Use of fluorine-19 nuclear magnetic resonance spectroscopy and hydralazine for measuring dynamic changes in blood perfusion volume in tumors in mice.

BACKGROUND: One method of evaluating the mechanism of action of agents which alter tumor oxygenation is to determine their effects on tumor blood flow. PURPOSE: This study tests applicability of a new approach using an emulsion of the inert fluorocarbon perfluorooctylbromide (PFOB) at nontoxic doses as a tracer in fluorine-19 (19F) nuclear magnetic resonance (NMR) spectroscopy to evaluate dynamic changes in vascular perfusion volume in transplanted tumors. METHODS: The PFOB emulsion (100% wt/vol) was injected into the tail vein in tumor-bearing C3H/He or nu/nu mice immobilized in a magnet interfaced to a spectrometer, either as a single bolus injection of 8 mL/kg body weight or in multiple injections to a total dose of 24 mL/kg. A 7-mm external surface coil was placed over the tumor. Signal from the PFOB in the tumor volume seen by the coil rapidly reached equilibrium and was maintained for at least 2 hours, and multiple doses of PFOB emulsion resulted in a linear increase in 19F signal strength. Since the 19F signal strength was directly proportional to the perfusion volume of the tumor vasculature, reduction of signal intensity should correspond directly to any reduction in volume caused by a change in the tumor blood flow. To investigate this hypothesis, the vasoactive agent hydralazine (5 mg/kg) was injected intravenously after administering the PFOB emulsion to induce changes in tumor blood supply. KHT and RIF-1 murine sarcomas, the HT29 human colon carcinoma, and the HX118 human melanoma tumors were studied. In a comparative analysis of changes in blood flow induced by hydralazine, we studied Xe-133 clearance in KHT murine sarcoma and SCCVII/Ha (SCCVII) murine squamous cell carcinoma. RESULTS: Hydralazine significantly reduced the 19F signal intensity in the murine tumors RIF-1 and KHT and in the HT29 human tumor, with little reduction in the SCCVII/Ha murine and HX118 human tumors. Hydralazine induced a statistically significant 64% decrease in mean clearance rate in the KHT tumor, while SCCVII/Ha tumors showed no significant change, indicating that hydralazine restricted blood flow to a greater extent in the tumor type that showed reduced 19F signal from the PFOB emulsion. CONCLUSION: These data demonstrate the potential of PFOB emulsion as a tracer in NMR spectroscopy for studying tumor vasculature.

Toxicity of nitro compounds toward hypoxic mammalian cells in vitro: dependence on reduction potential.

Fifteen nitroaromatic and nitroheterocyclic compounds that can act as "radiosensitizers" were tested for their cytotoxicity toward hypoxic Chinese hamster V79 cells in vitro. The cytotoxicity increased markedly as the electron affinity, measured as a one-electron reduction potential, increased. Non-nitro-containing compounds of similar electron affinities (such as quinones) that also act as radiosensitizers did not exhibit this specific toxicity toward hypoxic cells. The implications of the presence of the nitro group as a prerequisite for the hypoxic cell toxicity were discussed, and the mechanism of the cytotoxicity was compared with that of hypoxic cell radiosensitization.

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.

Effects of hypoxia and reoxygenation on the conversion of xanthine dehydrogenase to oxidase in Chinese hamster V79 cells.

The effects of hypoxia and reoxygenation on the conversion of xanthine dehydrogenase to the free radical-producing xanthine oxidase in Chinese hamster V79 cells have been investigated using a newly developed fluorimetric enzyme assay. Hypoxia caused an increase in xanthine oxidase activity from 25% to 80% of the total activity of xanthine oxidase and dehydrogenase. The ratio returned to normal levels within 24 h of aerobic incubation. Hypoxia caused the release of xanthine oxidase in the medium of V79 cells and an increase in total protein concentration in the medium. There was an early change induced in lipid peroxidation markers and this was inhibited by allopurinol. The effects of glucose deprivation and calcium blockers were also investigated. Fura-2 AM was found to interact with V79 cells, making it impossible to determine intracellular calcium levels in V79 cells by this reagent.

Induction of tumour hypoxia by a vasoactive agent. A combined NMR and radiobiological study.

