Autologous Bone Marrow Transplantation - Blog Science Connections

Chemopurging 145
incubation, the cells were washed three times, resuspended, and cultured in a
soft bilayer agar system at 1 x 10 5
cells/plate. The number of CFCIs-GM was
counted on each plate after 10 days of incubation at 37°C in a humidified 5%
C0 2
,12% 0 2
atmosphere. The dose-response curve was plotted and the IC 90
dose was determined from its intercept of the 10% CFCI-GM survival line.
After determining the 1C 90
dose for each drug, we investigated the kill of
leukemic colony-forming cells. Limiting dilution analysis was used to
establish the leukemic cell kill of each drug on four ALL and two AML cell
lines. This assay has been described elsewhere (11). With an initial cell
concentration of 10 5
cells/well, serial dilution factor of 5-6 wells used at each
dilution and 10 serial dilutions per experiment, the maximum detectable log
kill under our experimental conditions was 5.6 logs. In this system, a log kill
difference of 0.9 is statistically significant (P < .05).
L-Asparaginase, bleomycin, spirogermanium, taxol, and dexamethasone
were eliminated from further investigations either because of lack of
leukemic cell kill or because of solubility problems. After comparing the
leukemic cell kills of the other drugs tested at their IC 90
dose, we concluded
that none of these drugs was any more effective than 4-HC alone.
We thereupon chose to investigate drug combinations. Vincristine was
selected to be part of each combination because of its known clinical activity
in ALL and its ability to kill up to 2.5 logs on ALL cell lines in vitro while not
exceeding even its IC 70
dose. The following combinations were tested: VCR
plus HY, VCR plus ADR, VCR plus VP-16-213, and VCR plus 4-HC. Table 1
shows the changes in calculated ICgo doses for these drugs as increasing
doses of VCR are added. The interaction of VCR in the drug combinations was
evaluated in greater detail by the isobologram analysis of Steel and Peckham
(12). The toxicity of combinations of VCR plus HY, VCR plus ADR, and VCR
plus VP-16-213 were either additive or superadditive on the normal bone
marrow; even a small dose of VCR (0.1 /ug/ml) could necessitate the other
drug's being given at below its IC 90
dose in order not to exceed the IC 90
Table 1. Percent of Granulocyte-Macrophage Colony-Forming Unit Survival
Following Ex Vivo Chemotherapy on Normal Bone Marrow 8
VCR (jug/ml)
Added 4-HC VP-16-213 ADR HY
None 4.9 24.0 1.85 18.0
0.1 — 20.2 1.5 8.5
1 6.5 16.5 1.15 6.8
5 4.4 12.8 0.9 8.5
"Based on five experiments, IC go dose of drugs alone reflect only the patient
population tested with combination drugs, and may differ somewhat from the
overall population average.
Abbreviations: VCR, vincristine; 4-HC, 4-hydroperoxycyclophosphamide; VP-16-
213, etoposide; ADR, Adriamycin; HY, hydrocortisone.