the cause of the cytostatic effect. Thus, for therapeutic
purposes, there is no need to separate the epimeric DFMO-
peptide conjugates.
phalan and nitrosourea congeners of melanotropin frag-
ments,17,28 the DFMO-peptide conjugates have a higher
cytotoxic effect than the parent drug itself, which seems to
be influenced at least to some extent by the nature of the
carrier peptide.
All the DFMO-peptide derivatives exerted inhibition of cell
proliferation. The cell growth inhibition is obviously due to
the enzymatic release of DFMO from the carrier peptide
because, according to the experimentally proved mechanism
of ODC inhibition,24 a free R-carboxyl group is required on
the inhibitor to achieve irreversible inhibition. On the other
hand, according to our previous investigations MSH fragments
do not inhibit cell proliferation. On the contrary, MSH has a
mitogenic effect on normal melanocytes but not on melanoma
cells.25
Comparing the cytotoxic effects of the conjugates with one
another, the best results were achieved with II, although all
conjugates are more effective in inhibiting cell proliferation
than DFMO itself. A possible explanation for the increased
cytotoxicity is that the carrier peptide enhances the binding
of the conjugate to the tumor cell surface, where DFMO is
generated enzymatically in situ by a cell membrane peptidase.
In this way, a higher concentration and a higher uptake of
the drug can be achieved than in cases where DFMO is
applied alone. The differences in the cytotoxic effects of the
conjugates on various cell lines may be the consequence of
the local peptidase activity. Another explanation for the
different sensitivity of the various types of human neoplastic
cells to the DFMO-peptide conjugates might be, as in the case
of DFMO itself, that the various types of human neoplastic
cells may regulate polyamine biosynthesis differently. As a
consequence, the differential sensitivity to the compounds
tested may be a result of fundamental differences in the
control of the polyamine biosynthetic pathway and in the
variations in the specific polyamine requirements of human
cancer cell types.26 It has also been shown that normal and
neoplastic cells regulate polyamine biosynthesis differently,4
and the role of polyamines in cell differentiation seems also
to vary with the model system studied.27
The rationale for using melanotropin fragments as carriers
for DFMO was not only the favorable cytotoxic effect of DFMO
on melanoma cells, but also the fact that in our previous
investigations with melanotropin conjugates containing Mel-
phalan we demonstrated a specific receptor-mediated cytotoxic
effect on them.17 In the case of the present conjugates, we
also performed receptor binding investigations. Although the
DFMO derivatives of the central hormone fragments are
capable of specifically displacing the natural hormone from
its receptor, no preferred cytotoxic action was observed on the
receptor-positive cells. A possible reason for this may be that
receptor-mediated transport of the conjugates into the cell
decreases the potential DFMO concentration on the cell
surface where peptidases are acting. Therefore, we tested the
cell growth inhibition of the conjugates on melanoma cells
containing melanotropin receptor either in the presence or in
the absence of MSH, and found no significant difference in
the cytotoxicities. This result means that either no receptors
are involved in the cytotoxic action or that their mediated
cytotoxic effect could not be observed under the current
experimental conditions.
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In conclusion it may be stated that whatever mechanism(s)
might be responsible for our findings, the increased cytotox-
icity caused by coupling DFMO to carrier peptides may be a
successful way for increasing the therapeutic efficacy of
DFMO. Although no receptor-specific targeting effect was
demonstrated, it is worth mentioning that contrary to Mel-
Acknowledgments
We thank Professor P. Soha´r for the interpretation of the NMR
measurements. This work was supported by the Hungarian National
Research Fund (OTKA T 015709).
J S970080D
1000 / Journal of Pharmaceutical Sciences
Vol. 86, No. 9, September 1997