Increase in pO2 and radiosensitivity of tumors by Fluosol-DA (20%) and carbogen

The potential usefulness of i.v. injection of perfluorochemicals and breathing carbogen (95% O2 and 5% CO2) to improve the radiation-induced control of tumors was investigated. When C3H mice, bearing RIF-1 tumors in the legs, were given i.v. injections of Fluosol-DA (20%) at 12 ml/kg, and allowed to breathe carbogen for 1 h before and during a single dose of X-irradiation, the curability of tumors increased by a dose modification factor of 1.47 +/- 0.03 (SE). Such a treatment also increased the radiation-induced skin damage by a factor of 1.15 +/- 0.12, resulting in a therapeutic gain of 1.28 +/- 0.04. Measurement of intratumor pO2 by oxygen microelectrodes demonstrated small increases in pO2 when the animals breathed carbogen, and marked increases in pO2 when Fluosol-DA (20%) was injected into the animals and the animals breathed carbogen. It was concluded that i.v. injection of Fluosol-DA (20%) followed by carbogen breathing significantly improved the oxygen supply to hypoxic cells in the RIF-1 tumors and thus increased the control of tumors by radiation.     

Increase in tumor pO2 by perfluorochemicals and carbogen

The effects of perfluorochemicals (Fluosol-DA) in combination with carbogen (95% O2 and 5% CO2) breathing on the tumor pO2 was investigated. The pO2 in RIF-1 tumors grown in the leg of C3H mice was determined by polarographic method using oxygen microelectrodes with diameters of 50-60 micron. The average and median pO2 in the control RIF-1 tumors was about 13 mm Hg and 6 mm Hg, respectively. Carbogen breathing alone could cause a significant increase in pO2 in some tumors and Fluosol-DA injection (i.v.) alone caused a slight change in tumor pO2. When the animals were treated with both Fluosol-DA injection and carbogen breathing, the pO2 markedly increased in most of the tumors resulting in an average and median tumor pO2 of 80 mm Hg and 60 mm Hg, respectively. Such an increase in tumor pO2 may account for the previous observations by us and others that the response of experimental tumors to radiation could be markedly increased by treating the tumor-bearing animals with carbogen and Fluosol-DA.     

Reactions of tumors and normal tissues in mice to irradiation in the presence and absence of a perfluorochemical emulsion

The effect of pre-treatment with the perfluorochemical emulsion, Fluosol-DA, on the radiation response of normal tissues and EMT6 mammary tumors in BALB/c mice was examined. Pre-treating tumor-bearing mice with .015 ml/g of Fluosol and 30 min of carbogen (95% O2/5% CO2) increased the number of tumor cells killed by irradiation with doses of 2.5-20 Gy; the change in the radiation dose-response curve was consistent with a reduction in the hypoxic fraction. Fluosol did not alter the response of tumors in air-breathing or N2-asphyxiated mice and carbogen alone did not alter the radiation response of this tumor significantly. Carbogen treatments 5-60 min in duration produced similar enhancements of tumor radiosensitivity in Fluosol-treated animals. Pre-treatment with Fluosol plus carbogen also increased the number of tumor cells killed by a fractionated regimen of four 2.5 Gy fractions given over 2 days. Pre-treatment with Fluosol-DA plus carbogen, therefore, increased the antineoplastic effects of radiotherapy in both single-dose and multi-fraction radiation regimens. In contrast, Fluosol did not increase the effect of radiation on the partially committed (CFU-GM) or pluripotent (CFU-S) stem cells of the bone marrow or on the CFU-GM of the spleen. The radiation response of the skin was only slightly enhanced by pre-treatment with Fluosol plus carbogen. These data show that treatment of mice with perfluorochemical emulsions plus carbogen can produce therapeutic gain by improving the radiation response of solid tumors, without producing an equivalent increase in the radiation response of potentially dose-limiting normal tissues. These findings encourage further evaluations of these agents as adjuncts to clinical radiotherapy.     