The effect of hydralazine treatment on 3 murine tumours (RIF-1, KHT and 16/C) was monitored using 31P-NMR. Changes in the 31P-NMR spectrum are compared with measurements of radiobiological hypoxic fraction (RHF) in the RIF-1 and KHT. Hydralazine is known to reduce temporarily blood flow in experimental tumours, and thus cause a transient increase in the RHF to 100% (in RIF-1 and KHT). This correlates with a decline in energy status as measured by 31P-NMR (i.e. there was an increase in Pi in all three tumours). Time-course data from the RIF-1 and KHT tumours show that maintenance of anaesthesia prolongs the hypoxia induced by hydralazine.

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.

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.

Potentiation of cyclophosphamide cytotoxicity in vivo: a study with misonidazole and fifteen other 1-substituted 2-nitroimidazoles.

It has recently been reported that compounds more lipophilic than misonidazole are better potentiators of CCNU tumor cytotoxicity in vivo. There is now a need to extend these studies to include other tumors and cytotoxic drugs. In the present study we have shown that misonidazole (MISO) can potentiate cyclophosphamide cytotoxicity in the Lewis lung carcinoma but not in the B16 melanoma. Further studies in the Lewis lung carcinoma, using 15 1-substituted 2-nitroimidazoles possessing a range of octanol:water partition coefficient (P) (from 0.18- greater than 100) have shown that potentiation increases with increasing lipophilicity. The most efficient compounds at administered dose levels of 1.0 and 2.0 mumol/g were Ro-07-1902, benznidazole (Ro-07-1051) and RSU 1050 which possess octanol:water partition coefficients in the range of 2.5-10. However, on the basis of an equitoxic administered dose (1/2LD50/2d), little difference in potentiation is seen over the range of P studied.

RSU 1069, a 2-nitroimidazole containing an alkylating group: high efficiency as a radio- and chemosensitizer in vitro and in vivo.

Electron affinity as measured by the one-electron reduction potentian, E7(1), is the major factor influencing radiosensitizing efficiency in vitro. RSU 1069 has an electron affinity (E7(1) = 398 mV) similar to misonidazole; however, the ability of this compound to sensitize hypoxic cells is considerably greater in vitro than that of misonidazole, e.g., 0.2 mM RSU 1069 gives an enhancement ratio of greater than 2.0 compared to 1.4 for the same concentration of misonidazole. Radiosensitization studies with the MT tumor in vivo also showed RSU 1069 to be a more efficient sensitizer than misonidazole. An administered dose of 0.08 mg/g RSU 1069 yielded an enhancement of 1.8 to 1.9 using tumor cell survival and tumor cure as end-points. At least a 10-fold higher dose of misonidazole is required for a similar degree of sensitization. Low doses of RSU 1069 also radiosensitize the Lewis lung and B16 experimental tumors. The ability of RSU 1069 to potentiate the cytotoxic action of melphalan and other cytotoxic drugs towards the MT tumor was also examined. RSU 1069 (0.08 mg/g) given to mice 1 hour before melphalan gave an enhancement of 2.8.

Evaluation of novel radiation sensitizers in vitro and in vivo.

In principle, radiation sensitizers with therapeutic ratios greater than that of misonidazole can be obtained either by increasing sensitizing efficiency, decreasing toxicity or preferably both. This paper illustrates, firstly that a 5-nitroimidazole (S73-0662) with an electron affinity close to that of metronidazole shows sensitizing efficiency similar to misonidazole both in vitro and in vivo. The suggestion is made that this compound should receive a detailed toxicological study to ascertain if its toxicity is lower than misonidazole. Secondly, Imuran, a 5-substituted 4-nitroimidazole and one of a series of compounds which show sensitizing efficiencies in vitro much greater than would be predicted from electron affinity considerations, also shows good sensitization in vivo. Compounds in this series are generally metabolically unstable and the positive results with Imuran in vivo provide a direction for future synthesis of novel sensitizers.

Chemosensitization in vitro: potentiation of melphalan toxicity by misonidazole, metronidazole and nitrofurazone.

