Biomedical subjects

I J Stratford

Publications and source records attributed to I J Stratford.

At least 19 recordsLinked to original sources

PubMed1995

Synthesis and biological activity of thiazolylindolequinones, analogues of the natural product BE 10988.

A number of analogues of the naturally occurring thiazolylindolequinone BE 10988, a reported potent inhibitor of topoisomerase II, have been prepared and evaluated. The compounds were synthesized from 4-(benzyloxy)-5-methoxy-1-methylindole by appropriate substitution at the indole 3-position followed by standard thiazole ring-forming reactions. The toxicity of these potentially bioreductively activated indolequinones was measured in Chinese hamster V79 cells under aerobic and hypoxic conditions. In addition, toxicity was measured in a human breast cancer cell line that shows amplification of the topo II alpha gene and hypersensitivity to known topo II inhibitors such as mAMSA and mitoxantrone. Using a DNA decatenation assay, a comparison was also made of the inhibitory effects of BE 10988 and mitoxantrone on topo II activity.

Aerobiosis↗

PubMed1995

Reduction of tumour intracellular pH and enhancement of melphalan cytotoxicity by the ionophore Nigericin.

Nigericin is an ionophore which permits the influx of H+ ions into cells down a concentration gradient, thus reducing intracellular pH (pHi) when extracellular pH is low. The effects of nigericin on the pHi of solid murine tumours in vivo were examined using 31P magnetic resonance spectroscopy. Nigericin at 2.5 mg/kg i.p. reduced pHi by 0.2-0.3 pH unit in the KHT and RIF-I tumours but had no effect on pHi in the SCCVII/Ha tumour. In vitro studies have shown that reduced pH can increase the toxicity of melphalan. Therefore, the anti-tumour effect of combining nigericin with melphalan was also examined. Nigericin at 2.5 mg/kg i.p. given before various doses of melphalan resulted in substantial delay in growth of the RIF-I tumour over that induced by melphalan alone. This observation was confirmed by an in vivo/in vitro excision assay, where nigericin given before melphalan produced a 30-fold increase in cell killing. By contrast, no enhancement of melphalan-induced cell killing by nigericin was observed in the KHT and SCCVII/Ha tumours, using growth delay and in vivo/in vitro excision assays, respectively.

Animals↗

PubMed1995

Increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine and modulation of 5-fluoro-2'-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase.

Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast cancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell lysates possessed elevated dThdPase activity and cells had up to 165-fold increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (5-FUdR) was unchanged. Recombinant dThdPase was shown to catalyse directly the phosphorolytic cleavage of 5'-DFUR to 5-FU. Exogenous thymidine (dThd) reversed the toxicity of 5-FUdR on the parental line (1 microM dThd increased the IC50 value 1000-fold), but the dThd rescue was substantially modulated in the dThdPase-expressing clone 4 (1 microM dThd raised the IC50 value 3-fold). We observed a substantial 'bystander' killing effect when small proportions of dThdPase-expressing cells were mixed with parental MCF-7 cells. dThdPase activity was on average 27-fold higher in breast tumours than in normal breast. The levels of wild-type MCF-7 are similar to the low end of the tumour expression. Thus, in some tumours resistance to 5'-DFUR therapy could be due to low dThdPase activity, and transfection to raise the dThdPase levels within the broad tumour range or above it should markedly enhance sensitivity to the prodrug. These results confirm that dThdPase is a major pathway in the metabolic activation of 5'-DFUR, and the bystander effect suggests that this may be a suitable enzyme for gene therapy-directed enzyme/prodrug activation therapy.

Antineoplastic Agents↗

PubMed1995

Importance of P450 reductase activity in determining sensitivity of breast tumour cells to the bioreductive drug, tirapazamine (SR 4233).

P450 reductase (NADPH:cytochrome P450 reductase, EC 1.6.2.4) is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines.

