Elizabeth Porter

ENHANCEMENT OF TUMOR RADIATION RESPONSE BY THE COMBINATION OF A PERFLUOROCHEMICAL EMULSION AND HYPERBARIC OXYGEN

The presence of radioresistant hypoxic cells in tumors is believed to be one of the limiting factors in achieving local tumor control by radiotherapy. Treatment with hyperbaric oxygen during irradiation has been shown to improve the radiation response of many solid tumors in rodents and of some tumors in patients. Intravenous administration of perfluorochemical emulsions combined with oxygen breathing at atmospheric pressure has also been shown to improve the radiation response of several rodent tumors. Theoretical considerations suggest that the combination of a perfluorochemical emulsion and hyperbaric oxygen should be significantly more effective than either agent alone. This hypothesis was tested by examining the radiation response of BA1112 rhabdomyosarcomas growing in WAG/rij-Y rats. Treatment with a perfluorochemical emulsion, Fluosol-DA, plus hyperbaric oxygen (3 Atmospheres O2) significantly increased the radiation response of the malignant cells in these solid tumors. The observed changes in the tumor cell survival curve suggest that the combination of Fluosol-DA and HBO decreases the proportion of severely hypoxic cells in the tumor to less than 1.5% of the original value. The effect of the Fluosol-DA dose and the duration of pretreatment with HBO are described.

Effect of a perfluorochemical emulsion on the radiation response of BA1112 rhabdomyosarcomas.

The effect of treatment with a perfluorochemical emulsion (Fluosol DA, 20%), carbogen, or the combination of these two agents on the radiation response of BA1112 tumors in WAG/rij rats was examined. Fluosol and carbogen as single agents had only small effects on the tumor cell survival curve. The combination of Fluosol plus carbogen had a larger effect on tumor cell survival, reducing the hypoxic fraction of the tumor from 23 to 1.6%. The amount of sensitization was a function of the Fluosol dose, with maximal augmentation of the radiation response obtained at doses of 7.5-15 ml/kg. Carbogen pretreatments ranging from 5 to 60 min in duration all had similar effects on tumor radiosensitivity. The effect of the perfluorochemical emulsion plus carbogen on the survival of irradiated tumor cells appears to reflect changes in tumor oxygenation, rather than cytotoxic or immunological effects, since the perfluorochemical emulsion (with or without carbogen) had no effect on the viability of cells in unirradiated tumors. These experiments extend previous studies by ourselves and others using mouse tumors to show that the combination of a perfluorochemical emulsion and carbogen breathing can also increase the radiation response of a nonimmunogenic rat tumor.

Modulation of tumor oxygenation and radio sensitivity by a perfluorooctylbromide emulsion

The effect of a concentrated perfluorooctylbromide emulsion (Oxygent) on the radiosensitivity and oxygenation of solid tumors was examined using EMT6 mammary tumors in BALB/c mice and BA1112 rhabdomyosarcomas in WAG/rij rats. Treatment with Oxygent plus carbogen or oxygen breathing increased the radiosensitivity of both tumors. Analysis of tumor cell survival data and polarographic measurements of intratumoral pO2 indicated that this potentiation reflected an increase in the proportion of well-oxygenated tumor cells. Treatments with carbogen breathing alone, with Oxygent plus air-breathing, or with a vehicle emulsion containing all the components except the perfluorocarbon did not produce comparable improvements in tumor radiosensitivity. Concentrated perfluorooctylbromide emulsions appear to warrant further development and preclinical testing as adjuncts to cancer therapy.

Effects of hyperbaric oxygen and a perfluorooctylbromide emulsion on the radiation responses of tumors and normal tissues in rodents

Perfluorochemical emulsions are being examined in many laboratory and clinical studies as possible adjuncts to radiotherapy and chemotherapy. The studies reported here examine the clinical potential of hyperbaric oxygen (HBO) in combination with a highly concentrated perfluorochemical emulsion (Oxygent) containing 100% w/v perfluorooctylbromide (PFOB). HBO alone produced only a small improvement in the radiation response of BA1112 tumors in WAG/rij rats, while regimens combining HBO with Oxygent produced much greater radiation sensitization. A sham emulsion, formulated without the O2-carrying PFOB, did not alter the radiation response of the tumors in comparison with that seen with HBO alone. Neither HBO nor Oxygent plus HBO altered the radiosensitivity of bone marrow progenitor cells in BALB/c mice. HBO alone augmented skin reactions in BALB/c mice, but addition of Oxygent did not alter the skin reactions in comparison to those seen with HBO alone. Regimens combining Oxygent with HBO selectively increased the radiation sensitivity of tumors relative to normal tissues, thereby enhancing the therapeutic ratio. These results support the potential usefulness of perfluorochemical emulsions and HBO in clinical radiation therapy.

Preclinical evaluation of Oxygent as an adjunct to radiotherapy.

