|
PROPOSED
SEQUENCE FOR THE EXPERIMENTAL TEST |
The guideline which addresses
the research activity of LINFA, follows
on the assioma "cytotoxic is
almost always devoid of selectivity".
With this principle, the experimental test
proposed by LINFA will guide to the identification
of compounds active on tumour growth by
a pharmacological rather than a chemotherapeutic
mechanism of action.
The method follows the principle
of exclusion: all compounds are subjected to
a preliminary test of activity on selected and
recognised cell lines; selection and recognition
are based on the experience that brought to
the identification of NAMI-A, the only anti-metastasis
agent based on ruthenium and free of direct
tumour cell cytotoxicity. Cytotoxicity, lack
of activity on test for tumour cell malignancy,
invasion and metastasis in the preliminary test
exclude any progression into the subsequent
studies.
In
vitro assays
The aim of the following
tests is to select compounds which interfere
with tumour malignancy without directly killing
tumour cells.
Initial tests:
a) and b) [all compound are eligible]
- cytotoxicity on KB, LoVo
and MCF7 cell cultures [cytotoxicity is
measured by the SRB and MTT tests to discriminate
between compounds active on protein synthesis
from those active on the mitochondria energy
producing system; flow cytometry analysis
following PI staining provides evidence of
cell cycle disruption and of cycling cells];
- tumour cell uptake of the
test compound [atomic absorption spectroscopy
provides the relationship between cytotoxicity
and tumour cell uptake of the compound during
in vitro challenge];
Compounds with
low cytotoxicity (possibly transient)
are eligible for subsequent tests: c), d),
e)
- activity on TS/A cells grown
on laminin and fibronectin [this test shows
whether the test compound inhibits TS/A adenocarcinoma
cells increased malignancy stimulated by extracellular
matrix components];
- invasion in the Boyden chamber
with TS/A cells [inhibition of crossing
of the matrigel barrier, as determined by
the Boyden chamber gives information on the
control of the spontaneous invasive potential
of tumour cells];
- inhibition of MMP2 and of
cyclin activity [preliminary tests for
determining drug target for anti-tumour action];
Compounds with
good activity on tests c), d), e)
are eligible for subsequent tests
- differential activity on
B16 F1 and F10 cell lines [preliminar test
of selective activity on low and high metastasising
tumours];
Compounds
with good activity on B16 F10 cell line
are eligible for in vivo tests
In
vivo
assays
The aim of the following
tests is to discriminate between compounds active
to preventing metastasis formation from those
which also inhibit those already formed.
- differential effects on
primary tumour and lung metastasis formation
[pilot tests with Lewis lung carcinoma
evaluate the antimetastatic effect with a
very common solid metastasising murine tumour];
- tumour cell uptake of the
test compound by tumour and host tissues [atomic
absorption spectroscopy provides the relationship
between activity, toxicity and cell uptake
of the compound by tumour cells at primary
and metastasis level and by host tissues after
in vivo treatment];
Compounds active on metastases
are eligible for further tests
- inhibition of early and
advanced metastases [treatment of metastases
of solid tumours, combined with surgical removal
of primary tumour, allows to discriminate
the capacity to prevent metastasis formation
from the activity on metastases in advanced
stage of growth];
Compounds that inhibit metastasis
growth, other than preventing metastasis formation,
are credited of the most favourable properties
of LINFA and subjected to further investigation
on the fine mechanism of metastasis inhibition
[i.e. activity in several models of metastases;
activity in combination with other treatments;
activity by the oral route.
|
