Indolequinone antitumor agents: correlation between quinone structure and rate of metabolism by recombinant human NAD(P)H:quinone oxidoreductase. Part 2.

A series of indolequinones bearing various functional groups has been synthesized, and the effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1) were studied. Indolequinones were selected for study on the basis of the X-ray crystal structure of the human enzyme, and were designed to probe the effect of substituents particularly at N-1. Metabolism of the quinones by NQO1 revealed that, in general, compounds with electron-withdrawing groups at the indole 3-position were among the best substrates, and that groups larger than methyl at N-1 are clearly tolerated. Compounds with a leaving group at the 3-indolyl methyl position generally inactivated the enzyme. The toxicity toward human colon carcinoma cells with either no detectable activity (BE-WT) or high NQO1 activity (BE-NQ) was also studied in representative quinones. The most toxic compounds were those with a leaving group at the C-3 position; these compounds were 1.1-5.3-fold more toxic to the BE-NQ than the BE-WT cells.

Topical delivery of 5-fluorouracil (5-Fu) by 3-alkylcarbonyl-5-Fu prodrugs.

The solubilities in isopropyl myristate (SIPM) and pH 4.0 buffer (SAQ) and the partition coefficients between IPM and pH 4.0 buffer (KIPM:AQ) have been measured for a series of 3-alkylcarbonyl-5-fluorouracil prodrugs (3-AC-5-FU). The 3-AC-5-FU prodrugs were all 100 times more soluble in IPM and the first two members of the series were also more soluble in pH 4.0 buffer than 5-FU. The abilities of the 3-AC-5-FU prodrugs to deliver total 5-FU species through hairless mouse skin from IPM suspensions (Ji) were also measured. The 3-propionyl derivative 3, which exhibited the highest SAQ in the series, gave the highest Ji value. The SIPM, SAQ and molecular weights (mw) of the 3-AC-5-FU series correctly predicted the rank order and very closely (0.10 log units) predicted the absolute values for logJi using the transformed Potts-Guy equation. Although the series of 3-AC-5-FU prodrugs was generally quite effective at increasing Ji (2-20 times), the best 3-AC-5-FU prodrug was not as effective as the best 1-alkylcarbonyl-5-FU prodrug (1-AC-5-FU) at increasing Ji and the ability of the 3-AC-5-FU prodrugs to increase the concentration of total 5-FU species in the skin was 2-5 times less than the 1-AC-5-FU prodrugs. Thus, the 1-AC-5-FU prodrugs remain as the best prodrugs with which to enhance the topical delivery of 5-FU.

Mechanisms of action of quinone-containing alkylating agents. I: NQO1-directed drug development.

Alkylating agents have been used to treat cancer since the 1940s. Quinone-containing alkylating agents represent a class of drugs called "bioreductive alkylating agents." These drugs require reduction of the quinone moiety for activation of their alkylating substituents. Despite active research in this area, mitomycin C is the only bioreductive alkylating agent approved for general use. The "enzyme-directed" approach to bioreductive drug development involves identification of reductases which are overexpressed in tumors when compared to uninvolved tissues. Bioreductive drugs which are substrates for these reductases should be selectively toxic to tumors with high reductase levels. NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) is a two-electron reductase found primarily in the cytosol. NQO1 has received considerable attention because of the high levels of this enzyme in tumors particularly in tumors of the lung, colon and breast. In this review, the current state of research on quinone-containing alkylating agents is discussed with the focus on NQO1-directed bioreductive drug development. Recent structure-activity studies on indolequinones, benzoquinones and other novel quinones are reviewed, and the status of drugs which have been studied in clinical trials is discussed. Finally, the limitations and possible future directions in this research area are presented.

NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is involved in chemoprotection and can also bioactivate certain antitumor quinones. This review focuses on detoxification reactions catalyzed by NQO1 and its role in antioxidant defense via the generation of antioxidant forms of ubiquinone and vitamin E. Bioactivation reactions catalyzed by NQO1 are also summarized and the development of new antitumor agents for the therapy of solid tumors with marked NQO1 content is reviewed. NQO1 gene regulation and the role of the antioxidant response element and the xenobiotic response element in transcriptional regulation is summarized. An overview of genetic polymorphisms in NQO1 is presented and biological significance for chemoprotection, cancer susceptibility and antitumor drug action is discussed.

Novel quinolinequinone antitumor agents: structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1).

The effects of functional group changes on the metabolism of novel quinolinequinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1) are described. Overall, the quinolinequinones were much better substrates for NQO1 than analogous indolequinones, with compounds containing heterocyclic substituents at C-2 being among the best substrates.

Indolequinone antitumor agents: correlation between quinone structure, rate of metabolism by recombinant human NAD(P)H:quinone oxidoreductase, and in vitro cytotoxicity.

A series of indolequinones bearing various functional groups has been synthesized, and the effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1) were studied. Thus 5-methoxyindolequinones were prepared by the Nenitzescu reaction, followed by functional group interconversions. The methoxy group was subsequently displaced by amine nucleophiles to give a series of amine-substituted quinones. Metabolism of the quinones by NQO1 revealed that, in general, compounds with electron-withdrawing groups at the indole 3-position were among the best substrates, whereas those with amine groups at the 5-position were poor substrates. Compounds with a leaving group at the 3-indolyl methyl position generally inactivated the enzyme. The toxicity toward non-small-cell lung cancer cells with either high NQO1 activity (H460) or no detectable activity (H596) was also studied in representative quinones. Compounds which were good substrates for NQO1 showed the highest selectivity between the two cell lines.

Indolequinone antitumor agents: relationship between quinone structure and rate of metabolism by recombinant human NQO1.

A series of indolequinones bearing various functional groups has been synthesized, and the effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1), and on the toxicity toward nonsmall cell lung cancer cells with either high NQO1 activity (H460) or with no detectable activity (H596) were studied.

Stability of sumatriptan succinate in extemporaneously prepared oral liquids.

The stability of sumatriptan succinate in extemporaneously prepared oral liquids was studied. Suspensions of sumatriptan (as the succinate salt) in Ora-Sweet, Ora-Sweet SF, and Syrpalta syrups (Paddock Laboratories and Humco Laboratory) were extemporaneously compounded to produce a sumatriptan concentration of 5 mg/mL. Each suspension was prepared in triplicate. The suspensions were stored at 4 degrees C in amber glass bottles for 60 days. Two 1-mL samples were removed from each bottle initially and on days 2, 7, 14, 21, 28, 35, and 60. Sumatriptan concentrations were determined by high-performance liquid chromatography. The samples also underwent visual inspection and microbial testing. The mean concentration of sumatriptan in all suspensions remained above 90% of the initial concentration for up to 21 days. By day 28, the sumatriptan concentration of all suspensions had decreased to less than 90% of the initial concentration. None of the suspensions had microbial growth up to day 28, and there were no visible changes in the suspensions throughout the study period. Sumatriptan 5 mg/mL (as the succinate salt) in three oral suspensions was stable for up to 21 days when stored without light at 4 degrees C.

Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DT-diaphorase).

