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Biomedical subjects

P Workman

Publications and source records attributed to P Workman.

At least 73 records · Page 4Linked to original sources
PubMed1996

Synthesis and anti-cancer activity of 2,6-disubstituted N-methylpiperidine derivatives and their N-oxides.

Bioreducible anti-tumour agents are prodrugs which are intended to be inactive in normal cells, but are able to undergo metabolic reduction in cancer cells to produce toxic species that can damage biomolecules. A series of N-oxides of heterocyclic aliphatic amines were designed and prepared as mentioned below as bioreducible drugs based on the reported anti-cancer activity of 2,6-bis(halomethyl)piperidines. In order to study structure-activity relationships in these conformationally restricted nitrogen mustards, samples of cis- and trans-2,6-dihydroxymethyl-N-methylpiperidine were prepared and converted into a number of carbamate or halogen derivatives. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation. The corresponding N-oxides were also prepared for comparison in cytotoxicity tests. In total, 21 new compounds were synthesized plus cis-N-methyl-2,6-bis(chloromethyl)-piperidine (prepared previously but lacking spectroscopic data) and tested against two human colon carcinoma cell lines, HT 29 (high DT-diaphorase) and BE (no DT-diaphorase), under oxic and hypoxic conditions. The majority of the free bases were equally toxic against both cell lines. The most toxic compounds were cis- and trans-N-methyl-2,6-bis(bromomethyl)piperidine with oxic IC50 values between 6 and 11 microM against both cell lines. The N-oxides were relatively non-toxic under both oxic and hypoxic conditions apart from the N-oxide of trans-N-methyl-2,6-bis(bromomethyl)-piperidine. Their low toxicity suggested that the N-oxides are not reduced under hypoxic conditions. We conclude that: (i) 2,6-disubstituted N-methylpiperidine derivatives are chemically versatile cytotoxic entities that are suitable for prodrugging to enhance their therapeutic selectivity; and (ii) N-oxide prodrugs of these compounds are deactivated chemically and display reduced cytotoxicity compared to the parent amines but are apparently not reduced under hypoxic conditions. At least in the colorectal cell lines tested the latter issue would need to be addressed by modifying the redox properties in future work to progress this approach.

Antineoplastic Agents, Alkylating↗
PubMed1996

Synthesis and antiproliferative activity of tyrphostins containing quinoline moieties.

Tyrphostins are a series of benzylidenemalononitrile derivatives synthesized by condensing aromatic aldehydes with malononitrile derivatives. The use of heteroaromatic aldehydes in this process has received little attention. Accordingly, 27 tyrphostins containing a 2-, 3- or 4-substituted quinoline moiety were synthesized, of which 21 are novel compounds Compounds containing the 2-aminoethene-1, 1-dinitrile moiety in each series were the most potent inhibitors of the EGF receptor kinase in a cell-free enzyme assay (compounds 2, 11 and 20), having IC50 values of 1.7, 27.0 and 4.7 microM respectively. For each R group substitution the order of potency was 2-quinolines > 4-quinolines > 3-quinolines. Compounds 2, 11 and 20 were unable to inhibit the epidermal growth factor (EGF) receptor autophosphorylation in intact cells; however, they were able to inhibit the EGF-dependent phosphorylation of a 50 kDa protein. These three compounds were able to inhibit EGF-dependent proliferation in a fibroblast cell more efficiently than serum-stimulated proliferation, suggesting that their mechanism of action may be linked to the EGF receptor signalling pathway. Compound 2 exhibited a degree of cell line selectivity in the US National Cancer Institute in vitro human tumour cell line panel. The majority of non-small cell lung cancer lines were relatively resistant to compound 2, while most of the colon, CNS, melanoma and renal lines were relatively sensitive. Further work is required to elucidate the mechanism of action of this interesting group of substituted-quinoline compounds and to determine whether for compounds 2, 11 and 20 this is related to inhibition of EGF receptor function.

