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Biomedical subjects

I J Stratford

Publications and source records attributed to I J Stratford.

At least 19 recordsLinked to original sources
PubMed2000

The relative importance of NADPH: cytochrome c (P450) reductase for determining the sensitivity of human tumour cells to the indolequinone EO9 and related analogues lacking functionality at the C-2 and C-3 positions.

Analogues of EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2[1H-indole-4-7-dione]prop-2-e n-1-ol) which lack functionality at either the C-2 or C-3 position were synthesised. The aim was to establish the importance of each group towards toxicity and to give an indication as to whether substitution at either position altered activation and toxicity after metabolism by cellular NADPH: cytochrome c (P450) reductase (P450R). MDA231 breast cancer cells were transfected with the cDNA for human P450R and stable clones were isolated. These high P450R-expressing clones were used to determine the aerobic and hypoxic toxicity of EO9 and the two analogues that lacked functionality at either C-2 or C-3. The results showed that P450R was strongly implicated in the bioactivation of EO9 and its analogues under both of these conditions. This data also showed that the C-3 functionality was primarily implicated in hypoxic toxicity.

Antineoplastic Agents↗
PubMed2000

Evaluation of the alkaline comet assay and urinary 3-methyladenine excretion for monitoring DNA damage in melanoma patients treated with dacarbazine and tamoxifen.

PURPOSE: To develop, using dacarbazine as a model, reliable techniques for measuring DNA damage and repair as pharmacodynamic endpoints for patients receiving chemotherapy. METHODS: A group of 39 patients with malignant melanoma were treated with dacarbazine 1 g/m(2) i.v. every 21 days. Tamoxifen 20 mg daily was commenced 24 h after the first infusion and continued until 3 weeks after the last cycle of chemotherapy. DNA strand breaks formed during dacarbazine-induced DNA damage and repair were measured in individual cells by the alkaline comet assay. DNA methyl adducts were quantified by measuring urinary 3-methyladenine (3-MeA) excretion using immunoaffinity ELISA. Venous blood was taken on cycles 1 and 2 for separation of peripheral blood lymphocytes (PBLs) for measurement of DNA strand breaks. RESULTS: Wide interpatient variation in PBL DNA strand breaks occurred following chemotherapy, with a peak at 4 h (median 26.6 h, interquartile range 14.75-40.5 h) and incomplete repair by 24 h. Similarly, there was a range of 3-MeA excretion with peak levels 4-10 h after chemotherapy (median 33 nmol/h, interquartile range 20.4-48.65 nmol/h). Peak 3-MeA excretion was positively correlated with DNA strand breaks at 4 h (Spearman's correlation coefficient, r=0.39, P=0.036) and 24 h (r=0.46, P=0.01). Drug-induced emesis correlated with PBL DNA strand breaks (Mann Whitney U-test, P=0.03) but not with peak 3-MeA excretion. CONCLUSIONS: DNA damage and repair following cytotoxic chemotherapy can be measured in vivo by the alkaline comet assay and by urinary 3-MeA excretion in patients receiving chemotherapy.

Adenine↗
PubMed2000

NADPH:cytochrome c (P450) reductase activates tirapazamine (SR4233) to restore hypoxic and oxic cytotoxicity in an aerobic resistant derivative of the A549 lung cancer cell line.

Tirapazamine (TPZ, SR4233, WIN 59075) is a bioreductive drug that is activated in regions of low oxygen tension to a cytotoxic radical intermediate. This labile metabolite shows high selective toxicity towards hypoxic cells, such as those found in solid tumours. Under aerobic conditions, redox cycling occurs with subsequent generation of superoxide radicals, which are also cytotoxic. NADPH:cytochrome c (P450) reductase (P450R) is a one-electron reducing enzyme that efficiently activates TPZ. Recently a derivative of the A549 non-small cell lung cancer cell line (A549c50) was generated that showed substantially reduced P450R activity compared to its parental line (Elwell et al (1997) Biochem Pharmacol 54: 249-257). Here, it is demonstrated that the A549c50 cells are markedly more resistant to TPZ under both aerobic and hypoxic conditions. In addition, these cells have a dramatically impaired ability to metabolize TPZ to its two-electron reduction product, SR4317, under hypoxic conditions when compared to wild-type cells. P450R activity in the A549c50 cells was reintroduced to similar levels as that seen in the parental A549 cells by transfection of the full-length cDNA for human P450R. These P450R over-expressing cells exhibit restored sensitivity to TPZ under both aerobic and hypoxic conditions, comparable to that found in the original parental A549 cells. Further, the ability of the transfected cells to metabolize TPZ to SR4317 under hypoxic conditions is also shown to be restored. This provides further evidence that P450R can play an important role in the activation, metabolism and toxicity of this lead bioreductive drug.

