tirapazamine and Colonic-Neoplasms

Topics: Animals; Antineoplastic Agents; Biomimetic Materials; Cell Proliferation; Colonic Neoplasms; Drug Screening Assays, Antitumor; Erythrocyte Membrane; Female; Glucose Oxidase; Metal-Organic Frameworks; Mice; Mice, Inbred BALB C; Nanoparticles; Prodrugs; Tirapazamine; Tumor Hypoxia

The recent clinical successes of antiangiogenic drug-based therapies have also served to highlight the problem of acquired resistance because, similar to other types of cancer therapy, tumors that initially respond eventually stop doing so. Consequently, strategies designed to delay resistance or treat resistant subpopulations when they arise have assumed considerable importance. This requires a better understanding of the various possible mechanisms for resistance. In this regard, reduced oxygenation is thought to be a key mediator of the antitumor effects of antiangiogenic therapies; accordingly, increased hypoxia tolerance of the tumor cells presents a potential mechanism of resistance. However, hypoxia can also be exploited therapeutically through the use of hypoxic cell cytotoxins, such as tirapazamine. With this in mind, we measured the oxygenation of PC-3 human prostate cancer xenografts subjected to chronic low-dose metronomic (LDM) antiangiogenic chemotherapy using cyclophosphamide given through the drinking water. We found that LDM cyclophosphamide impairs the oxygenation of PC-3 xenografts even during relapse, coinciding with reduced microvessel density. Combination of LDM cyclophosphamide with tirapazamine results in significantly improved tumor control in the PC-3, HT-29 colon adenocarcinoma, and MDA-MB-231 breast cancer human xenograft models without having a negative effect on the favorable toxicity profile of LDM cyclophosphamide. These results provide further evidence that reduced vascular dependence/increased hypoxia tolerance may be a basis for eventual resistance of tumors exposed to long-term LDM chemotherapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Growth Processes; Cell Hypoxia; Cell Line, Tumor; Colonic Neoplasms; Cyclophosphamide; Female; Humans; Male; Mice; Mice, Inbred BALB C; Neoplasms; Neovascularization, Pathologic; Oxygen; Prostatic Neoplasms; Tirapazamine; Triazines; Xenograft Model Antitumor Assays

Topics: Animals; Antineoplastic Agents, Alkylating; Colonic Neoplasms; Disease Models, Animal; Drug Design; Drug Screening Assays, Antitumor; Humans; Prodrugs; Tirapazamine; Triazines; Tumor Cells, Cultured

Because of the inefficient vasculature of solid tumors, anticancer drugs must penetrate relatively long distances through the extravascular compartment. The requirement for such diffusion may limit their activity, especially that of hypoxia-targeted drugs. We tested whether a three-dimensional pharmacokinetic/pharmacodynamic (PK/PD) model based on a representative mapped tumor microvascular network could predict the therapeutic activity of anticancer drugs in mouse xenograft tumors.. Diffusion coefficients of the hypoxia-activated anticancer drug tirapazamine (TPZ) and of 15 TPZ analogs were estimated by measuring their transport through HT29 colon cancer multicellular layers (MCLs). Anoxic cytotoxic potency (by clonogenic assay) and metabolism of the TPZ analogs were measured in HT29 cell suspensions, and their plasma pharmacokinetics was measured in CD-1 nude mice. This information was used to create a spatially resolved PK/PD model for the tumor microvascular network. Model predictions were compared with actual hypoxic cell kill as measured by clonogenic assays on HT29 xenograft tumors 18 hours after treatment with each TPZ analog.. Modeling TPZ transport in the tumor microvascular network showed substantial drug depletion in the most hypoxic regions, with predicted maximum cell kill of only 3 logs, compared with more than 10 logs if there were no transport impediment. A large range of tissue diffusion coefficients (0.027 x 10(-6)-1.87 x 10(-6) cm2/s) was observed for the TPZ analogs. There was a strong correlation between model-predicted and measured hypoxic cell kill (R2 = 0.89) but a poor correlation when the model did not include extravascular transport (R2 = 0.32).. Extravascular transport in tumors, and its consequences for tumor cell killing, can be predicted by measuring drug penetration through MCLs in vitro and modeling pharmacokinetics at each position in three-dimensional microvascular networks.

