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Updating
of published results
| Antiviral
properties and cytotoxic activity of
platinum(II) complexes with 1,10-phenanthrolines
and acyclovir or penciclovir.
Margiotta N, Bergamo A, Sava G, Padovano
G, de Clercq E, Natile G.
Dipartimento Farmaco-Chimico, Universita
degli Studi di Bari, via E. Orabona
4, 70125 Bari, Italy. nmargiotta@farmchim.uniba.it
J Inorg Biochem.;98(8):1385-90,
ABSTRACT
Platinum compounds
containing an aromatic diimine (1,10-phenanthroline
or 2,9-dimethyl-1,10-phenanthroline;
phen and Me(2)phen, respectively) and
antiviral guanosine-type ligands (acyclovir
or penciclovir; acy and pen, respectively)
have been synthesised. These compounds
maintain the antiviral activity against
Herpes Symplex Virus (HSV) and have
greater efficacy than free acyclovir
or penciclovir against Cytomegalovirus
(CMV); in both cases the species with
Me(2)phen are more active. The same
complexes are effective against tumor
cell proliferation which also results
to be dependent upon the nature of the
diimine ligand: all compounds containing
Me(2)phen being more active than those
containing phen. Although in vivo
some complexes significantly reduce
tumor cell proliferation, nevertheless,
they do not appear to significantly
affect the life time expectancy of the
treated mice. The greater cytotoxicity
of compounds with Me(2)phen may result
from a higher reactivity towards cellular
components, such as glutathione, which
could cause release of the diimine,
known to be highly cytotoxic. |
| Inhibition
of hepatocellular carcinomas in
vitro and hepatic metastases in
vivo in mice by the histone deacetylase
inhibitor HA-But.
Coradini D, Zorzet S, Rossin R, Scarlata
I, Pellizzaro C, Turrin C, Bello M,
Cantoni S, Speranza A, Sava G, Mazzi
U, Perbellini A.
Unit of Biomolecular Determinants in
Prognosis and Therapy, Experimental
Department, Istituto Nazionale per lo
Studio e la Cura dei Tumori, Milan.
Clin Cancer Res.;10(14):4822-30,
ABSTRACT
PURPOSE: The purpose
is to evaluate the CD44-mediated cellular
targeting of HA-But, a hyaluronic acid
esterified with butyric acid (But) residues,
to hepatocellular carcinoma cell lines
in vitro and to hepatic tumor metastases
in vivo. EXPERIMENTAL DESIGN:
In vitro, the CD44-dependent
cytotoxicity in two human hepatocellular
carcinoma cell lines (HepB3 and HepG2)
with high and low CD44 expression was
investigated; in vivo, the
effect on liver metastases originating
from intrasplenic implants of Lewis
lung carcinoma (LL3) or B16-F10 melanoma
in mice was compared with the pharmacokinetics
of organ and tissue distribution using
different routes of administration.
RESULTS: HepB3 and HepG2 cell lines
showed different expression of CD44
(78 and 18%, respectively), which resulted
in a CD44-dependent HA-But inhibitory
effect as demonstrated also by the uptake
analysis performed using radiolabeled
HA-But ((99m)Tc-HA-But). Pharmacokinetic
studies showed different rates of (99m)Tc-HA-But
distribution according to the route
of administration (i.v., i.p., or s.c.):
very fast (a few minutes) after i.v.
treatment, with substantial accumulation
in the liver and spleen; relatively
slow after i.p. or s.c. treatment, with
marked persistence of the drug at the
site of injection. The effect of s.c.
and i.p. treatment with HA-But on liver
metastases originating from intrasplenic
implants of LL3 carcinoma or B16-F10
melanoma (both CD44-positive: 68 and
87%, respectively), resulted in 87 and
100% metastases-free animals, respectively
(regardless of the route of administration),
and a significant prolongation of the
life expectancy compared with control
groups. CONCLUSIONS: HA-But tends to
concentrate in the liver and spleen
and appears to be a promising new drug
for the treatment of intrahepatic tumor
lesions. |
| Cocultures
of metastatic and host immune cells:
selective effects of NAMI-A for tumor
cells.
