

 
We thank Dr. David E. Zembower for technical assistance and Wanda L. Rhodes for preparation of the manuscript.

	FOOTNOTES

 
Grant support: National Cancer Institute Contract CM5700 and Mass Spectrometry Grant, Mayo Clinic.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Matthew M. Ames, Department of Oncology, Mayo Clinic, 200 First Street S.W., Rochester, Minnesota 55905. Phone: (507) 284-2424; Fax: (507) 284-3906; E-mail: ames.matthew{at}mayo.edu

Received 4/ 5/03; revised 9/26/03; accepted 10/16/03.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS In Vitro Metabolism by... Urinary Excretion of PZA Metabolite Purification Analytical Methods Growth Inhibition Assay RESULTS DISCUSSION REFERENCES

 

1. Sebolt J. S., Scavone S. V., Printer C. D., Hamelehle K. L., Von Hoff D. D., Jackson R. C. Pyrazoloacridines, a new class of anticancer agents with selectivity against solid tumors in vitro. Cancer Res., 47: 4299-4304, 1987.[Abstract/Free Full Text]
2. LoRusso P., Wozniak A. J., Polin L., Capps D., Leopold W. R., Werbel L. M., Biernat L., Dan M. E., Corbett T. H. Antitumor efficacy of PD115934 (NSC 366140) against solid tumors of mice. Cancer Res., 50: 4900-4905, 1990.[Abstract/Free Full Text]
3. Sebolt J., Havlick M., Hamelehle K., Nelson J., Leopold W., Jackson R. Activity of the pyrazoloacridines against multidrug-resistant tumor cells. Cancer Chemother. Pharmacol., 24: 219-224, 1989.[Medline]
4. Keshelava N., Tsao-Wei D., Reynolds C. P. Pyrazoloacridine is active in multidrug-resistant neuroblastoma cell lines with nonfunctional p53. Clin. Cancer Res., 9: 3492-3502, 2003.[Abstract/Free Full Text]
5. Adjei A. A., Charron M., Rowinsky E. K., Svingen P. A., Miller J., Reid J. M., Sebolt-Leopold J., Ames M. M., Kaufmann S. H. Effect of pyrazoloacridine (NSC 366140) on DNA topoisomerases I and II. Clin. Cancer Res., 4: 683-691, 1998.[Abstract]
6. Grem J. L., Politi P. M., Berg S. L., Benchekroun N. M., Patel M., Balis F. M., Sinha B. K., Dahut W., Allegra C. J. Cytotoxicity and DNA damage associated with pyrazoloacridine in MCF-7 breast cancer cells. Biochem. Pharmacol., 51: 1649-1659, 1996.[CrossRef][Medline]
7. Adjei P. N., Kaufmann S. H., Leung W. Y., Mao F., Gores G. J. Selective induction of apoptosis in Hep 3B cells by topoisomerase I inhibitors: evidence for a protease-dependent pathway that does not activate CPP32. J. Clin. Investig., 98: 2588-2596, 1996.[Medline]
8. Rowinsky E. K., Noe D. A., Grochow L. B., Sartorious S. E., Bowling M. K., Chen T. L., Lubejko B. G., Kaufmann S. H., Donehower R. C. Phase I and pharmacologic studies of pyrazoloacridine, a novel DNA intercalating agent, on single-dosing and multiple-dosing schedules. J. Clin. Oncol., 13: 1975-1984, 1995.[Abstract/Free Full Text]
9. LoRusso P., Foster B. J., Poplin E., McCormick J., Kraut M., Flaherty L., Heilbrun L. K., Valdivieso M., Baker L. Phase I clinical trial of pyrazoloacridine NSC366140 (PD115934). Clin. Cancer Res., 1: 1487-1493, 1995.[Abstract]
10. Grem J. L., Harold N., Keith B., Chen A. P., Kao V., Takimoto C. H., Hamilton J. M., Pang J., Pace M., Jasser G. B., Quinn M. G., Monahan B. P. A Phase I pharmacologic and pharmacodynamic study of pyrazoloacridine given as a weekly 24-hour continuous intravenous infusion in adult cancer patients. Clin. Cancer Res., 8: 2149-2156, 2002.[Abstract/Free Full Text]
11. Berg S. L., Blaney S. M., Adamson P. C., O’Brien M., Poplack D. G., Arndt C., Blatt J., Balis F. M. Phase I trial and pharmacokinetic study of pyrazoloacridine in children and young adults with refractory cancers. J. Clin. Oncol., 16: 181-186, 1998.[Abstract/Free Full Text]