Addition of a hypoxic cell selective cytotoxic agent (mitomycin C or porfiromycin) to Fluosol-DA/carbogen/radiation

In an effort to develop effective combination treatments for use with radiation against solid tumors, the cytotoxic effects of the addition of mitomycin C or porfiromycin on treatment with Fluosol-DA/carbogen (95% O2/5% CO2) breathing and radiation in the FSaIIC tumor system were studied. In vitro mitomycin C and porfiromycin were both preferentially cytotoxic toward hypoxic FSaIIC cells. After in vivo exposure, however, the cytotoxicity of mitomycin C toward single cell tumor suspensions obtained from whole tumors was exponential over the dose range studied, but for porfiromycin a plateau in cell killing was observed. With Fluosol-DA/carbogen breathing and single dose radiation, addition of either mitomycin C or porfiromycin increased the tumor cell kill achieved at 5 Gy by approximately 1.2 and 1.0 logs, respectively. Less effect was seen with addition of the drugs at the 10 and 15 Gy radiation doses. In tumor growth delay experiments, the addition of either mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation resulted in primarily an additive increase in tumor growth delay. The survival of Hoechst 33342 dye-selected tumor cell subpopulations indicated that Fluosol-DA/carbogen breathing increased the cytotoxicity of radiation (10 Gy) more in the bright cell subpopulation (4-fold) than in the dim cell subpopulation (2-fold) resulting in an overall 4-fold sparing of the dim subpopulation. Mitomycin C and porfiromycin were both more toxic toward the dim cell subpopulations. Addition of mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation (10 Gy) resulted in a primarily additive effect of the drugs and radiation killing in both tumor cell subpopulations. Thus, with mitomycin C/Fluosol-DA/carbogen and radiation there was a 2-fold sparing of dim cells and with porfiromycin in the combined treatment a 1.6-fold sparing of the dim cell population. Our results indicate that treatment strategies directed against both oxic and hypoxic tumor subpopulations can markedly increase the tumor cell kill achieved by radiation.     

Changes in radiation sensitization induced by Fluosol-DA as measured by 31P nuclear magnetic resonance spectroscopy

Numerous agents have been studied in attempts to sensitize radioresistant hypoxic tumor cells. We have investigated the effect of Fluosol-DA plus carbogen (95% oxygen and 5% CO2) on the sensitivity of a radioresistant mammary carcinoma in C3H/He mice and also on tumor metabolism by 31P nuclear magnetic resonance spectroscopy. Statistically significant increases in phosphocreatine/Pi were noted for small- (150-350 mm3) and medium- (351-650 mm3) sized tumors treated with Fluosol-DA plus carbogen. Small tumors were shown to undergo significant radiosensitization in the presence of Fluosol-DA plus carbogen and medium-sized tumors showed a lesser degree of radiosensitization. Large tumors (greater than 900 mm3) showed no effect. Fluosol-DA or carbogen alone had no effects on animals with any tumor volume, as monitored by significant changes in radiosensitivity or nuclear magnetic resonance parameters. An approximately linear relationship was found between the decrease in the values for radiation dose which yields 50% tumor control and the increase in phosphocreatine/Pi, with a correlation of r = -0.93. 31P nuclear magnetic resonance spectroscopy may be useful for monitoring changes in radiosensitivity induced by agents which alter tumor oxygenation and subsequent metabolic status.     

Effect of Fluosol-DA on the response of intracranial 9L tumors to X rays and BCNU

Treatment with a perfluorochemical emulsion combined with breathing a 95% or 100% oxygen atmosphere has been shown to be an effective adjuvant to radiation therapy in several animal tumor systems. Similarly, the addition of treatment with a perfluorochemical emulsion combined with breathing a high oxygen atmosphere has been shown to improve the response of several animal tumor systems to treatment with BCNU. We now report results of the use of the perfluorochemical emulsion, Fluosol-DA, and carbogen breathing with single dose radiation treatment, BCNU and combined drug and radiation treatment in intracranially implanted 9L gliosarcoma. The median enhancement in life span produced by Fluosol-DA and carbogen breathing in addition to radiation was 2 days at 10 Gy and 6 days at 20 Gy compared to radiation treatment alone. In the group receiving 20 Gy with Fluosol-DA and carbogen breathing, 2 of 20 lived 120 days. Treatment with a single intraperitoneal injection of BCNU (10 mg/kg) on day 7 post tumor cell implantation produced an increase in life span of 2 days compared to untreated control animals. The combination of drug treatment with Fluosol-DA and carbogen breathing produced an increase in life span of 26 days, which was significantly different from BCNU treatment with air breathing (p less than 0.001). Finally, when BCNU and Fluosol-DA and carbogen were combined with radiation treatment (20 Gy), an increase in life span of nearly 85 days compared to untreated controls was produced, with 47% (9 or 19) surviving 120 days. These results suggest that this combination might be effective in the treatment of malignant brain tumors.     