Bacac M, Vadori M, Sava G, Pacor S.
Department of Biomedical Sciences, University
of Trieste, Via L. Giorgieri 7, 34127,
Trieste, Italy.
Cancer Immunol Immunother.; Jun 25.
ABSTRACT
The effects of NAMI-A,
[H(2)im][ trans-RuCl(4)(dmso-S)(Him)],
a new metal-based agent for treating
tumor metastases, have been investigated
in vitro on splenocytes, ConA-
or LPS-activated T and B lymphoblasts,
and thymocytes. Splenocytes and thymocytes
exposed for 1 h to 0.01-0.1-mM NAMI-A
do not change their mitochondrial functionality,
cell cycle distribution, protein synthesis,
and CD44 expression in comparison to
untreated control samples. Instead,
mitochondrial functionality increased
24 h after treatment in a fraction of
splenocytes. The same treatment reduced
mitochondrial functionality and S phase
of the cell cycle in T and B blasts
(already after 1 h treatment) and reduced
CD44 expression on B blasts, 24 h after
treatment. On cocultures of splenocytes
and metastatic cells (metGM) (1:1),
NAMI-A induces a selective depolarization
of mitochondrial membrane potential
of metGM cells, while it stimulates
splenocytes (mainly lymphocytes), as
shown by the increase of the S phase,
nitric oxide production, and adhesion
onto metastatic cells. This, in turn,
reduces the number of metastatic cells
and results in the increased ratio between
splenocytes and metGM in favor of diploid
cells (doubling from one to two). Rosetting
of leukocytes onto metastatic cells
correlates with induction of CD54 expression
on tumor cells after NAMI-A in vivo
treatment, which in turn, might contribute
to metastasis recognition by cytotoxic
lymphocytes. The overall antimetastatic
activity displayed by NAMI-A might therefore
be the result of complex interactions
with tumor cells, on which it displays
selective antitumor activity, and with
host immune cells through which it promotes
activation of host immune defenses involved
in tumor suppression. |
Ruthenium
anticancer drugs.
Alessio E, Mestroni G, Bergamo A, Sava
G.
Department of Chemical Sciences, University
of Trieste, Via L. Giorgieri 1, I-34127
Trieste, Italy. alessi@univ.trieste.it
Met Ions Biol Syst.;42:323-51, 2004.
ABSTRACT
A review paper. No
abstract available.
|
| Actin-dependent
tumour cell adhesion after short-term
exposure to the antimetastasis ruthenium
complex NAMI-A.
Sava G, Frausin F, Cocchietto M, Vita
F, Podda E, Spessotto P, Furlani A,
Scarcia V, Zabucchi G.
Department of Biomedical Sciences, University
of Trieste, Via L. Giorgieri 7, 34127
Trieste, Italy. g.sava@callerio.org
Eur J Cancer.; 40(9):1383-96,
ABSTRACT
Imidazolium trans-imidazoledimethylsulphoxidetrachlororuthenate
(NAMI-A) was tested in vitro
on the pro-adhesive properties, evaluated
as resistance to trypsin treatment,
which is a bona fide measure
of adhesion strength, of KB and HeLa
carcinoma cell lines and on human polymorphonuclear
neutrophils (HPMN). NAMI-A increased
the pro-adhesive activity of KB cells
at 0.001 mM concentration, after few
minutes incubation and this effect was
not influenced by the vehicle used for
cell challenge, neither did it depend
on NAMI-A concentration or on temperature.
The same effect occurred on HeLa cells
at 0.01 mM NAMI-A. This effect, detected
at concentrations up to 100 times lower
than those necessary to block cells
at the G(2)-M premitotic phase of cell
cycle, or to inhibit matrix metalloproteinase
release or cell invasion, was not related
to ruthenium uptake by tumour cells.