Antineoplastic Agents↗
PubMed2000

Pretreatment apoptosis in carcinoma of the cervix correlates with changes in tumour oxygenation during radiotherapy.

A relationship between hypoxia and apoptosis has been identified in vitro and in experimental tumours. The aim of this study was to investigate the relationship between apoptosis, hypoxia and the change in oxygenation during radiotherapy in human squamous cell carcinoma of the cervix. Forty-two patients with locally advanced disease underwent pretreatment evaluation of tumour oxygenation using an Eppendorf computerized microneedle electrode. Twenty-two of these patients also had a second evaluation of tumour oxygenation after receiving 40-45 Gy external beam radiotherapy. Paraffin-embedded histological sections were obtained from random pretreatment biopsies for all 42 patients. Apoptotic index (AI) was quantified by morphology on TUNEL stained sections. No correlation was found between pretreatment measures of AI and either the median pO2 (r = 0.12, P = 0.44) or percentage of values < 5 mmHg (r = -0.02, P = 0.89). A significant positive correlation was found between AI and the change in tumour oxygenation (ratio of pre:post-treatment % values < 5 mmHg) following radiotherapy (r = 0.61, P = 0.002). The lack of correlation between apoptosis and hypoxia may occur because the Eppendorf measures both acute and chronic hypoxia, and the relative ability of acute hypoxia to induce apoptosis is unknown. These results indicate that cell death via apoptosis may be a mechanism of tumour reoxygenation during radiotherapy.

Adult↗
PubMed1999

Bioreductive activation of a series of indolequinones by human DT-diaphorase: structure-activity relationships.

A series of indolequinones including derivatives of EO9 bearing various functional groups and related indole-2-carboxamides have been studied with a view to identifying molecular features which confer substrate specificity for purified human NAD(P)H:quinone oxidoreductase (DT-diaphorase), bioreductive activation to DNA-damaging species, and selectivity for DT-diaphorase-rich cells in vitro. A broad spectrum of substrate specificity exists, but minor changes to the indolequinone nucleus have a significant effect upon substrate specificity. Modifications at the 2-position are favorable in terms of substrate specificity as these positions are located at the binding site entrance as determined by molecular modeling studies. In contrast, substitutions at the (indol-3-yl)methyl position with bulky leaving groups or a group containing a chlorine atom result in compounds which are poor substrates, some of which inactivate DT-diaphorase. Modeling studies demonstrate that these groups sit close to the mechanistically important amino acids Tyr 156 and His 162 possibly resulting in either alkylation within the active site or disruption of charge-relay mechanisms. An aziridinyl group at the 5-position is essential for potency and selectivity to DT-diaphorase-rich cells under aerobic conditions. The most efficient substrates induced qualitatively greater single-strand DNA breaks in cell-free assays via a redox mechanism involving the production of hydrogen peroxide (catalase inhibitable). This damage is unlikely to form a major part of their mechanism of action in cells since potency does not correlate with extent of DNA damage. In terms of hypoxia selectivity, modifications at the 3-position generate compounds which are poor substrates for DT-diaphorase but have high hypoxic cytotoxicity ratios.

Amino Acid Sequence↗
PubMed1999

Nitric oxide synthases catalyze the activation of redox cycling and bioreductive anticancer agents.

Nitric oxide synthases (NOSs) play a crucial role in the control of blood flow, memory formation, and the immune response. These proteins can be structurally divided into oxygenase and reductase domains. The reductase domain shares a high degree of sequence homology with P450 reductase, which is thought to be the major enzyme responsible for the one-electron reduction of foreign compounds, including bioreductive antitumor agents currently undergoing clinical trials. In view of the structural similarities between NOS and P450 reductase, we investigated the capacity of NOS to reduce the hypoxic cytotoxin tirapazamine, the antitumor agent doxorubicin, and also the redox cycling compound menadione. All three isoforms exhibited high levels of activity toward these compounds. In the case of doxorubicin and menadione, the activity of NOS II was 5-10-fold higher than the other enzymes, whereas with tirapazamine, the activities were broadly similar. NOS-mediated metabolism of tirapazamine resulted in a large increase in plasmid DNA strand breaks, demonstrating that the reduction was a bioactivation process. In addition, tirapazamine inhibited NOS activity. Because nitric oxide is implicated in maintaining tumor vascular homeostasis, it is conceivable that tirapazamine could potentiate its own toxicity by increasing the degree of hypoxia. This study suggests that the NOSs could play a key role in the therapeutic effects of tirapazamine, particularly because NOS activity is markedly increased in several human tumors. In addition, the presence of NOS in the heart indicates that these enzymes may contribute to the cardiotoxicity of redox cycling drugs, such as doxorubicin.