Modification of photodynamic therapy-induced hypoxia by fluosol-DA (20%) and carbogen breathing in mice

The administration of a perfluorochemical emulsion and carbogen (95% O2, 5% CO2) breathing before photodynamic therapy (PDT) was studied to determine how increased levels of tumor oxygenation may affect PDT-induced tumor destruction. C3H/HeJ mice bearing the RIF tumor were given injections of 5 to 10 mg/kg of dihematoporphyrin ethers 24 h prior to treatment. Animals were given injections of 12 ml/kg of Fluosol-DA (20%) followed by carbogen breathing or 12 ml/kg of saline and air breathing (controls) 1 h before tumors were exposed to 135 J/cm2 of 630-nm light treatment. Changes in the hypoxic fraction of tumors, the time course for decreases in tumor cell clonogenicity, and tumor response were measured immediately and at various times after treatment. The administration of Fluosol-DA (20%) and carbogen breathing was found to delay the onset of PDT-induced hypoxia through the first hour posttreatment. Progressive tumor hypoxia was observed after 4 h posttreatment. The time period in which tumors remained well oxygenated coincided with observations of increased tumor cell survival. Decreases in tumor cell clonogenicity were observed only after tumor cells became hypoxic. These findings were consistent with the 24-h delay in complete tumor response in animals given Fluosol-DA (20%) and carbogen breathing before PDT. There were only minor variations in long-term tumor response and cure observed between the two groups tested. A second series of experiments was done to assess any treatment advantage of the adjuvant use of Fluosol-DA (20%) and carbogen breathing with PDT at high tumor photosensitizer levels. At an injected dose of 50 mg/kg of dihematoporphyrin ethers, no such advantage was observed. The administration of Fluosol-DA (20%) and carbogen breathing did not reduce the extent of PDT-induced microvascular damage, maintain high levels of tumor oxygenation through light treatment, or modify the extent of tumor cell kill following treatment.     

Response of subpopulations of the FSall C fibrosarcoma to low dose x-rays and various potential enhancing agents.

The effectiveness of various oxygen carrying treatments in sensitizing subpopulations of the FSallC fibrosarcoma to low doses of radiation was assessed, and compared with survivals obtained with the same cells after in vivo irradiation under normally oxygenated or hypoxic conditions. FSallC tumors were treated with 2-10 Gray then the Hoechst 33342 dye diffusion method was used to separate the tumor into bright (enriched in normally oxygenated cells) and dim (enriched in hypoxic cells) subpopulations. There was good agreement between the survival of normally oxygenated cells in culture and bright cells from tumors and between hypoxic cells in culture and dim cells from tumors over a radiation dosage range of 2-5 Gray. At 10 Gray bright cells from tumors were minimally less sensitive to the radiation dose than normally oxygenated cells in vivo. When maximally effective doses of perfluorochemical emulsions (F44E at 4 g PFC/kg or Fluosol-DA at 2.4 g PFC/kg) or a purified bovine hemoglobin solution (PBHS at 1.32 g protein/kg) were administered 1 hr. prior to radiation therapy with carbogen (95% O2, 5% CO2) breathing prior to and during radiation delivery, low single doses of x-ray (2-5 Gray) were measurably more cytotoxic toward both FSallC tumor cell subpopulations. These results indicate that perfluorochemical emulsions or purified bovine hemoglobin preparations along with carbogen breathing may be able to increase tumor radiosensitivity to the relatively low radiation doses per fraction used in the clinic.     

Addition of misonidazole, etanidazole, or hyperthermia to treatment with fluosol-DA/carbogen/radiation

The antitumor efficacy of adding the nitroimidazole radiosensitizing drugs misonidazole and etanidazole or hyperthermia (43 degrees C for 30 min) to Fluosol-DA/carbogen (95% O2/5% CO2) and irradiation was tested in the FSaIIC tumor system. Both the nitroimidazole drugs and hyperthermia produced additional tumor growth delays and tumor cell cytotoxicity when given with Fluosol-DA/carbogen, either before or after irradiation. For each of the modalities tested, the dose-modifying effect was greater when that therapy preceded rather than followed irradiation (misonidazole 2.7 vs. 1.9, etanidazole 2.4 vs. 1.7, hyperthermia 4.0 vs. 1.7 relative to the effect of radiotherapy alone). Because the nitroimidazole drugs must be present before radiation is administered to exert their radiosensitizing effect, the increase in tumor growth delay observed when these drugs cytotoxic to hypoxic cells were administered following Fluosol-DA/carbogen and irradiation suggests that Fluosol-DA/carbogen could not fully oxygenate the tumors and that the nitroimidazole drugs were effectively toxic to residual hypoxic cells. The treatment Fluosol-DA/carbogen----hyperthermia----irradiation produced a marked increase in tumor growth delay not seen with the sequence Fluosol-DA/carbogen----irradiation----hyperthermia. The results indicate that a treatment combination of radiation sensitizers may be more effective than irradiation plus Fluosol-DA with oxygen breathing alone.     