Adenocarcinoma↗
PubMed1995

Development and validation of a solid-phase extraction and high-performance liquid chromatographic assay for a novel fluorinated 2-nitroimidazole hypoxia probe (SR-4554) in Balb/c mouse plasma.

N-(2-Hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide, a novel 2-nitroimidazole, is currently being developed as a non-invasive probe for tumour hypoxia. A sensitive (minimum quantifiable level = 25 ng/ml; C.V. = 6.01%) and selective assay has, therefore, been developed for the analysis of this compound in mouse plasma. The assay employed a solid-phase extraction followed by a rapid (10 min) HPLC analysis with UV-photodiode-array detection. No drug-related metabolites were observed in plasma when mice were treated with 180 mg/kg of the drug. The assay has proved to be suitable for studying the plasma pharmacokinetics of this fluorinated 2-nitroimidazole in mice.

Animals↗
PubMed1995

Development and validation of a sensitive solid-phase-extraction and high-performance liquid chromatography assay for the bioreductive agent tirapazamine and its major metabolites in mouse and human plasma for pharmacokinetically guided dose escalation.

A sensitive solid-phase-extraction and high-performance liquid chromatography (HPLC) method has been developed to investigate the pharmacokinetics and metabolism of the hypoxic-cell cytotoxic agent tirapazamine (1,2,4-benzotriazine-3-amine 1,4-di-N-oxide; WIN 59075, SR 4233), currently in phase I/II studies in the United Kingdom and United States. A sample extraction and concentration process was devised using strong cation-exchange Bond Elut cartridges. Tirapazamine, the mono and zero-N-oxide metabolites (WIN 64012, WIN 60109) were isocratically resolved using a microBondapak phenyl HPLC column and measured using photodiode-array detection. The minimal quantifiable level (MQL) of tirapazamine was 40 ng/ml in mouse plasma and 20 ng/ml in human plasma. Recovery was consistently greater than 80% for all compounds over the concentration range of 20 ng/ml to 20 micrograms/ml. No significant decomposition was observed following up to three freeze/thaw cycles and storage at -70 degrees C for 52 days. The assay was accurate and reproducible, with measured values lying within the limits of defined acceptance criteria. Additional studies to investigate the degree of plasma protein binding showed that tirapazamine did not bind extensively to plasma proteins (binding, 9.7% +/- 0.1% and 18.7% +/- 1.3% in mouse and human plasma, respectively). These small species differences in protein binding are unlikely to have any major impact on the extrapolation of pharmacokinetic data from mice to humans. The assay has now been successfully applied to investigate the pharmacokinetics and metabolism of tirapazamine in mice and patients as part of a pharmacokinetically guided dose-escalation strategy for phase I clinical trials.

Animals↗
PubMed1995

Growth arrest vs direct cytotoxicity and the importance of molecular structure for the in vitro anti-tumour activity of ether lipids.

A panel of 25 different lipid agents was evaluated for in vitro activity against HT29 human colon carcinoma and HL60 promyelocytic leukaemia cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The structure-activity relationships seen with this series, including those for four sets of positional or stereoisomers, indicate that specific receptor proteins are unlikely as targets for anti-tumour lipid (ATL) action. Additional data confirm the lack of involvement of the platelet-activating factor receptor in particular and suggest that metabolic stability is a most important determinant of ATL activity. More detailed studies, with 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET18-OCH3) and (+/-)-2-(Hydroxy[tetrahydro-2-(octadecyloxy)methylfuran-2- yl]methoxyphosphinyloxy)-N,N,N,-trimethylethaniminium hydroxide (SRI 62-834), suggest three different modes of activity, depending on drug concentration and exposure time. Low doses of up to 5 microM in standard serum-containing medium cause population growth arrest after prolonged exposure. Growth arrest was associated with a leaky G2/M block as determined by flow cytometry. These effects are reversible. Intermediate concentrations (5-40 microM) were cytotoxic, causing a net reduction in cell numbers after 2-3 days. At even higher concentrations, all lipids caused rapid, direct membrane lysis. When the clonogenic assay was used to assess the effects of ATLs, most agents reduced colony formation at concentrations above 5 microM. However, some compounds proved stimulatory at nanomolar concentrations, suggesting that they might possess mitogenic properties. These results, particularly those concerning the concentration and time dependence, may be relevant to current clinical trials with ether lipids.

