Biomedical subjects

R H Hargreaves

Publications and source records attributed to R H Hargreaves.

9 recordsLinked to original sources

PubMed2001

Relationship between NAD(P)H:quinone oxidoreductase 1 (NQO1) levels in a series of stably transfected cell lines and susceptibility to antitumor quinones.

To investigate the importance of NAD(P)H:quinone oxidoreductase 1 (or DT-diaphorase; NQO1) in the bioactivation of antitumor quinones, we established a series of stably transfected cell lines derived from BE human colon adenocarcinoma cells. BE cells have no NQO1 activity due to a genetic polymorphism. The new cell lines, BE-NQ, stably express wild-type NQO1. BE-NQ7 cells expressed the highest level of NQO1 and were more susceptible [determined by the thiazolyl blue (MTT) assay] to known antitumor quinones and newer clinical candidates. Inhibition of NQO1 by pretreatment with an irreversible inhibitor, ES936 [5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione], protected BE-NQ7 cells from toxicity induced by streptonigrin, ES921 [5-(aziridin-1-yl)-3-(hydroxymethyl)-1,2-dimethylindole-4,7-dione], and RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone]. RH1 was evaluated further by clonogenic assay for cytotoxic response and was more cytotoxic to BE-NQ7 cells than to BE cells. Cytotoxicity was abrogated by inhibition of NQO1 with ES936 pretreatment. Using a comet assay to evaluate DNA cross-linking, BE-NQ7 cells demonstrated significantly higher DNA cross-links than did BE cells in response to RH1 treatment. DNA cross-linking in BE-NQ7 cells was observed at very low concentrations of RH1 (5 nM), confirming that NQO1 activates RH1 to a potent cross-linking species. Further studies using streptonigrin, ES921, and RH1 were undertaken to analyze the relationship between NQO1 activity and quinone toxicity. Toxicity of these compounds was measured in a panel of BE-NQ cells expressing a range of NQO1 activity (23-433 nmol/min/mg). Data obtained suggest a threshold for NQO1-induced toxicity above 23 nmol/min/mg and a sharp dose-response curve between the no effect level of NQO1 (23 nmol/min/mg) and the maximal effect level (>77 nmol/min/mg). These data provide evidence that NQO1 can bioactivate antitumor quinones in this system and suggest that a threshold level of NQO1 activity is required to initiate toxic events.

Antibiotics, Antineoplastic↗

PubMed2000

Mechanisms of action of quinone-containing alkylating agents: DNA alkylation by aziridinylquinones.

Aziridinyl quinones can be activated by cellular reductases eg. DT-diaphorase and cytochrome P450 reductase to form highly reactive DNA alkylating agents. The mechanisms by which this activation and alkylation take place are many and varied. Using clinically relevant and experimental agents this review will describe many of these mechanisms. The agents discussed are Mitomycin C, EO9 and analogues, diaziridinylbenzoquinones and the pyrrolo[1, 2-alpha]benzimidazolequinones.

Alkylation↗

PubMed2000

The abnormal cytotoxicities of 2,5-diaziridinyl-1,4-benzoquinone-3-phenyl esters.

Several derivatives of 2,5-diaziridinyl-3-phenyl-1,4-benzoquinone have been synthesized and their cytotoxicities in six different human cancer cell lines (H460, H596, HT29, BE, K562 and A2780) have been determined. It was observed that certain phenol-ester derivatives were significantly more cytotoxic in all of the cell lines investigated. These esters were shown to be cleaved by esterases to form a stable meta-phenol and an unstable para-phenol. The meta-phenol was also highly cytotoxic. Several of these compounds were studied in detail using DNA cross-linking, clonogenic, apoptosis and flow cytometry assays. It is proposed that although the phenol-esters and the phenols can efficiently cross-link DNA, this mechanism alone is not sufficient to explain the toxicities of these compounds.

Antineoplastic Agents↗

PubMed1999

Cross-linking and sequence-specific alkylation of DNA by aziridinylquinones. 3. Effects of alkyl substituents.

The cytotoxicities and DNA cross-linking abilities of several alkyl-substituted diaziridinylquinones have been investigated. The cytotoxicities were determined in DT-diaphorase-rich (H460 and HT29) and -deficient (H596 and BE) cell lines. It was shown that the cytotoxicities in these cell lines correlated with the relative rates of reduction by the purified human enzyme and with the cross-linking efficiencies. The rates of reduction by DT-diaphorase were more dependent on the structures of the compounds than the reduction potentials, as determined by cyclic voltammetry. A computer model was also used to explain high efficiency of cross-linking and the GNC sequence selectivity of the reduced methyl-substituted diaziridinylquinones.

Antineoplastic Agents, Alkylating↗

PubMed1999

Development and validation of a sensitive solid-phase extraction and high-performance liquid chromatographic assay for the novel bio-reductive anti-tumor agent RH1 in human and mouse plasma.