Antineoplastic Agents↗

PubMed1995

Fused pyrazine mono-N-oxides as bioreductive drugs. III. Characterization of RB 90740 in vitro and in vivo.

RB 90740 is the lead compound in a series of aromatic mono-N-oxide bioreductive drugs. The compound shows considerably greater toxicity towards hypoxic verses aerobic mammalian cells in vitro. The differential in concentration required to give the same level of cell killing under these conditions ranges from 3.5 in a human bronchio-alveolar tumor cell line up to 120 in a rodent cell line defective in the repair of DNA strand breaks. The ability of RB 90740 to cause DNA strand breaks under hypoxic conditions was confirmed by alkaline sucrose gradient and pulsed field gel electrophoresis techniques. Despite these properties demonstrated in vitro, RB 90740 was shown not to be cytotoxic to hypoxic cells in experimental murine tumors in vivo. This may be due, in part, to the level of hypoxia (< 0.02% O2) necessary to produce toxicity in vitro.

Animals↗

PubMed1994

Induction of hypoxia in experimental murine tumors by the nitric oxide synthase inhibitor, NG-nitro-L-arginine.

The nitric oxide synthase inhibitor NG-nitro-L-arginine (NOARG) was examined for its ability to alter energy metabolism in three murine tumors using 31P magnetic resonance spectroscopy. NOARG (10 mg/kg, i.v.) increased the inorganic phosphate:total phosphate ratio (Pi:total) 2-3-fold in the KHT, RIF-1, and SCCVII/Ha intradermal back tumors from 30 min to 6 h after injection, but the 31P magnetic resonance spectrum from normal tissue on the mouse back was unchanged after this treatment. NOARG (10 mg/kg, i.v.) injected 30 min before X-rays increased tumor cell survival 3-5-fold in SCCVII/Ha and 50-200-fold in RIF-1, measured using an in vivo/in vitro clonogenic assay. These effects were equivalent to those obtained from clamped tumors, indicating full radiobiological hypoxia. In KHT, only a 2-fold increase in radioresistance was observed after NOARG, which was less than the response of clamped tumors. In RIF-1 tumors, NOARG induced full radiobiological hypoxia when given from 30 min to 6 h prior to X-rays, consistent with the time course for the increase in Pi:total, measured by 31P magnetic resonance spectroscopy. Pi:total after NOARG doses of 0.1-10 mg/kg, i.v., increased in a dose-dependent manner in this tumor. Increased RIF-1 tumor radioresistance was similarly dependent on NOARG dose. The combination of the bioreductive agent RB6145 (300 mg/kg, i.p.) 15 min prior to NOARG (10 mg/kg, i.v.) produced greater than 5 decades of KHT tumor cell killing at 24 h after treatment. This combination also increased Pi:total 4.5-fold over the control value at 24 h in the KHT tumor. Histological examination of tumors at this time indicated extensive necrosis.

Animals↗

PubMed1994

Cyclopropamitosenes, novel bioreductive anticancer agents. Synthesis, electrochemistry, and biological activity of 7-substituted cyclopropamitosenes and related indolequinones.

The synthesis of the indolequinones 8 and 9 starting from methyl 4-(benzyloxy)-5-methoxy-indole-2-carboxylate (10) is described. The methoxy group in the indolequinones 1, 2, 4, 5, and 7-9 can be displaced by various nitrogen nucleophiles (ammonia, 2-methoxyethylamine, aziridine, 2-methylaziridine, pyrrolidine) in 22-88% yield. The resulting amino-substituted quinones, together with their methoxy precursors, were studied by cyclic voltammetry to determine their reduction potentials, which, in DMF solution, lie in the range -1.355 to -1.597 V (vs ferrocene). The cytotoxicity of the compounds towards aerobic and hypoxic mammalian cells was also determined; in general, under aerobic conditions, the cyclopropamitosenes are more toxic than the corresponding pyrrolo[1,2-a]indolequinones, which are in turn more toxic than the simple 1,2-dimethylindolequinones, with many of the compounds in each series showing greater toxicity toward hypoxic cells.