HeLa cells and healthy HPMN, following
short exposure to 0.1 mM NAMI-A, assumed
a different shape, with the extrusion
of filopodia (HeLa) and of large lamellopodia
(HPMN), which increased their interactions
with the substrate. This effect was
attributed to stabilisation, altered
turnover and sensitivity to cytochalasin
D of actin filaments. Provided that
adhesion is associated with cell motility
and invasion, these data suggest that
NAMI-A may exert antimetastatic properties
at concentrations lower than those observed
in the lungs at the end of a conventional
intraperitoneal treatment in vivo. |
| Electrochemical
measurements confirm the preferential
bonding of the antimetastatic complex
[ImH][RuCl(4)(DMSO)(Im)] (NAMI-A) with
proteins and the weak interaction with
nucleobases.
Ravera M, Baracco S, Cassino C, Colangelo
D, Bagni G, Sava G, Osella D.
Dipartimento Scienze dell'Ambiente e
della Vita, Universita del Piemonte
Orientale Amedeo Avogadro, Spalto Marengo
33, 15100 Alessandria, Italy.
J Inorg Biochem.; 98(6):984-90,
ABSTRACT
An electrochemical
and biological study of interaction
between the prototypical antimetastatic
drug imidazolium trans-tetrachlorodimethylsulfoxideimidazoleruthenate
(III) complex, [ImH][RuCl(4)(DMSO)(Im)]
(DMSO = dimethylsulfoxide, Im = imidazole),
nicknamed NAMI-A, and several biomolecules,
namely DNA, bovine (BSA) and human (HSA)
serum albumin, is reported. Electrochemistry
offers great advantages over the existing
devices based on optical techniques,
since it provides rapid, simple, and
low-cost information whether the interaction
occurs or not. Moreover, we describe
some biochemical assays to test the
interaction of NAMI-A with ribonucleoprotein
telomerase and protein Taq polymerase.
All the data confirm the preferential
interaction of NAMI-A with proteins
with respect to nucleotides, especially
when compared with the behaviour of
the well-known alkylating drug cisplatin
in the presence of the same targets. |
| Hyaluronic-acid
butyric esters as promising antineoplastic
agents in human lung carcinoma: a preclinical
study.
Coradini D, Pellizzaro C, Abolafio
G, Bosco M, Scarlata I, Cantoni S, Stucchi
L, Zorzet S, Turrin C, Sava G, Perbellini
A, Daidone MG.
Unit of Biomolecular Determinants in
Prognosis and Therapy, Experimental
Department, Istituto Nazionale per lo
Studio e la Cura dei Tumori, Milano,
Italy.
danila.coradini@istitutotumori.mi.it
Invest New Drugs.; 22(3):207-17.
ABSTRACT
New promising compounds,
derived from the esterification of hyaluronic
acid with butyric acid, were investigated
in vitro on a non-small cell lung carcinoma
cell line (NCI-H460) and an its metastatic
subclone (NCI-H460M). All new compounds
exerted a dose-dependent inhibitory
effect on both cell lines, which expressed
CD44, the specific surface receptor
for hyaluronic acid, in a very high
percentage of cells (90%). HE1, the
most effective of these compounds, was
10-fold more effective than sodium butyrate
(NaB) in inhibiting cell proliferation.
Similarly to NaB, after 24 hours of
treatment, HE1 affected the expression
of three cell cycle-related proteins
(p27(kip1), p53 and p21(waf1)) responsible
for growth arrest, indicating that the
presence of the hyaluronic acid backbone
does not interfere with the biologic
activity. Intratumoral treatment with
HE1 demonstrated a marked efficacy on
primary tumor growth and on lung metastases
formation of the murine Lewis Lung Carcinoma
model. Altogether, present findings
suggest a possible clinical application
of these novel butyric pro-drugs in
primary and metastatic lung cancer.
|
| Intratumoral
NAMI-A Treatment Triggers Metastasis
Reduction, Which Correlates to CD44
Regulation and Tumor Infiltrating Lymphocyte
Recruitment.
Pacor S, Zorzet S, Cocchietto M, Bacac
M, Vadori M, Turrin C, Gava B, Castellarin
A, Sava G.