NAD(P)H:quinone oxidoreductase (NQO1, EC 1.6.99.2) is an obligate two-electron reductase that can either bioactivate or detoxify quinones and has been proposed to play an important role in chemoprevention. We have previously characterized a homozygous point mutation in the BE human colon carcinoma cell line that leads to a loss of NQO1 activity. Sequence analysis showed that this mutation was at position 609 of the NQO1 cDNA, conferring a proline to serine substitution at position 187 of the NQO1 enzyme. Using polymerase chain reaction (PCR) analysis, we have found that the H596 human non-small-cell lung cancer (NSCLC) cell line has elevated NQO1 mRNA, but no detectable enzyme activity. Sequencing of the coding region of NQO1 from the H596 cells showed the presence of the identical homozygous point mutation present in the BE cell line. Expression and purification of recombinant wild-type and mutant protein from E. coli showed that mutant protein could be detected using immunoblot analysis and had 2% of the enzymatic activity of the wild-type protein. PCR and Northern blot analysis showed moderate to low levels of expression of the correctly sized transcript in the mutant cells. Immunoblot analysis also revealed that recombinant mutant protein was immunoreactive; however, the mutant protein was not detected in the cytosol of either BE or H596 cells, suggesting that the mutant proteins were either not translated or were rapidly degraded. The absence of any detectable, active protein, therefore, appears to be responsible for the lack of NQO1 activity in cells homozygous for the mutation. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for the mutation at position 609 conducted on 90 human lung tissue samples (45 matched sets of tumour and uninvolved tissue) revealed a 7% incidence of individuals homozygous for the mutation, and 42% heterozygous for the mutation. These data suggest that the mutation at position 609 represents a polymorphism in an important xenobiotic metabolizing enzyme, which has implications for cancer therapy, chemoprevention and chemoprotection.

Expression of human NAD(P)H: quinone oxidoreductase (DT-diaphorase) in Chinese hamster ovary cells: effect on the toxicity of antitumor quinones.

Previous studies have indicated that NAD(P)H: quinone oxidoreductase [DT-diaphorase (NQO1)] plays an important role in the bioreductive activation of quinone-containing antitumor agents. Although these studies demonstrated that purified NQO1 can reduce these compounds in vitro, the importance of NQO1 in the intracellular activation of quinone-containing antitumor agents remains controversial. In our study, we transfected human NQO1 into Chinese hamster ovary cells that do not normally express NQO1 activity and obtained stable clones that expressed NQO1 activity of 19-3527 nmol of 2,6-dichlorophenolindophenol reduced/min/mg of protein. The level of NQO1 expression correlated with an increased killing by streptonigrin, EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)-propen ol), and 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone, but mitomycin C sensitivity was independent of this activity. NQO1 expression also led to a slight decrease in the sensitivity of cells to menadione. Our data demonstrate that compounds that are efficient substrates for NQO1 in vitro are also bioactivated in cultured mammalian cells when they are transfected with human NQO1. These results are consistent with the relative abilities of mitomycin C, streptonigrin, EO9, and 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone to serve as substrates for bioreduction by human NQO1, and show that NQO1 levels are not necessarily predictive in terms of sensitivity to mitomycin C.

Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin.

The metabolism, cytotoxicity, and genotoxicity of streptonigrin (SN) w ere determined in two human colon carcinoma cell lines: HT-29 with high NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, DTD) activity and BE with undetectable DTD activity. Dicumarol-sensitive oxidation of NADH was observed with HT-29 cytosol, but not with BE cytosol. Oxygen consumption was also observed using HT-29 cytosol, but was absent with BE cytosol. Dicumarol inhibited oxygen consumption with HT-29 cytosol, but deferoxamine had no effect, suggesting that divalent metal cations were not necessary for efficient auto-oxidation of SN hydroquinone. In cytotoxicity studies, SN was much more toxic to the DTD-rich HT-29 cells than to the DTD-deficient BE cells. Deferoxamine decreased toxicity in both cell lines, implicating hydroxyl radicals produced during Fenton-type reactions as the toxic species. In the genotoxicity assay, SN induced a much higher incidence of DNA strand breaks in HT-29 cells than in BE cells, and deferoxamine protected against DNA strand breaks in both cell lines. Some evidence of DNA repair was also observed in the two cell lines. These results support an important role for DTD in the cytotoxicity of SN in the high DTD HT-29 colon carcinoma cell line.

Nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase (DT-diaphorase) as a target for bioreductive antitumor quinones: quinone cytotoxicity and selectivity in human lung and breast cancer cell lines.