Topics: Analysis of Variance; Animals; Antineoplastic Agents; Biological Transport, Active; Chromatography, High Pressure Liquid; Colonic Neoplasms; Disease Models, Animal; Humans; Hypoxia; Mice; Radiation-Sensitizing Agents; Reproducibility of Results; Tirapazamine; Transplantation, Heterologous; Triazines; Tumor Cells, Cultured; Tumor Stem Cell Assay

Tirapazamine (TPZ), a bioreductive drug with selective toxicity for hypoxic cells in tumors, is currently in Phase III clinical trials. It has been suggested to have a dual mechanism of action, both generating DNA radicals and oxidizing these radicals to form DNA breaks; whether the second (radical oxidation) step is rate-limiting in cells is not known. In this study we exploit the DNA radical oxidizing ability of the 1-N-oxide metabolite of TPZ, SR 4317, to address this question. SR 4317 at high, but nontoxic, concentrations potentiated the hypoxic (but not aerobic) cytotoxicity of TPZ in all four of the human tumor cell lines tested (HT29, SiHa, FaDu, and A549), thus providing a 2-3-fold increase in the hypoxic cytotoxicity ratio. In potentiating TPZ, SR 4317 was 20-fold more potent than the hypoxic cell radiosensitizers misonidazole and metronidazole but was less potent than misonidazole as a radiosensitizer, suggesting that the initial DNA radicals from TPZ and radiation are different. SR 4317 had favorable pharmacokinetic properties in CD-1 nude mice; coadministration with TPZ provided a large increase in the SR 4317 plasma concentrations relative to that for endogenous SR 4317 from TPZ. It also showed excellent extravascular transport properties in oxic and anoxic HT29 multicellular layers (diffusion coefficient 3 x 10(-6) cm(2)s(-1), with no metabolic consumption). Coadministration of SR 4317 (1 mmol/kg) with TPZ at a subtherapeutic dose (0.133 mmol/kg) significantly enhanced hypoxic cell killing in HT29 tumor xenografts without causing oxic cell killing, and the combination at its maximum tolerated dose was less toxic to hypoxic cells in the retina than was TPZ alone at its maximum tolerated dose. This study demonstrates that benzotriazine mono-N-oxides have potential use for improving the therapeutic utility of TPZ as a hypoxic cytotoxin in cancer treatment.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Drug Synergism; Female; Humans; Lung Neoplasms; Mice; Pharyngeal Neoplasms; Tirapazamine; Triazines; Uterine Cervical Neoplasms

The hypoxic cytotoxin tirapazamine (TPZ) is currently in phase III clinical trial and appears to have clinical activity. One hypothesis as to why TPZ has been used more successfully in the clinic than most other bioreductive drugs is that its unusual O(2) dependence allows killing of radioresistant cells at "intermediate" O(2) concentrations. We have determined the O(2) dependence of the metabolism of TPZ to its reduction product SR 4317, and its cytotoxicity, in stirred suspensions of HT29 colon carcinoma cells while monitoring O(2) in solution with an Oxylite trade mark probe. The O(2) dependence of the cytotoxicity of TPZ is entirely accounted for by its inhibition of the metabolism of TPZ, with a K(O(2)) value (O(2) concentration for 50% inhibition) of 1.21 +/- 0.09 (SEM) microM. We used this experimental O(2) dependence to extend a recent (Hicks et al., Cancer Res. 63, 5970-5977, 2003) pharmacokinetic/pharmacodynamic model for the cytotoxicity of TPZ in anoxic HT29 multicellular layers to model cell killing in tumors. The model indicates that the O(2) dependence of killing by TPZ complements that of radiation well during fractionated radiotherapy. It predicts that lowering K(O(2)) would decrease killing in radioresistant cells at intermediate O(2) concentrations, while higher K(O(2)) values would exacerbate metabolic consumption of TPZ and thus further impede its penetration into hypoxic regions. Raising K(O(2)) would also increase metabolic activation at physiological O(2) concentrations, thereby compromising hypoxic selectivity. We conclude that the K(O(2)) value of TPZ is indeed close to the optimum for a bioreductive drug of this class (i.e. one that kills only cells in which it is reduced).