Department of Biomedical Sciences, University
of Trieste, via L. Giorgieri 7-9, 34127
Trieste, Italy. pacorsab@univ.trieste.it
J Pharmacol Exp Ther.; 310(2):737-44,
Aug 2004.
ABSTRACT
Intratumor (i.t.) injection
of 35 mg/kg/day NAMI-A for six consecutive
days to CBA mice bearing i.m. implants
of MCa mammary carcinoma reduces primary
tumor growth and particularly lung metastasis
formation, causing 60% of animals to
be free of macroscopically detectable
metastases. The i.t. treatment allows
study of the effects of NAMI-A on in
vivo tumor cells exposed to millimolar
concentrations for a relatively prolonged
time. Under these conditions, NAMI-A
reduces the number of CD44+ tumor cells
and changes tumor cell phenotype to
a lower aggressive behavior, as shown
by scanning electron microscopy analysis.
On primary tumor site, NAMI-A causes
unbalance between 2n and aneuploid cells
in favor of lymphocytes. Furthermore,
in tumor tissue, nitric oxide production
is increased and active matrix metalloproteinase
9 is decreased, and these effects are
accompanied by a reduced hemoglobin
concentration. These data are in agreement
with the reduction of tumor invasion
and metastasis and suggest the therapeutic
usefulness of NAMI-A in neoadjuvant
or tumor reduction treatments for preventing
metastasis formation. These data further
stress the usefulness of intratumor
treatments as experimental preclinical
model for studying in vivo the mechanism
of tumor cell interactions after prolonged
exposure to ruthenium-based compounds
to be developed for metastasis inhibition. |
| Structure-dependent
in vitro cytotoxicity of the
isomeric complexes [Ru(L)(2)Cl(2)] (L=
o-tolylazopyridine and 4-methyl-2-phenylazopyridine)
in comparison to [Ru(azpy)(2)Cl(2)].
Hotze AC, Caspers SE, de Vos D, Kooijman
H, Spek AL, Flamigni A, Bacac M, Sava
G, Haasnoot JG, Reedijk J.
Leiden Institute of Chemistry, Gorlaeus
Laboratories, Leiden University, PO
Box 9502, 2300 RA, Leiden, The Netherlands.
J Biol Inorg Chem.; 9(3):354-64, Apr
2004.
ABSTRACT
The dichlorobis(2-phenylazopyridine)ruthenium(II)
complexes, [Ru(azpy)(2)Cl(2)], are under
renewed investigation due to their potential
anticancer activity. The three most
common isomers alpha-, beta- and gamma-[RuL(2)Cl(2)]
with L= o-tolylazopyridine (tazpy) and
4-methyl-2-phenylazopyridine (mazpy)
(alpha indicating the coordinating Cl,
N(pyridine) and Nazo atoms in mutual
cis, trans, cis positions, beta indicating
the coordinating Cl, N(pyridine) and
Nazo atoms in mutual cis, cis, cis positions,
and gamma indicating the coordinating
Cl, N(pyridine) and Nazo atoms in mutual
trans, cis, cis positions) are synthesized
and characterized by NMR spectroscopy.
The molecular structures of gamma-[Ru(tazpy)(2)Cl(2)]
and alpha-[Ru(mazpy)(2)Cl(2)] are determined
by X-ray diffraction analysis. The IC(50)
values of the geometrically isomeric
[Ru(tazpy)(2)Cl(2)] and [Ru(mazpy)(2)Cl(2)]
complexes compared with those of the
parent [Ru(azpy)(2)Cl(2)] complexes
are determined in a series of human
tumour cell lines (MCF-7, EVSA-T, WIDR,
IGROV, M19, A498 and H266). These data
unambiguously show for all complexes
the following trend: the alpha isomer
shows a very high cytotoxicity, whereas
the beta isomer is a factor 10 less
cytotoxic. The gamma isomers of [Ru(tazpy)(2)Cl(2)]
and [Ru(mazpy)(2)Cl(2)] display a very
high cytotoxicity comparable to that
of the gamma isomer of the parent compound
[Ru(azpy)(2)Cl(2)] and to that of the
alpha isomer. These biological data
are of the utmost importance for a better
understanding of the structure-activity
relationships for the isomeric [RuL(2)Cl(2)]
complexes. |
| Synthesis
and chemical-pharmacological characterization
of the antimetastatic NAMI-A-type Ru(III)
complexes (Hdmtp)[trans-RuCl4(dmso-S)(dmtp)],
(Na)[trans-RuCl4(dmso-S)(dmtp)], and
[mer-RuCl3(H2O)(dmso-S)(dmtp)] (dmtp
= 5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine).