These studies examine the potential value of a concentrated emulsion of perfluorooctylbromide (perflubron; Oxygent, Alliance Pharmaceutical Corp.) as an adjunct to radiotherapy. The effects of Oxygent on solid tumors were examined using EMT6 mammary tumors in BALB/c mice and BA1112 rhabdomyosarcomas in WAG/rij rats. Treatment with Oxygent plus O2, carbogen (95% O2/5% CO2), or hyperbaric oxygen (HBO) increased the effects of radiation on the tumors. Analyses of tumor cell survival curves and measurements of intratumor pO2 showed that this potentiation reflected an increase in the proportion of well-oxygenated tumor cells. Neither treatment of the animals with carbogen, O2, or HBO alone nor treatment of air-breathing rodents with Oxygent produced changes of similar magnitude. Treatment with a vehicle emulsion containing all the components of Oxygent except the perflubron did not alter tumor radiosensitivity, showing that tumor radiosensitization required the oxygen-transporting perfluorocarbon, and did not result from any biologic or physiologic effects of other components of the emulsion. These studies also examined the effects of Oxygent on the radiation responses of mouse skin and bone marrow. Oxygent selectively increased the radiation sensitivity of tumors relative to these normal tissues, thereby increasing the therapeutic ratio and producing therapeutic gain. Oxygent appears to warrant further testing as an adjunct to cancer therapy.

Effects of a Perfluorochemical Emulsion on the Response of BA1112 Rat Rhabdomyosarcomas to Continuous Low-Dose-Rate Irradiation

The effects of the combination of a perfluorochemical emulsion (Fluosol DA, 20%) and carbogen (95% O2, 5% CO2) on the response of BA1112 rat rhabdomyosarcomas to continuous low-dose-rate irradiation were examined. Tumors were irradiated locally in unrestrained, unanesthetized rats at a dose rate of 0.98 Gy/h, using a specially designed 241Am irradiator system. Cell survival was measured using a colony formation assay. The tumor cell survival curves were fitted to linear relationships of the form ln S = - alpha D, where alpha for air-breathing rats was 0.104 +/- 0.005 Gy-1, as compared to 0.137 +/- 0.009 Gy-1 for rats treated with Fluosol plus carbogen. The increase in the slope of the survival curve produced by the treatment with Fluosol and carbogen was highly significant with a P value of 0.0015. The radiosensitization factor for the combination of Fluosol/carbogen plus continuous low-dose-rate irradiation was 1.32 +/- 0.11. Slightly less radiosensitization was observed with continuous low-dose-rate irradiation than in previous experiments using acute high-dose-rate irradiation. The diminished sensitization with Fluosol/carbogen during continuous low-dose-rate irradiation probably reflects the intrinsically lower oxygen enhancement ratio (OER) of low-dose/low-dose-rate irradiation, reoxygenation of the tumors during the prolonged treatment times used for continuous low-dose-rate irradiation, and the decrease in the levels of circulating perfluorochemicals during the 30-h irradiations. More importantly, the significant level of radiosensitization observed in the experiments with continuous low-dose-rate irradiation suggests that hypoxic cells persist in BA1112 tumors during continuous low-dose-rate irradiations and that the response of these tumors to continuous low-dose-rate irradiation can be improved by adjunctive treatments which oxygenate these radioresistant hypoxic tumor cells.

Relative biological effectiveness of 241Am relative to 192Ir for continuous low-dose-rate irradiation of BA1112 rat sarcomas

Sealed sources of 241Am have been developed for intracavitary irradiation of gynecological cancers. Relative to conventional isotopes (that is, 226Ra, 137Cs, 192Ir), 241Am allows for better shielding of dose-limiting normal tissues in the patient. In addition, the long half-life of 241Am (432 years) makes it an attractive isotope both for clinical use and for long-term radiobiology studies. Using a previously developed in vivo applicator system, BA1112 sarcomas on WAG/Rij Y rats were irradiated using 241Am or 192Ir at three different dose rates. Following in vivo treatment of the sarcomas with graded doses of radiation, cell survival curves were determined using an in vitro colony formation assay. The slopes of the resulting cell survival curves were observed to increase significantly as the dose rate increased from 0.30 to 0.60 Gy/h, then to decrease slightly as the dose rate increased from 0.60 to 0.95 Gy/h. The relative biological effectiveness (RBE) of 241Am relative to 192Ir was observed to increase linearly with increasing dose rate; the RBEs were 0.96 +/- 0.009, 1.09 +/- 0.12, and 1.17 +/- 0.11 at dose rates of 0.30, 0.60, and 0.95 Gy/h, respectively.

Quantitative Transplantation Assays of the Rat Rhabdomyosarcoma Ba1112 Isografts Into the Wag Rij-Y Rat and Xenotransplantation Into the Athymic Ncr(Nu-Nu) Nude-Mouse

Quantitative tumor cell transplantation assays have been performed to compare the transplantability of rat rhabdomyosarcoma BA1112 into isologous WAG/Rij Y rats and athymic NCr(nu/nu) nude mice. The end-point was the TD50 or the number of viable tumor cells which would transplant the tumors into half of the recipients. At Yale, two sets of 2-fold dilutions were prepared, one was sent to the MGH by Air Express. That afternoon, concurrent assays were performed at Yale using the WAG/Rij Y rat and at MGH using the NCr(nu/nu) mouse. The TD50 values were the same for iso- and xenotransplantation. Furthermore, the TD50s in rats and mice were unaffected by standard immunization procedures prior to challenge of the TD50 assay. The BA1112 (10(7) trypan blue excluding cells) grew to 10-12 mm and then completely regressed if transplanted into NCr(nu/+) mice which had received 6 Gy whole body irradiation but did not grow in control NCr(nu/+) mice. The times for the BA1112 to grow to 10 mm were the same in normal or preimmunized WAG/Rij Y rats or NCr(nu/nu) mice and in 6 Gy WBI NCr(nu/nu) mice. All of the experimental data show that the xenogenic NCr(nu/nu) mice accept the BA1112 as readily as do the isologous WAG/Rij Y rats.