Misonidazole, metronidazole and nitrofurazone can enhance the cytotoxicity of melphalan in vitro if the cells are subjected to a hypoxic pretreatment with the drug prior to exposure of melphalan in air. Potentiation of melphalan is dependent upon the concentrations of the nitro compound and the duration of the hypoxic pretreatment. On a concentration basis, nitrofurazone was most effective at enhancing melphalan toxicity and metronidazole was the least effective. This potentiating activity correlates with the electron affinity of each drug.

Enhancement of the anti-tumor effect of melphalan in experimental mice by some vaso-active agents.

A comparison is made between the vaso-active agents hydralazine, nifedipine, and verapamil for their ability to increase the anti-tumor effectiveness of melphalan. Treatment of mice with hydralazine (5 mg/kg) 15 mins after melphalan increases by a factor of approximately 2.5 melphalan-induced delay in growth of either the RIF-1 or KHT tumors. Similar enhancements are obtained when measurement is made of the surviving fraction of tumor cells in vitro following treatment in vivo with hydralazine and melphalan. Further, tumor cell kill is also increased when nifedipine is administered with melphalan compared with the effect of melphalan alone. These enhanced effects are observed if the vaso-active agents are given before or after melphalan. Hydralazine (5 mg/kg) induces close to 100% radiobiological hypoxia in the RIF-1 and KHT tumors. In contrast, Nifedipine has no effect on tumor hypoxic fraction at a dose of 10 mg/kg although the anti-tumor effectiveness of melphalan is substantially increased. However, a higher dose of 50 mg/kg nifedipine causes a large increase in tumor hypoxic fraction, an effect that persists for several hours. Verapamil causes no change in the fraction of hypoxic cells in the KHT tumor and increases, only slightly, the anti-tumor effect of melphalan.

Bioreductive drugs as post-irradiation sensitizers: comparison of dual function agents with SR 4233 and the mitomycin C analogue EO9.

Various bioreductive drugs that are potent hypoxic cell cytotoxins can also function as effective potentiators of radiation action when administered in vivo post irradiation. There is evidence that a contributory mechanism to this potentiation is enhanced sensitivity to the bioreductive drugs exhibited by cells that are damaged sublethally by radiation.

The measurement of radiosensitizer-induced changes in mouse tumor metabolism by 31P magnetic resonance spectroscopy.

Flunarizine and nicotinamide have previously been shown to increase blood perfusion to experimental mouse tumors and consequently, to increase their sensitivity to X rays. These agents were examined for their ability to alter metabolism, measured by 31P magnetic resonance spectroscopy, in the SCCVII/Ha carcinoma and the KHT sarcoma. Flunarizine at 5 mg/kg I.P. produced a 45% reduction in the ratio of inorganic phosphate to total phosphate (Pi/total) in the SCCVII/Ha tumor but only a 24% reduction in this ratio in the KHT tumor. These effects were seen 45 min after drug administration, and ratios returned to control levels by 90 min. In the SCCVII/Ha tumor, nicotinamide at 1000 mg/kg I.P. reduced Pi/total by 56% from 30 min to at least 2 hr after injection, and the ratio was reduced by 59% in the KHT tumor at 30 min after injection, returning to control levels by 2 hr. For the SCCVII/Ha tumor, the time course for the effects of flunarizine and nicotinamide on the inorganic phosphate ratio coincided with that previously reported for radiosensitization.

Induction of severe tumor hypoxia by modifiers of the oxygen affinity of hemoglobin.

Methods have been compared for inducing severe hypoxia in experimental tumors. Hypoxic fractions in the tumors were obtained from measurements of the displacement of cellular survival plots in vitro following tumor irradiation in vivo. Two compounds that displace to the left, oxygen-hemoglobin association curves greatly increase the hypoxic fractions in the tumors. The compound BW12C increases the hypoxic fractions in the KHT and Lewis-Lung tumors from about 10% to between 50-100%. The longer acting analogue BW589C increases hypoxic fraction in the KHT tumor to the same level achievable by treatment with the vaso-active drug hydralazine. The effect is also observed in the RIF-1 tumor even though the hypoxic fraction in this tumor is normally only about 1-3%. The kinetics for hypoxia induction by BW589C and its subsequent return to normal levels are comparable to those for the left-shifting of the oxy-hemoglobin association curve observable up to about 2 days post treatment.

Magnetic resonance spectroscopy studies on experimental murine and human tumors: comparison of changes in phosphorus metabolism with induced changes in vascular volume.