Effect of oxygen on the cytotoxicity and antitumor activity of etoposide

The selective cytotoxicity of the epipodophyllotoxin etoposide toward normally oxygenated and hypoxic EMT6 mouse mammary tumor cells in culture was examined. Etoposide was much more toxic to normally oxygenated cells. The ratio (hypoxic to oxygenated) of drug concentrations producing 1 log of cell kill was approximately 30:1. Established FSa-11C fibrosarcomas of C3HeB/FeJ mice were treated with 10, 15, or 20 mg etoposide/kg body weight in a 6-day protocol. Fluosol-DA with or without breathing of carbogen (i.e., 95% O2-5% CO2) was added to the treatment program on days 1, 3, and 5. The combination of etoposide-Fluosol-DA-carbogen markedly enhanced tumor growth delay compared to the result with etoposide alone. The dose-modifying effect observed was 1.9 +/- 0.3. With the use of both single-dose and multiple-dose protocols for etoposide and Fluosol-DA with air or carbogen breathing, the survival of bone marrow cells was measured by colony formation in vitro (granulocyte-monocyte colony-forming units). Fluosol-DA and carbogen breathing did not increase the toxicity of etoposide to the bone marrow. Thus the enhancement in antitumor activity produced by the addition of Fluosol-DA and carbogen breathing to etoposide treatment was not accompanied by a concomitant increase in normal tissue toxicity and represents an increase in the therapeutic efficacy of etoposide.     

Effect of various oxygenation conditions and fluosol-DA on cytotoxicity and antitumor activity of bleomycin in mice

In an attempt to improve the antitumor efficacy of bleomycin, the effects of the oxygen-carrying emulsion Fluosol-DA and increased levels of inspired oxygen were tested in the mouse FSaIIC fibrosarcoma system. The dose-dependent cytotoxicity of bleomycin toward the FSaIIC cells in vitro was significantly decreased under hypoxic conditions, but it increased in a 95% O2-5% CO2 (carbogen) atmosphere as compared with the cytotoxicity of bleomycin in normally oxygenated cells. Investigations on the FSaIIC tumor in vivo also demonstrated that growth delays induced by bleomycin (10 mg/kg ip given on days 6, 10, 13, and 16) were significantly increased when one of the following treatments was given with each bleomycin injection: carbogen breathing for 2 hours (4.7 days), carbogen breathing for 6 hours (5.7 days), and breathing 3 atm of hyperbaric oxygen (6.3 days) versus normal air (3.3 days). When Fluosol-DA (12 mL/kg iv) was administered just before each bleomycin injection, the following growth delays were produced: 4.8 days with air breathing, 14.6 days with carbogen breathing for 2 hours, 14.9 days with carbogen breathing for 6 hours, and 19.7 days with breathing 100% O2 at 3 atm for 1 hour. Excision studies on the FSaIIC tumor also demonstrated that the cytotoxicity increased approximately fivefold when Fluosol-DA and carbogen breathing for 2 hours were combined with a single treatment with 10 mg of bleomycin/kg. In contrast, no measurable bone marrow toxicity was evident with this combined regimen. These results suggest that the use of Fluosol-DA plus carbogen breathing could add substantially to the clinical antitumor effects of bleomycin.     