Animals↗
PubMed1999

Prodrugs for targeting hypoxic tissues: regiospecific elimination of aspirin from reduced indolequinones.

A series of regioisomeric derivatives of a 1-methylindole-4,7-dione were synthesised, substituted with a 2-acetoxybenzoate leaving group linked through the (indol-2-yl)methyl or (indol-3-yl)methyl (or propenyl) positions. Reductive elimination of the leaving group occurred from the (indol-3-yl)methyl derivatives but not the 2-substituted regioisomers, indicating that only the C-3 position may be utilised in bioreductively-activated drug delivery, which was demonstrated with an aspirin prodrug.

Animals↗
PubMed1999

Heterocyclic analogues of L-citrulline as inhibitors of the isoforms of nitric oxide synthase (NOS) and identification of N(delta)-(4,5-dihydrothiazol-2-yl)ornithine as a potent inhibitor.

L-Thiocitrulline is a known potent inhibitor of several isoforms of nitric oxide synthase (NOS). To explore the structure-activity relationships (SARs) for this molecule in more depth than has previously been reported, three analogues substituted at the sulphur of the isothioureas have been synthesised. In two of these, the S-substituent was 'tied back' sterically by cyclisation to the nitrogen remote from the amino-acid unit. N(delta)-(4,5-Dihydrothiazol-2-yl)ornithine was identified as an inhibitor of rat inducible and constitutive isoforms of NOS and of a constitutive NOS derived from a human tumour xenograft. Analogous N(delta)-(thiazol-2-yl)ornithines were less active, whereas the corresponding N(delta)-(oxazol-2-yl)ornithine and N(delta)-(pyrimidin-2-yl)ornithine failed completely to inhibit NOS. A new efficient preparation of the critical synthetic intermediate, N(alpha)-Boc-thiocitrulline t-butyl ester, has been developed. Further exploration of the SAR with 2-amino-5-(heterocyclylthio)pentanoic acids (synthesised from 2-(Boc-amino)-5-bromopentanoic acid t-butyl ester), with N-(4-aminobutyl)thiourea and with 2-(4-aminobutylamino)-4,5-dihydrothiazole enabled refinement of our previous model for binding of the substrate, L-arginine, and the inhibitors to NOS.

Animals↗
PubMed1999

Does reductive metabolism predict response to tirapazamine (SR 4233) in human non-small-cell lung cancer cell lines?

The bioreductive drug tirapazamine (TPZ, SR 4233, WIN 59075) is a lead compound in a series of potent cytotoxins that selectively kill hypoxic rodent and human solid tumour cells in vitro and in vivo. Phases II and III trials have demonstrated its efficacy in combination with both fractionated radiotherapy and some chemotherapy. We have evaluated the generality of an enzyme-directed approach to TPZ toxicity by examining the importance of the one-electron reducing enzyme NADPH:cytochrome P450 reductase (P450R) in the metabolism and toxicity of this lead prodrug in a panel of seven human non-small-cell lung cancer cell lines. We relate our findings on TPZ sensitivity in these lung lines with our previously published results on TPZ sensitivity in six human breast cancer cell lines (Patterson et al (1995) Br J Cancer 72: 1144-1150) and with the sensitivity of all these cell types to eight unrelated cancer chemotherapeutic agents with diverse modes of action. Our results demonstrate that P450R plays a significant role in the activation of TPZ in this panel of lung lines, which is consistent with previous observations in a panel of breast cancer cell lines (Patterson et al (1995) Br J Cancer 72: 1144-1150; Patterson et al (1997) Br J Cancer 76: 1338-1347). However, in the lung lines it is likely that it is the inherent ability of these cells to respond to multiple forms of DNA damage, including that arising from P450R-dependent TPZ metabolism, that underlies the ultimate expression of toxicity.

Antineoplastic Agents↗
PubMed1999

Synthesis and cytotoxic activity of N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates.

As analogs of the widely used anti-tumor agents, N-(2-chloroethyl)-N-nitrosoureas, N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates were synthesized by nitration following the reaction of the appropriate amines or alcohols with 2-chloroethyl isocyanate. All tested compounds exert cytotoxic effect with IC50 values of 10(-4) to 10(-6) M and most of them show somewhat higher cytotoxicity in nitrogen than in air.

Air↗
PubMed1999

A similarity model for the human angiogenic factor, thymidine phosphorylase/platelet derived-endothelial cell growth factor.