Animals↗

PubMed1994

Potential antitumour mitosenes: relationship between in vitro DNA interstrand cross-link formation and DNA damage in Escherichia coli K-12 strains.

This investigation was aimed at determining the possible relationship between DNA interstrand cross-linking and the cytotoxic activity of potential antitumour mitosene compounds. Mitosenes, possessing two good leaving groups at C-1 and C-10, were found to be able to cross-link calf thymus DNA under hypoxic conditions following sodium dithionite (Na2S2O4) reduction at pH 7.0 and pH 5.5. DNA interstrand cross-linking was pH dependent for most of the mitosenes used, with a higher amount of cross-links formed at pH 5.5 compared to pH 7.0. Without reduction or under aerobic conditions no cross-link formation was detected. The importance of DNA damage for the toxic effect of these mitosenes was assayed by comparing the survival in a DNA repair deficient and a DNA repair proficient E. coli K-12 strain. A correlation between the number of cross-links formed in calf thymus DNA in vitro and the IC50 values in the DNA repair deficient E. coli strain was found. The effect of hypoxia on toxicity of mitosenes was studied in Chinese hamster V79 cells. In these cells, mitosenes appeared to be very active. Under severe hypoxic conditions toxicity of these mitosenes increased, most likely due to the increased lifetime of the activated mitosene species as compared to aerobic conditions. The results suggest that DNA cross-linking following reductive activation is important for the eventual activity of mitosenes in a bacterial system. Increased activity of mitosenes under hypoxic conditions in the V79 cells indicates that these mitosenes may be more active in hypoxic parts of tumours.

Animals↗

PubMed1994

Reversal of P-glycoprotein-mediated multidrug resistance by pure anti-oestrogens and novel tamoxifen derivatives.

In this study the ability of five novel anti-oestrogens [4-iodotamoxifen, pyrrolidino-4-iodotamoxifen, ethyl bromide tamoxifen (EBTx), ICI 164,384 (ICI 164) and ICI 182,780] to alter drug toxicity to multidrug resistant cell lines have been compared. The effect of these compounds on ATP-dependent vinblastine (VBL) transport was also tested using inside-out vesicles (IOV) prepared from highly P-glycoprotein (Pgp)-expressing CCRF-CEM/VBL1000 cells. The pure anti-oestrogen ICI 164 was most effective, enhancing doxorubicin and VBL toxicity to MCF-7Adr cells 25- and 35-fold, respectively, and was also the best inhibitor of ATP-dependent [3H]VBL accumulation by IOV. Pure anti-oestrogens, tamoxifen and iodotamoxifens completely reversed VBL resistance in the mdr1 transfected lung cancer cell line, S1/1.1, where resistance relative to wild-type cells was mediated solely by Pgp. The membrane impermeant tamoxifen derivative EBTx did not modify drug resistance, yet was as effective an inhibitor of VBL accumulation by inside-out Pgp-positive vesicles as tamoxifen. This indicates an intracellular role for tamoxifen and its derivatives in the modulation of Pgp-mediated drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Modification of metabolism of transplantable and spontaneous murine tumors by the nitric oxide synthase inhibitor, nitro-L-arginine.

PURPOSE: To determine the effects of the nitric oxide synthase inhibitor, nitro-L-arginine on energy metabolism in transplantable and spontaneous murine tumors. METHODS AND MATERIALS: The responses of the transplantable murine tumor SCCVII/Ha and a range of spontaneously arising murine mammary adenocarcinomas to 10 mg/kg IV nitro-L-arginine were examined using in vivo 31P magnetic resonance spectroscopy (MRS). The influence of Hypnorm/Hypnovel anesthesia on the response to nitro-L-arginine was also determined in the SCCVII/Ha tumors. Data were expressed as changes in the inorganic phosphate peak area relative to the sum of all peak areas from the 31P MR spectrum, or Pi/total. RESULTS: Nitro-L-arginine at 10 mg/kg IV increased Pi/total 2-3-fold in the SCCVII/Ha tumors for at least 2 h after administration, in both anesthetized and nonanesthetized mice, consistent with increased tumor hypoxia. Similar increases in Pi/total were observed after 10 mg/kg IV nitro-L-arginine in 13 spontaneous murine tumors from three different mouse strains, where anesthetic was used. CONCLUSION: The results indicate that tumor metabolism may be modified by an inhibitor of nitric oxide synthesis, that this modification occurs in both transplantable and spontaneous murine tumors and is not affected by anesthetic.