Bioreductive antitumor quinones require reductive metabolism to produce their cytotoxic effects. A series of these compounds was screened for relative rates of reduction by the two-electron reductase, NAD(P)H:quinone oxidoreductase (DTD). The antitumor quinones streptonigrin (SN), 2,5-diaziridinyl-3-phenyl-1,4-benzoquinone (PDZQ), 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinine (MeDZQ), and [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)-propen ol] (EO9) were all excellent substrates for recombinant rat and human DTD. All four compounds were reduced by DTD at least 100 times faster than the clinically important bioreductive alkylating agent, mitomycin C (MC). Reduction of the antitumor quinones was generally 4-5 times more efficient by rat DTD than by human DTD. The exception was EO9, which, surprisingly, was reduced 23 times faster by rat DTD than by human DTD. The rate of reduction of each individual quinone was similar under either aerobic or anaerobic conditions, suggesting that DTD may be an important activating enzyme in the hypoxic fraction of solid tumors. The cytotoxicity of MeDZQ and MC was examined in a panel of human breast and lung cancer cell lines. The data showed good correlations between DTD activity and toxicity for both MeDZQ (r = 0.57, p = 0.054) and MC (r = 0.69, p = 0.020), confirming biochemical data that both compounds are bioactivated by DTD. In addition, IC50 values were in general lower for MeDZQ than for MC in cell lines containing elevated DTD, a finding that was consistent with metabolic data that indicated that MeDZQ was a better substrate for DTD than MC. SR, defined as the ratio of the IC50 value for the H596 NSCLC cell line (undetectable DTD activity) to the IC50 value for the H460 NSCLC cell line (high DTD activity), were determined for all five antitumor quinones. SN was the most selective (SR = 86) followed by EO9 (SR = 62), MeDZQ (SR = 17), and MC (SR = 11). Surprisingly, PDZQ, an excellent substrate for DTD, was toxic to both cell lines (SR = 1.8). These data suggest that antitumor quionones that are substrates for DTD may be selectively toxic to tumors with high DTD activity and may be useful in the treatment of those tumors.

Noncovalent binding of a mitomycin C metabolite, 2,7-diaminomitosene, to duplex DNA.

The major metabolite of mitomycin C, 2,7-diaminomitosene (DAM), interacts noncovalently with DNA. This was supported by ultraviolet-visible spectrum changes upon mixing with DNA and ethidium bromide displacement from DNA, measured as fluorescence changes. Moreover, DAM bound to DNA sufficiently strongly to hold DNA in a double stranded conformation under denaturing gel electrophoresis conditions commonly used to measure mitomycin C cross-links. These data show that generation of DAM and interaction with DNA represent a potential additional mechanism of DNA damage induced by mitomycin C.

Metabolism of bioreductive antitumor compounds by purified rat and human DT-diaphorases.

The metabolisms of two standard electron acceptors and a series of bioreductive antitumor compounds by purified rat and human DT-diaphorases (DTD) were compared. DTD was purified from rat liver cytosol and from Escherichia coli in which rat liver or human lung tumor DTD complementary DNA was expressed. Km and kcat values for menadione and 2,6-dichlorophenolindophenol reduction were similar for the three enzyme preparations except that rat E. coli DTD had 2-3-fold higher kcat values for both menadione and 2,6-dichlorophenolindophenol and a 2-3-fold higher Km for menadione than either rat liver or human E. coli DTD. Reduction of the antitumor compounds was 1.9-4.9 times faster with rat E. coli DTD than with human E. coli DTD. The antitumor compounds were reduced in the following order by rat E. coli DTD: 2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone > streptonigrin > mitomycin A > diaziquone > mitomycin C (MC) > 5-(aziridin-1-yl)-2,4-dinitrobenzamide. The order was the same for human E. coli DTD with one exception; diaziquone was reduced slightly faster than mitomycin A. Metabolism of doxorubicin could not be detected using rat or human E. coli DTD. MC-induced DNA cross-linking was also more efficient using rat E. coli DTD relative to human E. coli DTD. Metabolism of MC by rat and human E. coli DTD was also compared under aerobic and hypoxic conditions. Rates of reduction of MC and metabolite formation were similar under aerobic and hypoxic conditions, and the toxicity of MC to DTD-rich HT-29 cells was also similar in aerobic and hypoxic conditions. In contrast, the toxicity of MC to DTD-deficient BE cells was potentiated markedly under hypoxia. These data show that although small catalytic differences between rat and human E. coli DTD can be observed, compounds such as 2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone and streptonigrin are still excellent substrates for the human enzyme and may be useful in the therapy of tumors high in DTD activity. In addition, metabolism of MC by rat and human E. coli DTD was similar in aerobic and hypoxic conditions; in agreement with these data, cytotoxicity of MC to a DTD-rich cell line was oxygen independent. Increased MC cytotoxicity under hypoxia appears to be mediated by enzymes other than DTD.