Animals↗
PubMed1995

The novel fluorinated 2-nitroimidazole hypoxia probe SR-4554: reductive metabolism and semiquantitative localisation in human ovarian cancer multicellular spheroids as measured by electron energy loss spectroscopic analysis.

The novel fluorinated 2-nitroimidazole SR-4554 is undergoing preclinical development as a magnetic resonance spectroscopy and imaging probe for hypoxic tumour cells. We have used electron energy loss spectroscopic analysis (EELS) to show selective reduction and differential subcellular localisation of SR-4554 in human ovarian multicellular spheroids. SR-4554 was demonstrated to be metabolised by these A2780 cells under hypoxic but not under normal aerobic cell culture conditions. The EELS technique illustrated that the relative amount of drug within the cytoplasm of cells from both the inner region (150-160 microns from edge) and outer edge of the spheroid did not differ significantly after an initial 3 h incubation with drug. In contrast, an 8-fold differential between the amount of drug retained in the cytoplasm (primarily ribosomes and endoplasmic reticulum) of cells from the inner vs outer regions of the spheroids was observed following a subsequent 2 h 'chase' culture in drug-free medium. Within cells from the hypoxic region of the spheroid, SR-4554 was mainly associated with the endoplasmic reticulum, nucleus and the cytoplasmic side of intracellular vesicles and also to a lesser extent with the nuclear periphery. Interestingly, the drug was only weakly associated with the mitochondria and plasma membrane of the cells. The characteristics of cellular and subcellular distribution of SR-4554 are consistent with the hypothesis that 2-nitroimidazole compounds undergo hypoxia-mediated enzymatic reduction to reactive species. These reactive species are selectively retained in the cells in which they are metabolised through covalent association with subcellular components. These findings provide additional support for the clinical development of the drug as a non-invasive probe for tumour hypoxia and at the same time illustrate the utility of the EELS technique for examining the heterogeneity of drug distribution both between and within cells.

Aerobiosis↗
PubMed1995

Catalytic properties of NAD(P)H:quinone acceptor oxidoreductase: study involving mouse, rat, human, and mouse-rat chimeric enzymes.

NAD(P):quinone acceptor oxidoreductase (quinone reductase) (DT-diaphorase, EC 1.6.99.2) is involved in the process of reductive activation of cytotoxic antitumor quinones and nitrobenzenes. In this study, we initially examined the relative abilities of mouse, rat, and human quinone reductases to reduce two prodrugs, CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] and EO9 [5-(1-aziridinyl)-3-(hydroxymethyl)-2-(3-hydroxy-1-propenyl)-1- methyl-1H-indole-4,7-dione]. By using Escherichia coli-expressed quinone reductases and evaluating them under identical conditions, we confirmed previous finding showing that the human enzyme is not as effective as the rat enzyme in reducing CB 1954 and EO9, although the two enzymes have similar NAD(P)H-menadione reductase activities. Interestingly, although the amino acid sequence of mouse quinone reductase is more homologous to that of the rat enzyme, we found that the mouse enzyme behaves similarly to the human enzyme in its ability to reduce these compounds and to generate drug-induced DNA damage. To determine the region of quinone reductase that is responsible for the catalytic differences, two mouse-rat chimeric enzymes were generated. MR-P, a chimeric enzyme that has mouse amino-terminal and rat carboxy-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of rat quinone reductase, and RM-P, a chimeric enzyme that has rat amino-terminal and mouse carboxyl-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of mouse quinone reductase. In addition, MR-P and RM-P were found to be inhibited by flavones with Ki values similar to those for rat and mouse quinone reductases, respectively. Based on these results, we propose that the carboxyl-terminal portion of the enzyme plays an important role in the reduction of cytotoxic drugs and the binding of flavones.