Amino Acid Oxidoreductases↗

PubMed1994

Combination of photodynamic therapy (PDT) and melphalan in experimental tumors.

PURPOSE: To investigate whether application of "early" photodynamic therapy (PDT) using a disulphonated aluminium phthallocyanine photosensitizer can potentiate the action of melphalan in experimental RIF-1 tumors in vivo. METHODS AND MATERIALS: Tumors were irradiated with laser light of wavelength 675 nm 60 min after treatment with the photosensitizer and 15 min after melphalan. Melphalan pharmacokinetics were measured using high performance liquid chromatography with optical detection. RESULTS: Melphalan and PDT when given alone, caused a significant delay in tumor growth. This was increased for the combined treatment. Pharmacokinetic analyses showed that levels of free, unreacted melphalan in freely circulating blood are unaffected by combined treatment. However, significant differences in tumor levels were observed between treatment with melphalan alone or in combination. Whereas in the former, melphalan is still present in tumors after 2 h, it was not detectable even at the earliest time of 15-23 min for the combined treatment. CONCLUSION: The antitumor effects were additive with no evidence of significant potentiation.

Animals↗

PubMed1994

Bioreductive drugs for cancer therapy: the search for tumor specificity.

The activity of three different classes of bioreductive drug, i.e., heterocyclic nitro compounds, N-oxides and quinones are compared. The major characteristics of RB-6145, tirapazamine and E09 are summarized and future directions for development of new bioreductive drugs are outlined. The concept of potentiating bioreductive drug activity by increasing tumor hypoxia is described and illustrated in particular by the use of photodynamic therapy (PDT) in combination with RSU-1069. Examples of how the therapeutic effectiveness of this approach can be studied by the use of 31P magnetic resonance spectroscopy is described. The effects of manipulation of nitric oxide (NO) levels in tumors by the use of modifiers of NO-synthase activity is illustrated by studies with the inhibitor nitro-L-arginine in experimental tumors. Associated changes in tumor physiology indicate promise for potential applications in therapy. Finally, changes in expression of reductase enzyme levels are considered in the context of the heterogenous nature of the tumor microenvironment.

Animals↗

PubMed1994

31P MRS to monitor the induction of tumor hypoxia by the modification of the oxygen affinity of hemoglobin using BW 589C.

PURPOSE: BW 589C induces severe tumor hypoxia by modifying the affinity of oxyhemoglobin, causing a left shift of the oxygen-hemoglobin dissociation curve. 31P magnetic resonance spectra (MRS) was used to monitor the effects of BW 589C on tumor energy metabolism in three experimental tumor models. METHODS AND MATERIALS: HT-29 colon xenograft, murine transplantable RIF-1 fibrosarcoma and KHT sarcoma were studied in unanesthetised mice. 31P MR spectra were acquired on a 4.7 Tesla magnet before administering oral BW 589C (250 mg/kg) and after 3, 6, and 24 h. Samples of tail vein blood were then taken for 2,3 DPG levels for RIF-1 and HT-29 tumors. RESULTS: Doubling of inorganic phosphorus (Pi) to total phosphorus was observed 5-6 h after BW 589C for all three tumor types. Although the left shift due to BW 589C persists at 24 h, the level of Pi to total phosphorus returned to baseline with no significant difference from control values for the RIF-1 and HT-29 tumors. These results suggest that there was cellular metabolic adaptation to the reduction of oxygen delivery by BW 589C. This does not appear to involve 2,3 DPG as there was no significant alteration in tumor levels. The death of hypoxic cells may, also, have contributed to the recovery of Pi to total phosphorus. CONCLUSION: The efficacy of bioreductive drugs can be enhanced by increasing the severity of tumor hypoxia. 31P MRS in conjunction with other techniques for assessing the intratumor environment could play an important role in planning cancer therapy.