Preclinical studies of a perfluorochemical emulsion as an adjunct to radiotherapy

Tumor growth and tumor cell survival endpoints were used to examine the effects of a perfluorochemical emulsion, Fluosol-DA, 20%, and carbogen (95% O2/5% CO2) on EMT6 mouse mammary tumors in BALB/c mice. These studies defined the effects of the Fluosol dose on the hematocrit and fluorocrit of the mice and on the radiation response of the tumors. The effect of varying the duration of carbogen breathing before irradiation was examined; times of 5-60 min gave similar enhancements of tumor radiosensitivity. Potentiating effects were not observed when the tumors were irradiated 1-3 days after Fluosol injection, probably reflecting the observed clearance of the perfluorochemicals from the circulating blood. Fluosol injected 30 min-2 days before irradiation did not alter the radiation response of tumors in air-breathing or N2-asphyxiated mice. Together, these studies provided additional support for the hypothesis that the potentiation of tumor radiation response observed after treatment with Fluosol plus carbogen results from changes in O2 delivery to the hypoxic tumor cells by oxygenated perfluorochemical particles. This confirms the conclusion drawn on the basis of the observed changes in the tumor cell survival curve. Studies of tumor cell survival, tumor cell yield, tumor growth, and artificial lung metastasis formation revealed no effects of Fluosol treatment (without irradiation) on tumor progression or metastasis. Studies examining the effects of Fluosol plus carbogen on the growth of tumors irradiated with 5 Gy showed that the changes in tumor radiosensitivity observed using cell survival endpoints also occurred in tumors left in situ after irradiation with a radiation dose similar to those used in some clinical trials.     

Potentiation of rat brain tumor therapy by fluosol and carbogen

We have been using the 9L rat brain tumor model to investigate the effect of the combination of a perfluorochemical emulsion, Fluosol-DA 20%, and carbogen breathing on the therapy of brain tumors. The combination of Fluosol, carbogen breathing, and carmustine (BCNU) was more effective at prolonging survival than was BCNU alone. This difference was small but significant (P less than 0.25). neither Fluosol without carbogen nor carbogen without Fluosol significantly altered the effect of BCNU. Fluosol and carbogen alone did not affect the survival of tumor-burdened rats. Fluosol and carbogen breathing did not alter the effect of single doses of radiation on these tumors. This result supports the hypothesis that 9L brain tumors contain few, if any, critical hypoxic cells. However, these tumors may contain cells which are oxygen deficient but not radiobiologically hypoxic. The Fluosol-carbogen combination may be changing the intratumor environment in such a way that the metabolism or activity of BCNU is altered.     

Differential enhancement of melphalan cytotoxicity in tumor and normal tissue by Fluosol-DA and oxygen breathing

The addition of Fluosol-DA carbogen breathing to melphalan treatment of the FSa-IIC fibrosarcoma was assessed by tumor growth delay and cell survival assays. Melphalan (10 mg/kg) administered intraperitoneally (i.p.) was preceded by Fluosol-DA (0.3 ml) administered intravenously (i.v.) and followed by 1 hr of carbogen breathing; this resulted in a tumor growth delay of 9.5 +/- 1.4 days or an approximately 3-fold increase compared to melphalan alone. Melphalan produced about 1.7 logs of cell killing; neither carbogen breathing nor Fluosol-DA pretreatment altered the cell killing observed. There was a 10-fold increase in tumor-cell killing when Fluosol-DA was administered immediately prior to melphalan administration followed by carbogen breathing for 1 hr. Density gradient separation identified a population of denser FSa-IIC cells which showed increased sensitivity to melphalan after Fluosol-DA administration. There was no additional toxicity to bone marrow as measured by CFU-GM with the combination of melphalan/Fluosol-DA/O2 compared to melphalan alone.     

Effects of dose and scheduling on growth delay of the Lewis lung carcinoma produced by the perfluorochemical emulsion, Fluosol-DA

The perfluorochemical emulsion, Fluosol-DA, combined with breathing a 95% oxygen/5% carbon dioxide atmosphere enhances the response of several rodent tumors. B6D2F1/J mice bearing Lewis lung tumors, measuring 50-100 mm3 were treated with 4, 8 and 12 ml/kg of Fluosol-DA intravenously each morning. Three Gy fractions twice per day were employed and carbogen breathing was maintained 1 hr prior to and during each X ray treatment. The dose modifying factors were 1.42 +/- 0.16 at 4 ml/kg, 1.85 +/- 0.23 at 8 ml/kg, and 2.17 +/- 0.34 at 12 ml/kg. In a second experiment, a single dose of Fluosol-DA (12 ml/kg) was administered on day 1 to B6D2F1/J male mice, bearing established subcutaneous Lewis lung tumors, as described above. X rays were delivered in 2, 3, or 4 Gy fractions once per day for five days. The dose modifying effect was 2.60 +/- 0.54. The effect of this treatment program was the same as that seen with single dose radiation. These experiments demonstrate that Fluosol-DA need not be administered with every fraction to produce an improved treatment outcome with fractionated X rays.     