Amino Acid Sequence↗
PubMed1994

Differential intracellular processing of the anthracycline drug ME2303 in doxorubicin-sensitive (A2780) and -resistant (A2780AD) human ovarian cancer cells as studied with confocal laser scanning microscopy and image analysis.

Laser scanning confocal microscopy has been used to follow the uptake and efflux of the 2-fluoroglycoside of doxorubicin, ME2303, in live cultures of the human ovarian cancer cell line A2780 and its doxorubicin-resistant variant A2780AD. Our methods combine confocal laser scanning microscopy and image analysis to examine the dynamics of anthracycline drugs in cancer cells. Cytotoxicity determined by MTT dye reduction showed that A2780AD cells were more than 400 times less sensitive to doxorubicin compared to A2780 cells but almost 9 times more sensitive to ME2303 compared to doxorubicin. The naturally fluorescent drug was tracked within live cells at 37 degrees C to provide time-course information in relation to nuclear and Golgi-associated cellular domains, as indicated by BODIPY FL ceramide-associated fluorescence. In both cell types, ME2303 was characterised by strong nuclear membrane and perinucleolar fluorescence and as a localised punctate pattern within the nucleus. A2780AD cells accumulated ME2303 in their nuclei at a much reduced rate compared to the doxorubicin-sensitive cells, and ME2303 efflux from resistant cell nuclei was approximately twice as fast as from A2780 cells. The relative uptake of ME2303 into Golgi-associated domains and the nucleus were monitored simultaneously during the initial 35 min of exposure to 10 microM ME2303 and during the first 45 min of a "chase" culture following exposure to 20 microM ME2303. ME2303 was detectable within the Golgi-associated domains of A2780 cells several minutes later and in less relative concentration than in A2780AD cells 15 min into a chase culture. Our results suggest the direct involvement of differences in drug processing via the Golgi apparatus in the expression of P-glycoprotein-related drug resistance.

Coloring Agents↗
PubMed1994

Direct measurement of pO2 distribution and bioreductive enzymes in human malignant brain tumors.

PURPOSE: To measure the oxygen status of human malignant brain tumors in vivo and to determine the activities and expression of bioreductive enzymes in these same human brain tumor samples, as a means of assessing their suitability as targets for bioreductive drug therapy. METHODS AND MATERIALS: A polarographic oxygen electrode was used to measure the intratumoral oxygen tension in twenty patients with malignant brain tumors during open brain surgery, performed under standard anaesthetic conditions. Six different tracks, each with a path length of 22 mm, were recorded per patient representing 192 readings. Following pO2 measurements the tumors were resected and stored in liquid N2 for subsequent bioreductive enzyme analysis. Eight human malignant brain tumors were assessed, by enzyme activity and western blot expression, for the presence of various bioreductive enzymes. These enzymes included DT-diaphorase, NADH cytochrome b5 reductase, and NADPH cytochrome P-450 reductase. Of these eight gliomas analyzed six samples were incubated with the bioreductive drug tirapazamine, in the presence of cofactor(s), to establish whether human brain tumors could metabolize this compound. RESULTS: Both the high grade intrinsic and metastatic brain tumors showed significant regions of hypoxia. All the tumors subjected to enzyme profiling contained the bioreductive enzymes, DT-diaphorase, NADH cytochrome b5 reductase and NADPH cytochrome P-450 reductase. Also all six of the brain tumors investigated could metabolize tirapazamine to the two-electron reduction product. CONCLUSION: These findings would favor primary brain tumors as suitable targets for bioreductive therapy.

Brain Neoplasms↗
PubMed1994

Relative importance of DT-diaphorase and hypoxia in the bioactivation of EO9 by human lung tumor cell lines.