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the experimental anticancer agent, RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) which is currently being evaluated by the CRC phase I/II committee. A 500 mg amino propyl solid-phase extraction cartridge was used to isolate RH1 from human plasma. Analysis was performed on a reversed-phase chromatography system using a 15 cm cyanopropyl column and isocratic elution with a 10% methanol-90% water (double distilled) solution. The lower limit of quantitation for RH1 was found to be 0.00375 microg/ml (3.75 ng/ml+/-8.3%) in water and following extraction from plasma. Recovery of >80%(+/-11.9%) was achieved over a five-day validation study. This method was used to carry out pre-clinical studies in BDF mice (standard strain of hybrid mice) at three dose levels (2, 5 and 10 mg/kg of RH1 in 0.9% (w/v) saline via an intraperotoneal injection). Standard Version of PC Winnonlin pharmacokinetic modelling software was used to model the data. A none-compartmental model was used to describe the disposition of RH1 in mice plasma. RH1 was rapidly eliminated from plasma with a mean plasma clearance of 23.4 ml/min, mean volume of distribution of 321.6 ml and mean t(1/2) alpha and beta decays of 4.8 and 9.6 min, respectively. RH1 in human and mouse whole blood and plasma was found to be stable up to 2 h.

Animals↗

PubMed1998

A new screening system for NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones: identification of a new aziridinylbenzoquinone, RH1, as a NQO1-directed antitumor agent.

NAD(P)H:quinone oxidoreductase (NQO1; DT-diaphorase) is elevated in certain tumors, such as non-small cell lung cancer (NSCLC). Compounds such as mitomycin C and streptonigrin are efficiently bioactivated by NQO1 and have been used in an enzyme-directed approach to chemotherapy. Previously, 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) was identified as a potential antitumor agent based on its high rate of bioactivation by human NQO1 and its selective cytotoxicity to cells containing elevated NQO1. RH1, a water-soluble analogue of MeDZQ synthesized in this work, was a better substrate for recombinant human NQO1 than the parent compound. RH1 was, correspondingly, more cytotoxic to human tumor cells expressing elevated NQO1 activity (H460 NSCLC and HT29 human colon carcinoma), as measured by 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium assay, than it was to cells deficient in NQO1 activity (H596 NSCLC and BE human colon carcinoma). RH1 exhibited a greater selective toxicity (ratio of IC50s in H596:H460 and BE:HT29) to cells with elevated NQO1 activity relative to MeDZQ. Additionally, we report the establishment of a stable line of BE human colon carcinoma cells transfected with wild-type human NQO1 (BE-NQ7). BE cells are devoid of NQO1 activity due to a homozygous point mutation in the NQO1 gene. In comparison to the parental cell line, RH1, MeDZQ, and mitomycin C were significantly more cytotoxic to BE-NQ7 cells (17-, 7-, and 3-fold, respectively), confirming that the presence of NQO1 is sufficient to increase cytotoxicity of these antitumor quinones. These data suggest that RH1 may be an effective NQO1-directed antitumor agent for the therapy of tumors with elevated NQO1 activity, such as NSCLC.

Antineoplastic Agents↗

PubMed1997

Cross-linking and sequence specific alkylation of DNA by aziridinyl quinones. 2. Structure requirements for sequence selectivity.

The cytotoxicities and DNA sequence selectivity for guanine-N7 alkylation of 22 mono- and disubstituted 2,5-diaziridinyl-1,4-benzoquinones have been investigated. Several quinones produced patterns of alkylation following reduction with a selectivity for 5'-TGC-3' sequences. This sequence selectivity appeared to be dependent only on the presence of a hydrogen in position-6 of the quinone. A computer model, based on published crystallographic data, was used to explain this selectivity. The sequence selective quinones were generally more cytotoxic that the quinones which reacted randomly.

Alkylation↗

PubMed1996

Cross-linking and sequence specific alkylation of DNA BY aziridinylquinones. 1. Quinone methides.

The cytotoxicities and DNA cross-linking abilities of 16 1,4-benzoquinones have been investigated. All of the alkylmonoaziridinyl-1,4-benzoquinones were able to interstrand crosslink DNA after reduction and were cytotoxic in vitro. Compounds lacking an aziridine group were unable to cross-link DNA and were less cytotoxic. The methyl analogues were shown to preferentially react at TGC sequences. From comparing the structural requirements for crosslinking and the cytotoxicities, a mechanism has been proposed wherein some hydroquinones can associate and react at TGC sequences in DNA. These hydroquinones can subsequently autoxidize to form a reactive quinone methide which reacts at the opposite strand to form a cross-link.

Alkylation↗

PubMed1995

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.

Animals↗