Approaches to defining the mechanism of enhancement by Fluosol-DA 20% with carbogen of melphalan antitumor activity

Fluosol-DA with carbogen (95% oxygen and 5% carbon dioxide) breathing can increase the efficacy of melphalan. Addition of Fluosol-DA to treatment with melphalan leads to a greater increase in tumor growth delay under conditions of air breathing and carbogen breathing than does the fat emulsion Intralipid. The ability of melphalan to kill tumor cells increased with dose over the range of drug examined. At the lower doses of drug there is some increase in tumor cell killing seen with the addition of carbogen breathing or Fluosol-DA and air breathing; however, at the highest dose of the drug this difference disappeared. Throughout the melphalan dosage range examined there is approximately 1 log greater tumor cell kill observed with the addition of Fluosol-DA and carbogen breathing compared to the drug treatment alone. There was no significant difference in the survival of bone marrow cells under any of the treatment conditions. Fluosol-DA itself with air or carbogen breathing produced no detectable cross-links in DNA from tumors treated in vivo. The cross-linking factors for melphalan with air or carbogen breathing and for melphalan plus Fluosol-DA and air breathing were similar; when carbogen breathing was added to the treatment combination, the cross-linking factor increased almost 3-fold. When melphalan was dissolved in Fluosol-DA, the melphalan moved quickly into the lipophilic perfluorochemical particles so that after 1 h 60% of the drug was in the perfluorochemical layer. At 24 h, 85-90% of the melphalan was sequestered in the perfluorochemical particles. The pharmacokinetics of [14C]melphalan alone, [14C]melphalan plus Fluosol-DA, and [14C]melphalan prepared in Fluosol-DA were studied in several tissues of FSaIIC fibrosarcoma-bearing mice. In general, the tissue absorption and distribution t1/2s for melphalan were shortened in the presence of Fluosol-DA (except for kidneys). Shifting the t1/2s for absorption and distribution to shorter times produces a much sharper and earlier peak in the drug exposure of the tumor. Fluosol-DA provides a relatively nontoxic means of increasing oxygen delivery to tumors and a therapeutically meaningful way of improving melphalan antitumor activity.     

The influence of Fluosol-DA on the occurrence of lung metastases in Lewis lung carcinoma and B16 melanoma

The development of lung metastases from subcutaneously implanted tumors or the development of lung nodules from intravenously injected tumor cells are model systems for metastases formation. Animals bearing subcutaneous Lewis lung tumors (50-100 mm3) were treated with a single dose of Fluosol-DA followed by 1 h of breathing carbogen or maintenance in air. Their lungs were examined for metastases 25 or 40 days after tumor cell implantation. Treatment with Fluosol-DA and carbogen or air breathing reduced by almost 4-fold the number of lung metastases seen. The addition of Fluosol-DA with air or carbogen breathing to treatment of the tumor-bearing limb with 20 Gy reduced the number of lung metastases by 2-fold compared to radiation treatment alone. If Fluosol-DA was administered immediately before or up to 3 days prior to an intravenous challenge with Lewis lung tumor cells, there was a 2- to 3-fold reduction in the number of lung nodules formed. Fluosol-DA administered immediately before or up to 4 days prior to B16 melanoma cells caused a 2- to 3-fold reduction in the number of lung nodules observed. The vascular endothelial cell monolayer adhesion assay was used to test the effects of prior exposure to Fluosol-DA on the attachment of radiolabelled B16 melanoma cells in vitro. There was a trend toward increasing attachment of B16 cells to the endothelial monolayer with prior exposure to increasing concentrations of Fluosol-DA; however, this difference did not reach statistical significance.     