Structure of 3-acetyl-5-fluorouracil (5-FU): implication for its rearrangements during hydrolysis and upon heating.

Single-crystal X-ray diffraction data show that the 3-acetyl group in 1,3-diacetyl-5-FU (FU = fluorouracil) is perpendicular to the plane of the 5-FU ring, while the 1-acetyl group is coplanar with the ring. Analyses of 1H NMR and IR spectra provide evidence that the 1- and 3-acyl groups are in different electronic environments, which is consistent with the X-ray diffraction structure. 3-Acetyl-5-FU is thermally unstable, giving mainly 1-acetyl-5-FU (80%) and 5-FU (20%) upon heating. The hydrolysis of 3-acyl derivatives of 5-FU showed a biexponential relationship between in concentration and time which had not been previously observed. The behavior of 3-acetyl-5-FU during hydrolysis can be explained by postulating its initial rapid equilibrium with an intermediate, 2-acetyl-5-FU, which subsequently hydrolyzes to 5-FU or rearranges to 1-acetyl-5-FU, which hydrolyzes to 5-FU. The 2-acetyl intermediate was trapped by its reaction with formaldehyde. The formaldehyde adducts of the symmetrical 2-acetyl intermediate rearranged to yield equal amounts of 1- and 3-acetyloxymethyl-5-FU.

Effect of aminomethyl (N-Mannich base) derivatization on the ability of S6-acetyloxymethyl-6-mercaptopurine prodrug to deliver 6-mercaptopurine through hairless mouse skin.

A series of 9-aminomethyl-S6-acetyloxymethyl-6-mercaptopurine (9-AM-6-AOM-6-MP) prodrugs have been synthesized and characterized, and their ability to deliver total 6-mercaptopurine (6-MP) through hairless mouse skin has been measured. The 9-AM-6-AOM-6-MP prodrugs are much more soluble in isopropyl myristate (IPM) than S6-acetyloxymethyl-6-MP (6-AOM-6-MP) itself or the corresponding 7-aminomethyl-6-MP (7-AM-6-MP) prodrugs. The 9-AM-6-AOM-6-MP prodrugs were all more effective (1.8-4 times) than 6-AOM-6-MP at delivering total 6-MP, except for the piperidylmethyl derivative which only gave a comparable rate of delivery. The 9-AM-6-AOM-6-MP prodrugs were also more effective (7-27 times) than the corresponding 7-AM-6-MP prodrugs at delivering 6-MP, except for the diethylaminomethyl derivative which only gave a comparable rate of delivery. In contrast to the 9-aminomethyl-S6-pivaloyloxymethyl-6-MP (9-AM-6-POM-6-MP) derivatives which generally delivered as much or more intact S6-pivaloyloxymethyl-6-MP (6-POM-6-MP) as 6-MP, the 9-AM-6-AOM-6-MP derivatives delivered mainly (80-95%) 6-MP from IPM. There was a direct correlation between log experimental permeability coefficients for delivery of total 6-MP (P sigma) and the calculated solubility parameter values for the 9-AM-6-AOM-6-MP prodrugs(delta j), with the P sigma generally decreasing as the value of delta j approached that of the vehicle, IPM.(ABSTRACT TRUNCATED AT 250 WORDS)