Animals↗

PubMed1994

Comparing the anti-tumor effect of several bioreductive drugs when used in combination with photodynamic therapy (PDT).

PURPOSE: To compare the effect on the RIF-1 murine sarcoma of nine bioreductive agents from five different classes when used in combination with either photodynamic therapy or clamping. METHODS AND MATERIALS: RIF-1 tumors implanted intradermally in C3H mice were treated with either 50J photodynamic therapy or with 120 min clamping in combination with either misonidazole, pimonidazole, metronidazole, nimorazole, RB6145, RSU1069, SR4233, mitomycin-C, or RB90740. The tumors were measured 3 times-per-week until reaching 4 x their initial treatment volume. RESULTS: RSU1069 produced the greatest anti-tumor activity in combination with both photodynamic therapy and clamping. RB6145 also substantially enhanced the effect of photodynamic therapy and clamping whereas misonidazole induced a smaller, but significant increase. Mitomycin-C had no effect under clamped conditions, but greatly increased the tumorcidal effect of photodynamic therapy. Mitomycin-C also induced an effect when given with light alone. None of the other agents showed any augmentation of the tumor cell killing induced by photodynamic therapy. CONCLUSION: Of the bioreductive agents studied RSU1069, RB6145 and mitomycin-C showed the greatest anti-tumor response in combination with photodynamic therapy.

Animals↗

PubMed1994

Radiolytic and photochemical reduction of the hypoxic cytotoxin 1,2-dihydro-8-(4-methylpiperazinyl)-4-phenylimidazo [1,2-a] pyrido [3,2-e] pyrazine 5-oxide (RB90740) and a potential mechanism for hypoxia-selective toxicity.

PURPOSE: To study the reduction of RB90740 (1), a fused pyrazine mono-N-oxide that has an oxic:hypoxic cytotoxicity ratio of > 10 in a range of murine and human tumor cells in vitro. METHODS AND MATERIALS: Reduction of 1 has been initiated radiolytically and photochemically in aqueous solution and the products isolated and characterized by high performance liquid chromatography (HPLC). RESULTS: Radiolytic reduction of 1 leads to the formation of the 2-electron reduced product, 2. The stoichiometry of the reduction is pH dependent, increasing from 1 to 2 with increasing pH, but independent of the presence of formate or 2-methyl 2-propanol in the reduction mixture. A dimerization product, 3, is also found, which is produced in greater yields at lower pH (< 6). Photochemical reduction of 1 to 2 was also found to be facile. Photolysis of 1 also leads to a deoxyribonucleic acid cleavage reaction. CONCLUSION: Since 2 is not cytotoxic towards hypoxic cells at concentrations at which 1 is toxic, a probable candidate as the cytotoxic species under hypoxic conditions is the 1-electron reduced intermediate species.

Animals↗

PubMed1994

Pharmacokinetics, metabolism and distribution of 1,2-dihydro-8-(4-methylpiperazinyl)-4-phenylimidazo [1,2-A] pyrido [3,2-E] pyrazine-5-oxide in C3H mice.