The responses of two experimental murine tumors and two human tumor xenografts to the vasodilator hydralazine were compared using two magnetic resonance spectroscopy endpoints. Changes in tumor metabolism were determined using 31P MRS where inorganic phosphate levels relative to total phosphate (Pi/total) were measured, and alteration in tumor blood volume was examined using 19F MRS with perfluorooctylbromide (PFOB) as tracer. The integrated 19F signal from PFOB is dose dependent and stable for at least 2 hr after injection. The murine tumors SCCVII/Ha and KHT both showed changes in tumor metabolism after hydralazine, as an increase in Pi/total. However, hydralazine reduced vascular volume in the KHT tumor, demonstrated by reduced 19F signal from PFOB, but no such reduction was seen in the SCCVII/Ha tumor. In contrast, hydralazine had no effect on phosphorus metabolism in the HT29 and HX118 human tumor xenografts, but reduced vascular volume in both tumors. These results demonstrate that the effects of vasoactive agents such as hydralazine on tumor phosphorus metabolism are only partially consistent with changes in vascular volume, measured by the 19F MRS technique.

The response of spontaneous and transplantable murine tumors to vasoactive agents measured by 31P magnetic resonance spectroscopy.

31P magnetic resonance spectroscopy has been used to compare the effects of the vasoactive agents hydralazine and flunarizine on the oxygenation of the transplantable tumors, SCCVII/Ha and 16C, and a range of spontaneous mammary tumors arising in the breeding stock in the Genetics Division at the Radiobiology Unit. The vasodilator hydralazine, previously shown to increase the radiobiological hypoxic fraction of transplantable murine tumors, increased inorganic phosphate to total phosphate (Pi/total) in SCCVII/Ha and 16C tumors. However, only two spontaneous tumors responded to this agent (2/12). The calcium antagonist flunarizine, which sensitizes the SCCVII tumor to X rays, consistent with a reduction in hypoxic fraction, reduced Pi/total in this and the 16C tumor. Further, most spontaneous tumors tested (8/10) responded to this agent, as measured by a reduction in Pi/total. These results point to fundamental differences between transplantable and spontaneously arising tumors in mice in their response to vasoactive agents.

Dual function nitroimidazoles less toxic than RSU 1069: selection of candidate drugs for clinical trial (RB 6145 and/or PD 130908.

Following the toxicity and synthetic difficulties encountered with the hypoxic cell radiosensitizer RSU 1069, efforts have focused on development of a superior analogue. Two compounds, RB 6145 and PD 130908, have emerged from this program which overcome the instability and synthetic problems associated with RSU 1069 while retaining favorable biological activity. Both agents show comparable radiosensitizing activity to RSU 1069 following oral or i.p. administration to mice bearing the KHT or RIF-1 tumors. Sensitizing efficiency is about 10 X greater than that observed for misonidazole or etanidazole. Toxicity toward hypoxic tumor cells in vivo is demonstrated by clamping tumors (for 60 min) following administration of PD 130908 or RB 6145. Both are effective hypoxic cytotoxins, but less potent than RSU 1069. Systemic toxicity is substantially reduced following oral drug administration. Further, doses achievable following fractionated drug treatments are sufficiently high to produce significant levels of radiosensitization.

Assessment of a range of novel nitro-aromatic radiosensitizers and bioreductive drugs.

In a directed search for the best compounds for clinical evaluation, some 150 selected nitroaromatic compounds, representing 6 distinct types, namely, furans, thiophenes, imidazoles, pyrazoles, pyrroles, and triazoles, have been synthesized and tested as hypoxic cell radiosensitisers and bioreductive drugs. These compounds have a wide range of one-electron redox potentials, ranging from -700 mV for 3-nitropyrroles to -250 mV for 5-nitrofurans. Within each series, those agents bearing alkylating moieties on the side chain are generally the more effective radiosensitisers in vitro. Studies in vivo demonstrated that the bifunctional nitroimidazoles were superior to the other nitroarenes tested. In terms of bioreductive cell killing, the best differential between oxic and hypoxic cell toxicity was shown for the bifunctional 2-nitroimidazoles, which had values greater than 20. In contrast, the other classes of nitroarines generally showed differential toxicities of less than 10.

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.