12. Small E. J., Fippin L. J., Whisenant S. P. Pyrazoloacridine for the treatment of hormone-refractory prostate cancer. Cancer Investig., 16: 456-461, 1998.[Medline]
13. Plaxe S. C., Blessing J. A., Morgan M. A., Carlson J. Phase II trial of pyrazoloacridine in recurrent platinum-resistant ovarian cancer: a Gynecologic Oncology Group study. Am. J. Clin. Oncol., 25: 45-47, 2002.[CrossRef][Medline]
14. Plaxe S. C., Blessing J. A., Bookman M. A., Creasman W. T. Phase II trial of pyrazoloacridine in recurrent platinum-sensitive ovarian cancer: a Gynecologic Oncology Group study. Gynecol. Oncol., 84: 32-35, 2002.[CrossRef][Medline]
15. Bastasch M., Panella T. J., Kretzschmer S. L., Graham D., Mayo M., Williamson S. Phase II trial of pyrazoloacridine in advanced non-small cell carcinoma of the lung: a Kansas Cancer Institute and Thompson Cancer Survival Center Study. Investig. New Drugs, 20: 339-342, 2002.[CrossRef][Medline]
16. Plaxe S. C., Blessing J. A., Olt G., Husseinzadah N., Lentz S. S., DeGeest K., Valea F. A. A Phase II trial of pyrazoloacridine (PZA) in squamous cell carcinoma of the cervix: a Gynecologic Oncology Group study. Cancer Chemother. Pharmacol., 50: 151-154, 2002.[CrossRef][Medline]
17. Plaxe S. C., Blessing J. A., Lucci J. A., Hurteau J. A. A Phase II trial of pyrazoloacridine (PZA) in squamous carcinoma of the cervix: a Gynecologic Oncology Group Study. Investig. New Drugs, 19: 77-80, 2001.[CrossRef][Medline]
18. Pelley R., Ganapathi R., Wood L., Rybicki L., McLain D., Budd G. T., Peereboom D., Olencki T., Bukowski R. M. A Phase II pharmacodynamic study of pyrazoloacridine in patients with metastatic colorectal cancer. Cancer Chemother. Pharmacol., 46: 251-254, 2000.[CrossRef][Medline]
19. Benson L. M., Tomlinson A. J., Reid J. M., Walker D. L., Ames M. M., Naylor S. Study of in vivo pyrazoloacridine metabolism by capillary electrophoresis using isotachophoresis preconcentration in non-aqueous separation buffer. J. High Res. Chromatogr., 16: 324-326, 1993.[CrossRef]
20. Naylor S., Benson L. M., Tomlinson A. J. Monitoring drug metabolism by capillary electrophoresis Landers J. P. eds. . CRC Handbook of Capillary Electrophoresis: A Practical Approach, 459-491, CRC Press Boca Raton, FL 1994.
21. Tomlinson A. J., Benson L. M., Naylor S. On-line capillary electrophoresis-mass spectrometry for the analysis of drug metabolite mixtures: practical considerations. J. Cap. Elec., 1: 127-135, 1994.
22. Elslager, E. F. Benzo and benzothiopyranoindazole N-oxides. United States Patent: 3,963,740, June 15, 1976.
23. Harris J. W., Rahman A., Kim B-R., Guengerich F. P., Collins J. M. Metabolism of Taxol by human hepatic microsomes and liver slices: participation of cytochrome P450 3A4 and an unknown P450 enzyme. Cancer Res., 54: 4026-4035, 1994.[Abstract/Free Full Text]
24. Ernster L., Siekevitz P., Palade G. E. Enzyme-structure relationships in the endoplasmic reticulum of rat liver. J. Cell Biol., 15: 541-562, 1962.[Abstract/Free Full Text]
25. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275, 1951.[Free Full Text]
26. Bratton A. C., Marshall E. K. A new coupling component for sulfanilamide determination. J. Biol. Chem., 128: 537-550, 1939.[Free Full Text]
27. Fouts J. R., Brodie B. B. The enzymatic reduction of chloramphenicol, P-nitrobenzoic acid and other aromatic nitro compounds in mammals. J. Pharmacol. Exp. Ther., 119: 197-207, 1957.[Abstract/Free Full Text]