Topics: Antineoplastic Agents; Carcinoma; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Computer Simulation; Dose-Response Relationship, Drug; Humans; Models, Biological; Oxygen; Radiation Tolerance; Radiation-Sensitizing Agents; Tirapazamine; Triazines

The poor blood supply to solid tumours introduces many factors that affect the outcome of chemotherapy, one of which is the problem of drug delivery to poorly vascularized regions of tumours. Whereas poor drug penetration has been recognized as a contributing factor to the poor response of many solid tumours, the question of drug penetration through multicell layers has not been thoroughly addressed, largely because of restrictions imposed upon these studies by the requirement for either radiolabelled or naturally fluorescent compounds. The aim of this study is to describe modifications made to a recently published assay that broadens the scope for assessing drug penetration during the early stages of drug development and to characterize the ability of various drugs to penetrate multicell layers. DLD-1 human colon carcinoma cells were cultured on Transwell-COL plastic inserts placed into 24-well culture plates so that a top and bottom chamber were established, the two chambers being separated by a microporous membrane. Drugs were added to the top chamber at doses equivalent to peak plasma concentrations in vivo and the rate of appearance of drugs in the bottom chamber determined by high-performance liquid chromatography (HPLC). Both 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine) and 7-[4'-(2-nitroimidazol-1-yl)-butyl]-theophylline (NITP) rapidly penetrated DLD-1 multicell layers (50.9 +/- 12.1 microm thick) with t(1/2) values of 1.36 and 2.38 h respectively, whereas the rate of penetration of 5-aziridino-3-hydroxymethyl-1-methyl-2-[1H-indole-4,7-dione] prop-beta-en-alpha-ol (EO9) and doxorubicin through multicell layers was significantly slower (t(1/2) = 4.62 and 13.1 h respectively). Inclusion of dicoumarol increases the rate of EO9 penetration, whereas reducing the oxygen tension to 5% causes a reduction in tirapazamine penetration through multicell layers, suggesting that the extent of drug metabolism is one factor that determines the rate at which drugs penetrate multicell layers. The fact that EO9 does not readily penetrate a multicell layer, in conjunction with its rapid elimination in vivo (t(1/2) < 10 min), suggests that EO9 is unlikely to penetrate more than a few microm from a blood vessel within its pharmacokinetic lifespan. These results suggest that the failure of EO9 in the clinic is due to a combination of poor drug penetration and rapid elimination in vivo.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Aziridines; Carcinoma; Cell Division; Chromatography, High Pressure Liquid; Colonic Neoplasms; Dicumarol; Doxorubicin; Humans; Indolequinones; Indoles; Oxygen; Tirapazamine; Triazines; Tumor Cells, Cultured

Local hyperthermia and the hypoxic cytotoxin SR 4233 were administered to nude mice with 693 +/- 47 mm3 (mean +/- SE) s.c. HCT-8 human colonic adenocarcinoma xenografts in an attempt to enhance the antitumor effects of radioimmunotherapy. Biodistribution studies revealed preferential binding of NR-Lu-10, a murine monoclonal antibody, to the tumors compared with an isotype-matched control antibody, CCOO16-3.A single injection of 25 microCi 90Y-NR-Lu-10 significantly inhibited tumor growth (control versus 90Y-NR-Lu-10: P = 0.048). The administration of hyperthermia at 41.5 degrees C for 1 h immediately following the injection of 111In-labeled NR-Lu-10 up-regulated tumor-associated antigen expression and increased antibody uptake in the tumors by 73% (P = 0.001) without significantly affecting antibody uptake in normal tissues. However, the heat treatment did not produce a more homogeneous distribution of the antibodies in the tumors and did not significantly enhance the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.07). The administration of local hyperthermia at 43.0 degrees C for 1 h, on the other hand, had direct cytotoxic effects (P = 0.03) and enhanced the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.01). SR 4233 also enhanced the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.03). The greatest antitumor effects were observed when both hyperthermia at 43.0 degrees C and SR 4233 were administered in combination with 90Y-NR-Lu-10 (P = 0.002). No toxicity was produced by the local hyperthermia, and the only toxicities produced by 90Y-NR-Lu-10 and SR 4233 were neutropenia and weight loss.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Autoradiography; Cell Division; Cell Hypoxia; Colonic Neoplasms; Combined Modality Therapy; Female; Humans; Hyperthermia, Induced; Immunotoxins; Indium Radioisotopes; Mice; Mice, Inbred BALB C; Mice, Nude; Radiation-Sensitizing Agents; Radioimmunoassay; Radioimmunotherapy; Tirapazamine; Tissue Distribution; Transplantation, Heterologous; Triazines; Yttrium Radioisotopes

We have observed that low pH can substantially potentiate the cytotoxic effect of the bioreductive drug SR4233 in aerobic HT-29 human tumour cells. No such potentiation was observed under hypoxic conditions. This pH effect might be relevant both to the therapeutic effectiveness and to the normal tissue toxicity of this new agent.