Thymidine phosphorylase (EC 2.4.2.4), identical to the angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), is up-regulated in several tumour types. A similarity model of human thymidine phosphorylase was built, based on the crystal structure of the Escherichia coli enzyme. The high residue conservation between the two enzyme sources (39% sequence identity and 53% sequence similarity) aided model building. The human model was very similar to the E. coli enzyme's crystal structure, with the main tertiary structure difference being the destruction of helix 15 in E. coli by the presence of a loop in the human model. The model was used to rationalize the nature of the binding of the substrates thymine and thymidine, and of known inhibitors using a quantitative docking algorithm. Ab initio calculations on the nM inhibitor 5-chloro-6-(1-(2-iminopyrrolidinyl)methyl)uracil hydrochloride gave its conformation and distribution of charge. Subsequent quantitative docking studies have led to the suggestion, for the first time, that this inhibitor behaves as an oxycarbenium ion transition-state analogue, explaining its strong reported inhibition.

Amino Acid Sequence↗
PubMed1998

The influence of elevated levels of platelet-derived endothelial cell growth factor/thymidine phosphorylase on tumourigenicity, tumour growth, and oxygenation.

PURPOSE: Investigation of the effect of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) on various aspects of tumour growth in a xenograft model, including growth rate, tumourigenicity and oxygenation levels. METHODS AND MATERIALS: MDA 231 breast cancer cells overexpressing PD-ECGF/TP protein were made by retroviral transduction. These cells were grown in vitro and in vivo as xenografts. Direct measurement of tumours was used to record growth parameters, while the comet assay with the bioreductive drug RSU 1069 was used to assess tumour cell oxygenation. RESULTS: We report that MDA 231 breast tumour cell lines expressing an increased range of levels of PD-ECGF/TP have increased tumourigenicity positively related to the level of PD-ECGF/TP when implanted in nude mice. As previously reported, tumours grown from these overexpressing cell lines grew faster than the parental line. These tumours expressed higher levels of TP activity and showed increased immunocytochemical staining for PD-ECGF. In addition, the rate of growth was found to be positively related to the level of PD-ECGF/TP expressed by the tumour cells. When the comet assay was used to compare the oxygenation status of cells between the parental and PD-ECGF/TP overexpressing tumours, the latter were found to have a larger proportion of well oxygenated cells. This is consistent with these tumours having an increased and functionally competent vascular supply in response to the expression of PD-ECGF/TP. CONCLUSION: PD-ECGF/TP appears to be capable of influencing tumourigenicity, angiogenesis and tumour growth in a proportional manner and can directly influence tumour oxygenation levels via its role in formation of functional vasculature.

Animals↗
PubMed1998

Indolequinone antitumor agents: reductive activation and elimination from (5-methoxy-1-methyl-4,7-dioxoindol-3-yl)methyl derivatives and hypoxia-selective cytotoxicity in vitro.

A series of indolequinones bearing a variety of leaving groups at the (indol-3-yl)methyl position was synthesized by functionalization of the corresponding 3-(hydroxymethyl)indolequinone, and the resulting compounds were evaluated in vitro as bioreductively activated cytotoxins. The elimination of a range of functional groups-carboxylate, phenol, and thiol-was demonstrated upon reductive activation under both chemical and quantitative radiolytic conditions. Only those compounds which eliminated such groups under both sets of conditions exhibited significant hypoxia selectivity, with anoxic:oxic toxicity ratios in the range 10-200. With the exception of the 3-hydroxymethyl derivative, radiolytic generation of semiquinone radicals and HPLC analysis indicated that efficient elimination of the leaving group occurred following one-electron reduction of the parent compound. The active species in leaving group elimination was predominantly the hydroquinone rather than the semiquinone radical. The resulting iminium derivative acted as an alkylating agent and was efficiently trapped by added thiol following chemical reduction and by either water or 2-propanol following radiolytic reduction. A chain reaction in the radical-initiated reduction of these indolequinones (not seen in a simpler benzoquinone) in the presence of a hydrogen donor (2-propanol) was observed. Compounds that were unsubstituted at C-2 were found to be up to 300 times more potent as cytotoxins than their 2-alkyl-substituted analogues in V79-379A cells, but with lower hypoxic cytotoxicity ratios.

Animals↗
PubMed1998

Thymidine phosphorylase moderates thymidine-dependent rescue after exposure to the thymidylate synthase inhibitor ZD1694 (Tomudex) in vitro.