PURPOSE: Although a number of bioreductive agents are substrates for purified DT-diaphorase the role of this enzyme in either activation or detoxification of these agents in the whole cell is unclear. The aim of this study was to determine the role of DT-diaphorase in the metabolic activation of EO9 under both aerobic and hypoxic conditions. METHODS AND MATERIALS: A panel of lung cancer cell lines was used and drug sensitivity was determined by clonogenic or tetrazolium-dye-based assays. Activities of DT-diaphorase, cytochrome P450 and cytochrome b5 reductase were determined spectrophotometrically by following the reduction of cytochrome c. RESULTS: Small-cell lung cancer cell lines showed a 600-fold range in DT-diaphorase activities but levels were much higher in three of the four non-small-cell lines. Activities of cytochromes P450 and b5 reductase were much lower than those of DT-diaphorase and showed much less variation between cell lines. There was no relationship between the activities of any of the enzymes and aerobic sensitivity to SR 4233, BCNU and cis-platin. Under aerobic conditions there was a clear correlation between DT-diaphorase activity and sensitivity to EO9. The small-cell lines were much more resistant to EO9 than the DT-diaphorase rich non-small-cell lines. A doxorubicin resistant variant of one of the small-cell lines (H69LX10) did not show cross resistance to EO9 but did show a small degree (3-fold) of cross resistance to SR 4233. Under hypoxic conditions, cell lines with high levels of DT-diaphorase showed only a small increase in sensitivity to EO9 (1.5-7 fold); cell lines with low levels of activity showed a 10-37-fold increase in sensitivity. CONCLUSION: These results suggest that under hypoxic conditions, EO9 is metabolized by 1-electron reducing enzymes to a toxic species. This reduction product is oxygen sensitive but a similar degree of activation is obtained under aerobic conditions in cell lines with high levels of 2-electron reducing DT-diaphorase.

Antineoplastic Agents↗
PubMed1994

Stimulation of intracellular free calcium increases by platelet-activating factor in HT29 colon carcinoma cells. Spectrofluorimetric and preliminary spatio-temporal analysis using confocal laser scanning fluorescence imaging microscopy.

We examined the ability of platelet-activating factor (PAF) and its lyso derivative (lyso-PAF) to elicit increases in intracellular free calcium concentration ([Ca2+]i) in HT29 human colon carcinoma cells. Using spectrofluorimetric analysis with indo-1 as the [Ca2+]i reporter molecule, we found that 1-10 microM concentrations of both lipids stimulated substantial, reversible, monophasic [Ca2+]i elevations. Evidence was obtained that the two lipids may act via specific receptors to release Ca2+ from internal stores. Homologous desensitization was observed in both cases and PAF and lyso-PAF were also able to desensitize cells reciprocally (heterologous desensitization). The potent PAF receptor antagonist WEB 2086 (3-[4-(chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2, 4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4-morpholinyl)-1-propano ne) successfully blocked PAF-induced [Ca2+]i elevations, but did not affect rises in response to lyso-PAF, suggesting that lyso-PAF may act through a different cellular receptor or mechanism. Higher concentrations (> 10 microM) of PAF resulted in non-reversible [Ca2+]i elevations which were caused by Ca2+ influx following membrane lysis. However, the WEB 2086 insensitivity of these effects and the resultant cellular toxicity clearly showed that such events were mechanistically distinct from the reversible [Ca2+]i elevations apparently operating via WEB 2086-sensitive receptors. Preliminary spatio-temporal observations, using confocal microscopy and fluo-3 as the [Ca2+]i reporter molecule, suggested that PAF can also induce [Ca2+]i elevations in the absence of cell lysis in monolayer HT29 cells. Visual impressions were obtained of cellular and subcellular heterogeneity and of [Ca2+]i oscillations in responding cells. However, these need to be interpreted with caution because of the intrinsic limitations of the methodology, particularly using non-ratiometric dyes. The significance of a receptor-mediated, reversible elevation of [Ca2+]i by sub-toxic concentrations of PAF in HT29 colon cancer cells remains to be elucidated, but it is tempting to speculate that PAF might function as a locally acting signalling mediator in these and possibly other tumour cells.