PURPOSE: 1,2-Dihydro-8-(4-methylpiperazinyl)-4-phenylimidazo [1,2-a] pyrido [3,2-e] pyrazine-5-oxide (RB90740) is a bioreductive drug with an oxic to hypoxic toxicity ratio of 16 in cultured V79 cells in vitro. The aim of this study was to examine the pharmacokinetics, metabolism and distribution of the drug in tumor bearing C3H mice. METHODS AND MATERIALS: A high pressure liquid chromatography assay for the quantitative determination of concentrations of the drug and its metabolites has been developed and used to examine their distribution in blood, RIF-1 and KHT tumors, brain, muscle, and liver tissue. Urine and feces collected for 24 h after drug administration have been examined for the drug and its metabolism products. RESULTS: Three metabolites, two of which have been identified, have been observed in mouse tissue. 1,2-Dihydro-8-(4-methylpiperazinyl)-4-phenylimidazo [1,2-a]pyrido[3,2-e]pyrazine (RB92815) is the two-electron reduced species, which is observed in liver, urine and occassionally in tumor samples. 1,2-dihydro-8-(4-piperazinyl)-4-phenylimidazo [1,2-a]pyrido[3,2-e]pyrazine-5-oxide (RB1739), the N-demethylated compound, is observed in urine and liver. Elimination of the drug after an intraperitoneal dose of 50 mg/kg is biphasic with t(1)2 alpha = 3 min and t(1)2 beta = 219 min. The area under the curve for blood concentration vs. time is 1.4 mg ml min-1. The drug is preferentially taken up into tumor tissue as is apparent from the area under the curve values for RIF-1 (28.3 mg ml min-1) and KHT (18.4 mg ml min-1) tumors. CONCLUSION: From these values of the area under the curve it is suggested that the drug is present in tumor tissue at concentrations sufficient to eliminate the hypoxic fraction provided reduction to a toxic species occurs. Bioreduction by the addition of two electrons to form RB92815 occurs in some tumors, but it is not known if this is due to an obligate two-electron detoxifying step or if reduction occurs by single electron additions via a toxic free radical species.

Animals↗

PubMed1994

The role of DT-diaphorase in determining the sensitivity of human tumor cells to tirapazamine (SR 4233).

PURPOSE: To determine the dependency of the aerobic and hypoxic toxicity of tirapazamine on the intracellular activity of DT-diaphorase. METHODS AND MATERIALS: A panel of 18 human cell lines comprising predominantly small cell and nonsmall cell lung cancer and breast cancer lines were used. The activity of DT-diaphorase was determined in cytosolic preparations from cell lysates. The toxicity of tirapazamine was determined using the MTT assay after either 96 or 3 h aerobic exposure or 3 h treatment in hypoxia. RESULTS: The cell lines exhibited a 5000-fold range in DT-diaphorase activity. In toxicity experiments, values of IC50 range from 10.2-120 microM and from 155-1230 for 96 and 3 h aerobic exposures, respectively. In N2, IC50s ranged from 8-55 microM. None of the toxicity values correlate with activity of DT-diaphorase, neither did the ratio of aerobic:hypoxic toxicity (differential toxicity). CONCLUSION: The expression of DT-diaphorase in human tumor cells does not affect the toxicity of tirapazamine.

Antineoplastic Agents↗

PubMed1994

Factors affecting sensitivity to EO9 in rodent and human tumour cells in vitro: DT-diaphorase activity and hypoxia.

Twenty-three human tumour cell lines (lung, breast, and colon) and eight rodent cell lines were evaluated for their sensitivity to the quinone-based anticancer drug EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H indole-4,7-dione)prop-beta-en-alpha-o1]. Sensitivity was compared with the intracellular levels of DT-diaphorase, and cell lines showing highest enzyme activity tended to be the most sensitive to EO9. The role of DT-diaphorase in determining drug sensitivity was confirmed by using the enzyme inhibitor dicoumarol, which protects cells containing high levels of DT-diaphorase from the cytotoxic action of EO9. Hypoxia increased the cytotoxicity of cells containing low but not high levels of DT-diaphorase, implying that both 1- and 2-electron reductive activation processes can be important for expression of EO9 toxicity. It is concluded that EO9 is a potentially useful agent in the enzyme directed approach to the use of bioreductive drugs in cancer therapy.

Animals↗