28. Kinsler S., Levi P. E., Hodgson E. Hepatic and extraheaptic microsomal oxidations of phorate by cytochrome P-450 and FAD monooxygenase systems in the mouse. Pestic. Biochem. Physiol., 31: 54-60, 1985.
29. Tassaneeyakul W., Birkett D. J., Veronese M. E., McManus M. E., Tukey R. H., Quattrochi L. C., Gelboin H. V., Miners J. O. Specificity of substrate and inhibitor probes for human cytochromes P450 1A1 and 1A2. J. Pharmacol. Exp. Ther., 265: 401-407, 1993.[Abstract/Free Full Text]
30. Le Bon A-M., Siess M-H., Suschetet M. Inhibition of microsome-mediated binding of benzo[]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats. Chem. Biol. Interact., 83: 65-71, 1992.[CrossRef][Medline]
31. Watkins P. B., Murray S. A., Winkelman L. G., Heuman D. M., Wrighton S. A., Guzelian P. S. Erythromycin breath test as an assay of glucocorticoid-inducible liver cytochromes P-450: studies in rats and patients. J. Clin. Investig., 83: 688-697, 1989.
32. Miniscalco A., Lundahl J., Regardh C. G., Edgar B., Eriksson U. G. Inhibition of dihydropyridine metabolism in rat and human liver microsomes by flavonoids found in grapefruit juice. J. Pharmacol. Exp. Ther., 261: 1195-1199, 1992.[Abstract/Free Full Text]
33. Guengerich F. P., Muller-Enoch D., Blair I. A. Oxidation of quinidine by human liver cytochrome P450. Mol. Pharmacol., 30: 287-295, 1986.[Abstract]
34. Mani C., Kupfer D. Cytochrome P-450-mediated activation and irreversible binding of the antiestrogen tamoxifen to proteins in rat and human liver: possible involvement of flavin-containing monooxygenases in tamoxifen activation. Cancer Res., 51: 6052-6058, 1991.[Abstract/Free Full Text]
35. Palomino E., Foster B., Kempff M., Corbett T., Wiegand R., Horwitz J., Baker L. Identification and antitumor activity of a reduction product in the murine metabolism of pyrazoloacridine (NSC-366140). Cancer Chemother. Pharmacol., 38: 453-458, 1996.[CrossRef][Medline]
36. Kinouchi T., Kataoka K., Miyanishi K., Akimoto S., Ohnishi Y. Biological activities of the intestinal microflora in mice treated with antibiotics or untreated and the effects of the microflora on absorption and metabolic activation of orally administered glutathione conjugates of K-region epoxides of 1-nitropyrene. Carcinogenesis (Lond.), 14: 869-874, 1993.[Abstract/Free Full Text]
37. Levin A. A., Dent J. G. Comparison of the metabolism of nitrobenzene by hepatic microsomes and cecal microflora from Fischer-344 rats in vitro and the relative importance of each in vivo. Drug Metab. Dispos., 10: 450-454, 1982.[Medline]
38. Weinshilboum R. Inheritance and drug response. N. Engl. J. Med., 348: 529-537, 2003.[Free Full Text]
39. Cashman J. R., Zhang J. Interindividual differences of human flavin-containing monooxygenase 3: genetic polymorphisms and functional variation. Drug Metab. Dispos., 30: 1043-1052, 2002.[Abstract/Free Full Text]
40. Berg S. L., Balis F. M., McCully C. L., Godwin K. S., Poplack D. G. Pharmacokinetics of pyrazoloacridine in the rhesus monkey. Cancer Res., 51: 5467-5470, 1991.[Abstract/Free Full Text]
41. Adjei A. A., Budihardjo I. I., Rowinsky E. K., Kottke T. J., Svingen P. A., Buckwalter C. A., Grochow L. B., Donehower R. C., Kaufmann S. H. Cytotoxic synergy between pyrazoloacridine (NSC 366140) and cisplatin in vitro: inhibition of platinum-DNA adduct removal. Clin. Cancer Res., 3: 761-770, 1997.[Abstract]

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