Calcium↗
PubMed1994

Unusually marked hypoxic sensitization to indoloquinone EO9 and mitomycin C in a human colon-tumour cell line that lacks DT-diaphorase activity.

Studies with purified DT-diaphorase have shown that the enzyme is capable of catalyzing a two-electron reduction of the novel indoloquinone EO9 to a DNA-damaging alkylating species. The aim of this study was to determine to what extent DT-diaphorase may be involved in the metabolic activation of EO9 and mitomycin C in both aerobic and hypoxic conditions. Two human colon-carcinoma cell lines were used; HT29 has high levels of DT-diaphorase whilst BE lacks this activity because of a point mutation in the NQOI gene. In aerobic conditions the 2 cell lines show similar sensitivities to a number of cytotoxic drugs including cisplatin, doxorubicin and etoposide. They are equally sensitive to the benzotriazine di-N-oxide SR 4233 but HT29 is more sensitive than BE to mitomycin C and EO9. Sensitivity to SR 4233 is increased by about 100-fold for both cell lines in hypoxic conditions. DT-diaphorase-deficient BE cells show markedly increased sensitivity to mitomycin C and particularly EO9 in hypoxic conditions, whereas DT-diaphorase-rich HT29 cells show little hypoxic sensitization to these agents unless exposed in the presence of dicoumarol. These results suggest that DT-diaphorase can reduce EO9 and mitomycin C to potent cytotoxic species in aerobic conditions, and this activity predominates over the one-electron-reducing enzymes even in hypoxic conditions. In the absence of DT-diaphorase activity, EO9 and mitomycin C are reduced in hypoxic conditions, presumably by one-electron-reducing enzymes, to a similar or greater extent than is achieved with DT-diaphorase.

Aerobiosis↗
PubMed1994

DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9.

Aerobic sensitivity to indoloquinone EO9 has been shown to correlate with cellular levels of the two-electron reducing enzyme DT-diaphorase. However, little is known about the relative roles of one- and two-electron reducing enzymes in the hypoxic cytotoxicity of EO9. We have characterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to EO9. Eight cell lines were then selected for a comparison of aerobic and hypoxic sensitivities. Activities of DT-diaphorase showed a wide range (> 10,000-fold), while activities of the one-electron reducing cytochrome b5 and cytochrome P450 reductases were generally lower and showed only a 15- and 25-fold range respectively. The aerobic cytotoxicity of EO9 was clearly related to the cellular levels of DT-diaphorase (r = 0.87), with higher levels giving increased sensitivity, but not to the levels of one-electron reducing enzymes. In contrast, there was no relationship between sensitivity to BCNU, cisplatin or the bioreductive agent SR 4233 (tirapazamine) and activities of any of these reducing enzymes. Under hypoxic conditions sensitivity to EO9 was markedly increased in cell lines with low levels of DT-diaphorase activity, while cell lines with high levels show only a small increase in sensitivity. This is reflected by a clear correlation (r = 0.98) between cellular DT-diaphorase activity and the ratio of aerobic to hypoxic sensitivity to EO9. However, we have now for the first time demonstrated an inverse correlation (r = 0.93) between the cellular activity of DT-diaphorase and hypoxic sensitivity to EO9, that is sensitivity decreases with increasing DT-diaphorase activity. Moreover, this correlation was lost when cells were exposed to drug in the presence of dicoumarol, supporting an involvement of DT-diaphorase in this relationship. These observations question the previously straightforward role for DT-diaphorase in the metabolic activation of EO9. Whereas DT-diaphorase is associated with increased toxicity in air, it appears to reduce the cytotoxicity of EO9 in hypoxic conditions. This suggests either that the one-electron reduction product of EO9 metabolism, the semiquinone, is more toxic than the two-electron reduction product, the hydroquinone, or that the hydroquinone is not cytotoxic and aerobic toxicity is due to the transient appearance of the semiquinone upon back oxidation of the hydroquinone.

Aerobiosis↗