The inhibition of de novo thymidine (dThd) synthesis by the novel folate-based thymidylate synthase (TS) inhibitor ZD1694 (Tomudex) can achieve tumor cell-specific cytotoxicity in vivo. However, nucleosides in the surrounding microenvironment of tumors may be used by the salvage pathway to regenerate any depleted pools, thus providing an efficient mechanism through which to circumvent the ZD1694-dependent toxicity. Anabolism of dThd to dTMP by dThd kinase (TK) is the first committed step in the dThd salvage pathway. However, dThd phosphorylase (dThdPase) can compete with TK by catalyzing the reversible phosphorolytic cleavage of dThd to thymine and deoxyribose 1-phosphate and rendering the salvaged dThd metabolically unavailable. Both TK and dThdPase are up-regulated in some tumors, and their relative importance is not fully defined. We have studied the influence of dThdPase expression on the capacity of exogenous dThd to reverse ZD1694-dependent growth inhibition and have shown that both intra- and extracellular dThdPase activity can effectively moderate dThd-rescue. This suggests that tumor levels of dThdPase may be an important factor in the outcome of ZD1694 therapy.

Antimetabolites, Antineoplastic↗
PubMed1998

S-2-amino-5-azolylpentanoic acids related to L-ornithine as inhibitors of the isoforms of nitric oxide synthase (NOS).

S-2-Amino-5-(2-aminoimidazol-1-yl)pentanoic acid and S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid have been used as weakly inhibitory lead compounds in the design of 2-amino-5-azolylpentanoic acids which are more potent in their inhibition of nitric oxide synthases. Treatment of 2-(Boc-amino)-5-bromopentanoic acid t-butyl ester with appropriate imidazoles and 1,2,4-triazoles and with tetrazole under basic conditions, followed by acidolytic deprotection, gave many of the required 2-amino-5-azolylpentanoic acids. Tetrazole was alkylated at 1-N and at 2-N in approximately equal amounts whereas the 1,2,4-triazoles reacted principally at 1-N. A nitrile was introduced at the 2-position of the imidazole by reaction of the 2-unsubstituted precursor with 1-cyano-4-dimethylaminopyridine. Of this series of compounds, 2-amino-5-(imidazol-1-yl)pentanoic acid was identified as the most potent member against rat iNOS, rat nNOS and a human-derived cNOS. Examination of the structure-activity relationships for the identity and substitution of the azoles has led to the proposal of a model for the binding of the inhibitors to the binding site for the natural substrate.

Animals↗
PubMed1998

5-substituted analogues of 3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]prop-2-en- 1-ol (EO9, NSC 382459) and their regioisomers as hypoxia-selective agents: structure-cytotoxicity in vitro.

A series of regioisomeric analogues of 3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]prop-2-en-1 -ol (EO9, NSC 382459) with the hydroxymethyl and hydroxypropenyl substituents situated at either the 2- or the 3-position of the indole ring were synthesized. The compound lacking the 2-hydroxypropenyl substituent (31) had similar properties to EO9 under both aerobic and hypoxic conditions against V79 cells and was more potent against a human tumour cell line (A549) than EO9. It was reduced by human DT-diaphorase (DTD) at more than double the rate of EO9, thus implicating the importance of the enzyme activation step. Compound 16 (lacking the 3-hydroxymethyl substituent) was a better substrate for human DTD than EO9, yet exhibited lesser toxicity under both aerobic and hypoxic conditions. The toxicity generated by 16 was attributed to the 5-aziridinyl moiety and suggests a greater contribution from the 3-substituent over the 2-substituent. The toxicity of EO9 was attributed to a combination of the aziridinyl group and the leaving group properties of the 3-hydroxymethyl substituent. In general, compounds with a 5-methylaziridinyl moiety, such as EO8, exhibited substantially better hypoxia-selectivity due to much slower reduction by DTD (20-fold), thus reducing aerobic potency. All compounds had similar electron affinities, as indicated by their one-electron reduction potentials.

Aerobiosis↗
PubMed1998

Enzymology of tirapazamine metabolism: a review.

The enzymology of triapazamine (TPZ, SR 4233, WIN 59075, 3-amino-1,2,4-benzotriazene 1,4-dioxide, Tirazone) has been extensively studied in rodents and to a lesser extent in human systems. While it is clear that the initial reductive step in TPZ activation is enzyme-mediated, there is limited consensus in the published literature as to the relative contributions of the cellular reductases involved. Moreover, not only is the importance of subcellular localization for these putative activating reductase(s) far from clear, but their activity profiles in vivo are poorly defined. The same might also be said of the potential detoxifying enzymes. This review will attempt to establish what common ground exists regarding the enzymology of TPZ metabolism, and will relate the available evidence to the enzyme profiles found in human cell lines in vitro, as well as in xenograft models and human solid tumours.

Animals↗