High-Performance Liquid Chromatographic–Fluorescent Method to Determine Chloroacetaldehyde, a Neurotoxic Metabolite of the Anticancer Drug Ifosfamide, in Plasma and in Liver Microsomal Incubations

Huang, Zeqi; Waxman, David J.

doi:10.1006/abio.1999.4197

Cited by 17 publications

(17 citation statements)

References 27 publications

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“…Two 80-l aliquots were removed from the supernatant of each sample and placed in separate screw-cap Ep- molpharm.aspetjournals.org pendorf tubes. One aliquot was used for derivatization of CAA to 1-N 6 -ethenoadenosine by addition of 10 l of 100 mM adenosine (dissolved in 0.25 N HCl) and 10 l of 2 M sodium acetate, pH 4.5 (final pH, 4.2) followed by heating for 2.5 h at 80°C (Huang and Waxman, 1999). The second aliquot was used to derivatize acrolein (breakdown product of 4-hydroxy-CPA and 4-hydroxy-IFA) to 7-hydroxyquinoline by addition of 40 l of 3-aminophenol (60 mg of 3-aminophenol and 60 mg of hydroxylamine HCl dissolved in 10 ml of 1 N HCl) followed by heating for 25 min at 90°C (Bohnenstengel et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics

Chen¹,

Lin²,

Goss³

et al. 2004

Mol Pharmacol

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Cyclophosphamide (CPA) and ifosfamide (IFA) are oxazaphosphorine anticancer prodrugs metabolized by two alternative cytochrome P450 (P450) pathways, drug activation by 4-hydroxylation and drug inactivation by N-dechloroethylation, which generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde. CPA and IFA metabolism catalyzed by P450s 2B1, 2B4, 2B5, and seven site-specific 2B1 mutants was studied in a reconstituted Escherichia coli expression system to identify residues that contribute to the unique activities and substrate specificities of these enzymes. The catalytic efficiency of CPA 4-hydroxylation by rat P450 2B1 was 10-to 35-fold higher than that of rabbit P450 2B4 or 2B5. With IFA, ϳ50% of metabolism proceeded via N-dechloroethylation for 2B1 and 2B4, whereas CPA N-dechloroethylation corresponded to only ϳ3% of total metabolism (2B1) or was absent (2B4, 2B5). Improved catalytic efficiency of CPA and IFA 4-hydroxylation was obtained upon substitution of 2B1 Ile-114 by Val, and replacement of Val-363 by Leu or Ile selectively suppressed CPA N-dechloroethylation Ն90%. P450 2B1-V367A, containing the Ala replacement found in 2B5, exhibited only ϳ10% of wild-type 2B1 activity for both substrates. Canine P450 2B11, which has Val-114, Leu-363, and Val-367, was therefore predicted to be a regioselective CPA 4-hydroxylase with high catalytic efficiency. Indeed, P450 2B11 was 7-to 8-fold more active as a CPA and IFA 4-hydroxylase than 2B1, exhibited a highly desirable low K m (80 -160 M), and catalyzed no CPA N-dechloroethylation. These findings provide insight into the role of specific P450 2B residues in oxazaphosphorine metabolism and pave the way for gene therapeutic applications using P450 enzymes with improved catalytic activity toward these anticancer prodrug substrates.The oxazaphosphorine cyclophosphamide (CPA) and its structural isomer ifosfamide (IFA) are DNA-alkylating agents commonly used in cancer chemotherapy (Sladek, 1994). These anticancer agents are administered as prodrugs that are activated by a liver cytochrome P450-catalyzed 4-hydroxylation reaction that yields active, cytotoxic metabolites. Several liver-expressed P450 enzymes catalyze this activation reaction; the phenobarbital-inducible rat P450 enzyme 2B1 (Clarke and Waxman, 1989) and its human counterpart 2B6 (Chang et al., 1993;Huang et al., 2000) show particularly high CPA 4-hydroxylase activity compared with other P450 forms. The primary metabolite, 4-OH-CPA or 4-OH-IFA, equilibrates with the ring-open aldophosphamide and undergoes ␤-elimination to yield the therapeutically active, DNA cross-linking phosphoramide mustard and the byproduct acrolein (4-hydroxylation pathway). CPA and IFA are also subject to an alternative, P450-catalyzed side chain oxidation that generates therapeutically inactive N-dechloroethylated metabolites and the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA; N-dechloroethylation pathway) (Furlanut and Franceschi, 2003).In patients with cancer, CPA and IFA are primarily activated...