Topics: Antineoplastic Agents; Cell Hypoxia; Colonic Neoplasms; Drug Screening Assays, Antitumor; Humans; Hydrogen-Ion Concentration; Tirapazamine; Triazines; Tumor Cells, Cultured

Radioimmunotherapy is hindered by the slow penetration of antibody molecules into tumors. Cells that are poorly targeted by antibody, because of their distance from feeding blood vessels, receive the lowest radiation dose, and this problem is compounded if there are radioresistant hypoxic cells present. It would be desirable to combine radioimmunotherapy with an agent that is preferentially toxic to these cells. SR 4233 is a potent hypoxic cytotoxin, and it was combined with 131I-NR-LU-10 to treat LS174T human colon adenocarcinoma multicell spheroids and nude mouse xenografts for these studies. Under conditions of severe hypoxia (< 0.01% O2), 2 h of pretreatment or 18 h of simultaneous treatment with SR 4233 did not significantly enhance the effectiveness of 131I-NR-LU-10 in spheroids. However, under aerobic conditions with a 10% fraction of hypoxic cells, there was more toxicity than would be predicted from simple additivity. Xenografts treated with 131I-NR-LU-10 + SR 4233 had a growth delay that was significantly longer than that achieved with 131I-NR-LU-10 alone. In both spheroids and xenografts, combined treatment produced about 10 times more cell killing than 131I-NR-LU-10 alone. The lack of enhancement in spheroids under complete hypoxia suggests that SR 4233 does not sensitize hypoxic cells to radiation damage. The results with aerobic spheroids and in vivo, where a portion of the cells were hypoxic, could be explained by the targeting of different cell populations (hypoxic and aerobic) by each therapeutic modality. This effect should also be enhanced by reoxygenation and reestablishment of the hypoxic fraction during treatment, thus allowing more than the initially hypoxic fraction of cells to be killed by the SR 4233.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Colonic Neoplasms; Combined Modality Therapy; Humans; In Vitro Techniques; Iodine Radioisotopes; Mice; Mice, Nude; Neoplasms, Experimental; Radiation-Sensitizing Agents; Radioimmunotherapy; Tirapazamine; Transplantation, Heterologous; Triazines; Tumor Cells, Cultured

DT-diaphorase is a unique two electron (2e) donating reductase catalyzing either bioactivation or bioprotection reactions. Using human and rodent DT-diaphorase preparations (cell extracts and purified enzyme) we have characterized the reductive metabolism of the hypoxic cell cytotoxins EO9, mitomycin C (MMC), CB 1954, and SR 4233 in vitro. Drug metabolism was assayed spectrophotometrically or by HPLC, with dicoumarol as a selective inhibitor. DNA damage was measured using an agarose gel mobility technique with plasmid pBR322 DNA. The developmental indoloquinone, EO9, was metabolized by both rat Walker and human HT29 tumor DT-diaphorases. Reduction proceeded 5-fold more efficiently with the rat than the human tumor enzyme and resulted in single-strand breaks in plasmid DNA. The structurally related MMC was metabolized much more slowly than EO9 by the rat Walker tumor enzyme and there was no detectable reaction with the human HT29 tumor DT-diaphorase. No DNA damage was seen with MMC for either enzyme. The dinitrophenylaziridine CB 1954 was reduced by both human and rat enzymes forming, preferentially, the highly toxic 4-hydroxylamine as a 4e reduction product. Rates were 3-fold lower than for the human tumor enzyme. SR 4233 was also reduced by the rat tumor enzyme predominantly via 4e reduction to the benzotriazine SR 4330, in a novel reaction mechanism. This appears to be a bioprotection pathway that bypasses the toxic 1e radical formed by other reductases. Such information may be valuable in the selection of hypoxic cell cytoxins to treat human tumors high or low in DT-diaphorase and should facilitate 'enzyme-directed' analogue development.

Topics: Animals; Antineoplastic Agents; Aziridines; Carcinoma 256, Walker; Cell Hypoxia; Colonic Neoplasms; Humans; In Vitro Techniques; Indolequinones; Indoles; Mitomycin; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Prodrugs; Rats; Tirapazamine; Triazines