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7 were measured. Each experiment was repeated three times. Using
the ORIGIN program,49 a plot of PLAP concentration (abscissa)
versus formation of 7 (ordinate) furnished a sigmoidal line from
which the IC50 value as well as the minimal concentration of PLAP
needed for any hydrolysis was calculated.
Examination by Autoradiography of Hydrolysis of 6 Incu-
bated with Human Tumor Cells. Human OVCAR3 ovarian
carcinoma cells (kindly provided by Thomas C. Hamilton, Fox
Chase Cancer Center, Philadelphia) were cultured in RPMI 1640
supplemented with fetal bovine serum (10%). Human TE671
rhabdomyosarcoma cells (ATCC) were cultured in DMEM supple-
mented with fetal bovine serum (10%). The cells were grown in
75-cm3 tissue-culture flasks and kept in a humidified atmosphere
of 5% CO2 at 37 °C, with the medium changed every other day.
Logarithmically growing cells were trypsinized, suspended in
medium, and seeded onto slides (Lab-Tek II Chamber Slide, Nalge
Nunc International Co.). After an overnight incubation at 37 °C,
the medium was removed, 6 (0.2 mL, ∼5 µCi) in PBS (0.01 mol/
L) was added, and the cells were re-incubated for 24 h. The slides
were then washed in 0.01 mol/L PBS (pH 7.4), and the cells were
fixed in ice-cold ethanol (-20 °C) for 10 min. After the slides were
dried, they were dipped vertically into Kodak NTB-2 emulsion
melted at 45 °C, withdrawn, and left to drain and gel. The slides
were stored at 4 °C in a black box containing Drierite (anhydrous
CaSO4). After exposure (3 days to 1 week), the emulsion-coated
slides were dipped at room temperature in Kodak developer D-19
for 5 min, washed in distilled water, immersed in Kodak fixer for
5 min, and rinsed again in distilled water. Finally, the nuclei were
stained with DAPI with mount (Vecta Shield) and the slides viewed
under a fluorescence (DAPI nuclei)/light (ARG grains) microscope.
Biodistribution of 6 in Normal Mice. To determine normal-
tissue localization of 6, each mouse (n ) 5/group, total three groups)
received an intravenous injection (lateral tail vein) of 5 µCi carrier-
free, radioiodinated prodrug in 0.1 mL PBS (0.01 mol/L, pH 7.4).
The mice were sacrificed at 24 h, the following organs and tissues
were dissected and weighed, and their radioactivity determined in
a gamma counter: blood, heart, lungs, liver, spleen, kidneys, fat,
bladder, urine, stomach, small intestine, large intestine, muscle, skin,
bone, thymus, and thyroid. Based on the injected activity and the
weight of these fluids, tissues, and organs, the percentage injected
dose per gram (% ID/g) was then calculated.
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Acknowledgment. This work was supported in part by the
US Department of Defense Grants W81XWH-04-1-0499 -
Radiodetection and Radiotherapy of Breast Cancer, W81XWH-
06-1-0043 - Radioimaging and Radiotherapy of Prostate
Cancer, and W81XWH-06-1-0204 - Radiodiagnosis and Ra-
diotherapy of Ovarian Cancer (to A.I.K.).
Supporting Information Available: Lineweaver-Burk plot of
PLAP-dependent conversion of prodrug 4 to drug 2. This material
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