Effect of Fluosol-DA/O2 on tumor-cell and bone-marrow cytotoxicity of nitrosoureas in mice bearing FSA-II fibrosarcoma

The perfluorochemical emulsion, Fluosol-DA, combined with carbogen breathing, potentiates the effects of radiation and a number of chemotherapeutic agents in several rodent tumors. The interaction of Fluosol-DA with drugs may be quite complex. In addition to increasing the oxygen supply in the tumor, Fluosol-DA may alter the pharmacokinetics of the drug and function as a drug delivery system. A series of 4 nitrosoureas of varying lipophilicity were administered as single doses intravenously (i.v.) to C3H/Be/FeJ mice bearing subcutaneous FSa-IIC fibrosarcomas. Doses of 40 mg/kg of CCNU, 15 mg/kg of BCNU, 20 mg/kg of MeCCNU and 15 mg/kg of chlorozoticin followed by 2 hr of breathing 95% oxygen produced tumor growth delays of 7.5, 4.0, 3.8 and 2.7 days, respectively. When the drug injection was followed immediately by 0.3 ml of Fluosol-DA and 2 hr of breathing 95% oxygen, the tumor growth delay produced by CCNU, BCNU, MeCCNU and chlorozoticin increased 2-fold, 10-fold, 4.5-fold and 3.5-fold, respectively. Administration of the drugs in Fluosol-DA followed by 2 hr of 95% oxygen breathing resulted in a 3.5-fold increase in tumor growth delay with CCNU, a 17-fold increase with BCNU, a 12.5-fold increase with MeCCNU and a 6-fold increase with chlorozoticin compared to drug and 95% oxygen breathing. These results are quantified in terms of cell survival by the tumor excision assay. Effects on the bone marrow from each treatment were measured using the granulocyte-monocyte colony-forming units (CFU-GM) assay. There was no correlation between the lipophilicity of the nitrosoureas tested and the tumor growth delay produced by each treatment.     

Tumor oxygenation and radiosensitization by pentoxifylline and a perflubron emulsion carbogen breathing

Oxygen tension measurements were made in three tumors: (i) the murine FSaII fibrosarcoma, (ii) the rat 9L gliosarcoma and (iii) the rat 13672 mammary adenocarcinoma using a pO2 histograph. Tumor oxygenation measurements were made while the animals breathed air or breathed carbogen (95% oxygen/5% carbon dioxide). Pentoxifylline or a perflubron emulsion was administered to the animals and tumor oxygen measurements were repeated under both breathing conditions. Both pentoxifylline and the perflubron emulsion improved the oxygenation of the FSaII fibrosarcoma under air breathing conditions but did not alter the oxygen profiles of either rat tumor compared with air breathing alone. Carbogen breathing increased the oxygenation of all tumors. Pentoxifylline administration did not change the oxygen profiles of the tumors under carbogen breathing conditions but administration of the perflubron emulsion increased the oxygenation of all three tumors under carbogen breathing conditions compared with carbogen breathing alone. Co-administration of pentoxifylline and the perflubron emulsion enhanced the radiation response of the Lewis lung tumor to daily fractionated radiation under air breathing conditions with a dose modifying factor of 1.65 and under carbogen breathing conditions with a dose modifying factor of 2.25. Over a range of perflubron emulsion doses, pentoxifylline increased the growth delay of the Lewis lung tumor in a constant manner. These results indicate that pentoxifylline and the perflubron emulsion have the largest impact on the oxygenation of more hypoxic tumors and that administration of the perflubron emulsion/carbogen breathing is the most effective means of increasing tumor oxygenation and radiation response.     

Minimal effective dose of perfluorochemical emulsion as a radiosensitizer

Perfluorochemical emulsion can dissolve large amount of oxygen, carry it into tissues, and consequently enhance the response of hypoxic tumor cells to radiation. Previously we reported on the radiosensitizing effect of perfluorochemical emulsion. In the present study, the minimal effective dose of Fluosol-DA Saline 20% (FDAS), one of preparations of perfluorochemical emulsion, was determined. Mice bearing Lewis lung carcinoma in their thighs were injected in single i.v. of FDAS (1.25-20 ml/kg) and were allowed to breath carbogen (95% O2 + 5% CO2) for 30 min. before and during irradiation (15 Gy). The effect of FDAS was measured by the growth delay of the treated tumor. Administration of FDAS at over 5 ml/kg together with carbogen breathing significantly (p less than 0.01) enhanced the tumor growth delay as compared with treatment of carbogen breathing without FDAS. There was a significant difference (p less than 0.05) in the radiosensitizing effect between injection of FDAS at 5 ml/kg and 20 ml/kg, while there were not significant differences between injection of FDAS at 5 ml/kg and 10 ml/kg, or 10 ml/kg and 20 ml/kg, respectively. These results indicate that the minimal effective dose of FDAS is 5 ml/kg, which could be applied to clinical fractionated irradiation therapy as a standard FDAS-dose.