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Section: Methodsmentioning

confidence: 99%

Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics

Chen¹,

Lin²,

Goss³

et al. 2004

Mol Pharmacol

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“…Samples were analyzed by highperformance liquid chromatography (HPLC) and quantified by comparison with standard curves of metabolite prepared using 4-OOH-cyclophosphamide dissolved in culture medium (43,44). In some cases, the derivatized samples were stored at À20jC in the dark before HPLC analysis.…”

Section: Retroviral Plasmid Constructionmentioning

confidence: 99%

Enhanced antitumor activity of P450 prodrug-based gene therapy using the low Km cyclophosphamide 4-hydroxylase P450 2B11

Jounaïdi

Chen

Veal

et al. 2006

Molecular Cancer Therapeutics

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Gene therapy using the prodrug-activating enzyme P450 2B6 has shown substantial promise in preclinical and initial clinical studies with the P450 prodrugs cyclophosphamide and ifosfamide. We sought to optimize this therapy using the canine P450 enzyme 2B11, which activates cyclophosphamide and ifosfamide with K m of 80 to 160 Mmol/L, f10-to 20-fold lower than the K m of P450 2B6. Retrovirus encoding a P450 2B11-internal ribosome entry signal-P450 reductase expression cassette induced marked cyclophosphamide and ifosfamide cytotoxicity toward 9L gliosarcoma cells and exhibited an impressive bystander killing effect at micromolar prodrug concentrations, where P450 2B6 displayed low activity. Adeno-2B11, a replication-defective, E1/E3 region-deleted adenovirus engineered to coexpress P450 2B11 and P450 reductase, dramatically increased tumor cell-catalyzed cyclophosphamide 4-hydroxylation and cytotoxicity compared with Adeno-2B6 and effected strong bystander killing at low (20 Mmol/L) cyclophosphamide concentrations. Further increases in cyclophosphamide cytotoxicity were obtained in several human cancer cell lines, including a 4-hydroperoxycyclophosphamide-resistant MCF-7 breast cancer cell line, when Adeno-2B11 was combined with Onyx-017, an E1b-55-kDa gene-deleted, tumor cellreplicating adenovirus that coamplifies and facilitates tumor cell spread of Adeno-2B11. To evaluate the therapeutic effect of P450 2B11 expression in vivo, 9L gliosarcoma cells transduced with P450-expressing retrovirus were grown as solid s.c. tumors in immunodeficient mice. Cyclophosphamide treatment on a metronomic, 6-day repeating schedule led to full regression of 9L/2B11 tumors but not P450-deficient control tumors, resulting in a tumor-free period lasting up to f100 days. 9L/2B6 tumors regressed more slowly and exhibited a tumor-free period of only 21 to 39 days. Thus, P450 genedirected enzyme prodrug therapy can be greatly improved by using the low K m P450 enzyme 2B11, which catalyzes intratumoral activation of cyclophosphamide and ifosfamide at pharmacologically relevant drug concentrations.

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“…Twenty-four h after infection with adenovirus, the cells were treated with 1 mM CPA in 3 ml of fresh RPMI 1640 containing 5% FBS and 0.5 mM semicarbazide to stabilize the 4OH-CPA metabolite. After 5 h of CPA treatment, 0.5 ml of culture supernatant was removed and assayed for the presence of 4OH-CPA released into the culture medium (33). The cells were then washed once with PBS, scraped from the wells, and lysed by sonication in 50 mM potassium phosphate buffer and 1 mM EDTA (pH 7.4) containing 20% glycerol.…”

Section: Introductionmentioning

confidence: 99%

Use of Replication-Conditional Adenovirus as a Helper System to Enhance Delivery of P450 Prodrug-Activation Genes for Cancer Therapy

Jounaïdi

Waxman

2004

Cancer Research

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Cytochrome P450 (CYP) gene transfer sensitizes tumor xenografts to anticancer prodrugs such as cyclophosphamide (CPA) without a detectable increase in host toxicity. Optimal prodrug activation is achieved when a suitable P450 gene (e.g., human CYP2B6) is delivered in combination with NADPH-cytochrome P450 reductase (P450R), which encodes the flavoenzyme P450 reductase. We sought to improve this gene therapy by coordinated delivery and expression of P450 and P450R on a single bicistronic vector using an internal ribosomal entry site (

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High-Performance Liquid Chromatographic–Fluorescent Method to Determine Chloroacetaldehyde, a Neurotoxic Metabolite of the Anticancer Drug Ifosfamide, in Plasma and in Liver Microsomal Incubations

Cited by 17 publications

References 27 publications

Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics

Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics

Enhanced antitumor activity of P450 prodrug-based gene therapy using the low Km cyclophosphamide 4-hydroxylase P450 2B11

Use of Replication-Conditional Adenovirus as a Helper System to Enhance Delivery of P450 Prodrug-Activation Genes for Cancer Therapy

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