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Difluoromethylornithine (DFMO) depletes endogenous putrescine and enhances the uptake of and retention of [3H] putrescine in vitro. To determine if DFMO also enhances uptake of [3H] putrescine in vivo, DFMO and trace doses of [3H] putrescine, dissolved in artificial CSF, were infused into growing (6-9 day) 9L brain tumors by means of osmotic pumps. When 7-day osmotic pumps were loaded with 1 microCi [3H] putrescine, with or without 10 or 100 mM DFMO, pumped at 1 microl/h, the mean uptake after 3 days was 168 +/- 62 cpm/mg tumor (17 rats) without DFMO, 300 +/- 197 cpm/mg tumor (11 rats) with 10 mM DFMO and 1088 +/- 421 cpm/mg tumor (11 rats) with 100 mM DFMO (p < or = 0.05 vs. control). Significantly less radioactivity was detected in the contralateral brain and in nonbrain tissues (0.5 +/- 0.1 to 14 +/- 5 cpm/mg). To measure the extent of [3H] putrescine distribution in the tumor, the same dose of drugs was delivered for a longer period of time, using 14-day pumps to allow tumors to become large enough to be divided into 1.4 mm thick transections. The mean radioactivity in the sections from eight control rats receiving [3H] putrescine without DFMO were not significantly different between the sections (174 +/- 61 cpm/mg tumor for sections containing the cannulas, 273 +/- 61 and 259 +/- 91 cpm/mg for adjacent sections). In the six rats given 100 mM DFMO there was a significant increase in mean radioactivity in the cannula containing section (2251 +/- 919 cpm/mg tumor). Mean counts from adjacent sections in these rats were 97 +/- 44 and 33 +/- 13 cpm/mg. Values for contralateral corpus striatum and nonbrain tissues ranged from 0.7 +/- 0.3 to 4.3 +/- 1.5 cpm/mg tissue. When DFMO was delivered directly to the tumors while [3H] putrescine was infused intraperitoneally, the uptake in the tumor slices was low (5-10 cpm/mg in different slices). These results demonstrate that infusion of DFMO directly into growing 9L brain tumors can selectively enhance the uptake of exogenous [3H] putrescine by rapidly dividing cells which are within a 1.4 mm diameter area at the cannula tip. Although these studies used [3H] putrescine at trace doses, it is estimated that infusion of higher doses of [3H] putrescine plus DFMO will selectively kill tumor cells.

Purpose: This study explored the possible use of D,L- alpha-difluoromethylornithine (DFMO) to enhance the uptake of [3H] putrescine in order to selectively kill brain tumor cells., Methods and Materials: Gliosarcoma cells (9L) were grown for 4 or 20 day periods in monolayer cultures with or without [3H] putrescine and/or DFMO. Cells in culture incubated for 20 days were replated at 4-day intervals. Cells were counted on a Coulter Electronic Particle Counter and percent viability was determined by eosin dye exclusion. Survival of cells with proliferative capacity was assayed by their colony. Forming ability and surviving fraction was calculated. The radioactive counts due to [3H] putrescine were measured in 9L cells and in medium and expressed as cpm/100 cells or cpm/ml, respectively., Results: As previously reported (15), DFMO treatment resulted in termination of cell proliferation that was reversible by the addition of exogenous putrescine. Specifically, after 4 days in culture, cell counts in groups exposed to 10 mM DFMO were 55% of those in control groups and addition of 3 mM putrescine reversed the DFMO effects. Uptake of [3H] putrescine into untreated cells increased in proportion to the amount of exogenous putrescine present during 4 days of culture (range 0.01 nmol to 100 nmol) and the presence of DFMO in the medium enhanced the uptake 9 fold throughout these ranges. At activities greater than 100 cpm/100 cells the cell count was reduced to 23 to 48% of control after 4 days in culture. Extending the treatment to 20 days of incubation increased the killing of 9L cells. During the 20-day incubation, control cells increased from 5 x 10(5) to 13 x 10(12) of which 90% were colony forming cells. Treatment with either 25 microCi [3H] putrescine or 1 mM DFMO for 4 days followed by removal of these agents and incubation for an additional 16 days for a total of 20 days resulted in 31 x 10(8) or 18 x 10(7) colony forming cells, respectively. Combining [3H] putrescine and DFMO treatments during the first 4 days of the 20 day incubation reduced the colony forming cells to 21 x 10(5) (surviving fraction to 67%). When the DFMO treatment was present during the entire 20 days, it became cytotoxic since the colony forming cells were reduced to 35 x 10(3) (surviving fraction was 17%). The combination of the 4-day [3H putrescine and the 20 day DFMO treatments resulted in only 1200 surviving colony forming cells (surviving fraction was only 2%)., Conclusion: DFMO treatment of 9L cells for 20 days resulted in increased uptake of [3H] putrescine, a 10(10) fold inhibition of colony forming cells and extensive 9L cell killing relative to untreated controls.

The efficacy of 5-iodo-2-deoxyuridine (IUDR) as a radiosensitizer when administered by continuous infusion into the cerebral spinal fluid (CSF) of the lateral cerebral ventricle was evaluated in a 9L gliosarcoma rat brain tumor model. Stereotactic implantation of a 5 x 10(4) tumor cell suspension into the left caudate nucleus was carried out in four groups of 10 rats each. Control animals had a median survival of 16.9 days (range 16-21 days). IUDR, 8.4 mg over 7 days administered by continuous infusion into the left lateral ventricle produced a slight survival advantage (median survival 21.5 days, range 12-56). Irradiation of the entire brain, 8 Gy on days 4, 6 and 7 after tumor cell implantation also produced a slight improvement in survival (median 19.5 days, range 17-34). The combination of radiation and IUDR infusion into the CSF produced a marked survival advantage (median 30.5, range 22-54) compared to the control and single modality treatment groups. This is the first demonstration of the effectiveness of IUDR as a radiosensitizer when administered into the lateral cerebral ventricle in the treatment of an intraparenchymal brain tumor.

We evaluated the effects of 16,16-dimethyl prostaglandin E2 (dm-PGE2), with and without syngeneic bone marrow transplantation (BMT) on the survival and hematopoietic recovery of mice given 14-20 Gy total body irradiation (TBI). Survival of mice given combined dm-PGE2 and BMT was improved significantly over that of mice given either treatment alone. The 30-day survival after 14, 15, 16 or 18 Gy TBI for combined treatment was 97, 90, 20 or 10 percent, respectively. The corresponding 30-day survival rates for mice given BMT alone were 69, 60, 7 or 0 percent, respectively. For dm-PGE2 alone, 30-day survival was 63, 20, 10 or 0 percent, respectively. Deaths in both dm-PGE2 treated groups generally occurred after day 10 whereas deaths in the BMT group occurred before day 10. All irradiated controls were dead on or before day 10; after larger doses, deaths clustered around day 5. After 20 Gy TBI, all mice in all groups were dead by day 7. Studies of white blood cell recovery 1-9 days after 14 Gy TBI showed improvement with BMT, whereas dm-PGE2 did not enhance recovery. Nucleated cells per humerus, spleen weight, and spleen iron uptake (erythropoiesis) were also improved by BMT but not dm-PGE2.