Velders AH, Bergamo A, Alessio E, Zangrando
E, Haasnoot JG, Casarsa C, Cocchietto
M, Zorzet S, Sava G.
Leiden Institute of Chemistry, Gorlaeus
Laboratories, Leiden University, P.O.
Box 9502, 2300 RA Leiden, The Netherlands.
a.velders@chem.leidenuniv.nl
J Med Chem.;47(5):1110-21., Feb 26 2004.
ABSTRACT
Ruthenium compounds
have gained large interest for their
potential application as chemotherapeutic
agents, and in particular the complexes
of the type (X)[trans-RuCl4(dmso-S)L]
(X = HL or Na, NAMI-A or NAMI, respectively,
for L = imidazole) are under investigation
for their antimetastatic properties.
The NAMI(-A)-like compounds are prodrugs
that hydrolyze in vivo, and the investigation
of their hydrolytic properties is therefore
important for determining the nature
of the potential active species. The
NAMI-A-type Ru(III) complex 1, (Hdmtp)[trans-RuCl4(dmso-S)(dmtp)]
(dmtp is 5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine),
and the corresponding sodium analogue
2, (Na)[trans-RuCl4(dmso-S)(dmtp)],
were synthesized. The hydrolyses of
1 and 2 in water as well as in buffered
solutions were studied, and the first
hydrolysis product, [mer-RuCl3(H2O)(dmso-S)(dmtp)].H2O
(3), was isolated and characterized.
The molecular structures of 1 and 3
were determined by single-crystal X-ray
diffraction analyses and prove the importance
of the hydrogen-bonding properties of
dmtp to stabilize hydrolysis products.
In vitro 1 (a) is not cytotoxic on tumor
cells, following challenges from 1 to
72 h and concentrations up to 100 microM,
(b) inhibits matrigel invasion at 0.1
mM and MMP-9 activity with an IC50 of
about 1 mM, and (c) is devoid of pronounced
effects on cell distribution among cell
cycle phases. In vivo compound 1, similar
to NAMI-A, significantly inhibits metastasis
growth in mice bearing advanced MCa
mammary carcinoma tumors. In the lungs,
1 is significantly less concentrated
than NAMI-A, whereas no differences
between these two compounds were found
in other organs such as tumor, liver,
and kidney. However, 1 caused edema
and necrotic areas on liver parenchyma
that are more pronounced than those
caused by NAMI-A. Conversely, glomerular
and tubular changes on kidney are less
extensive than with NAMI-A. In conclusion,
1 confirms the excellent antimetastatic
properties of this class of NAMI-A-type
compounds and qualifies as an interesting
alternative to NAMI-A for treating human
cancers. |
| The
hydrolysis of the anti-cancer ruthenium
complex NAMI-A affects its DNA binding
and antimetastatic activity: an NMR
evaluation.
Bacac M, Hotze AC, Schilden K, Haasnoot
JG, Pacor S, Alessio E, Sava G, Reedijk
J.
Leiden Institute of Chemistry, Gorlaeus
Laboratories, Leiden University, P.O.
Box 9502, 2300, RA Leiden, The Netherlands.
J Inorg Biochem.;98(2):402-12,Feb
2004.
ABSTRACT
The coordination of
the antimetastatic agent NAMI-A, [H(2)im][trans-RuCl(4)(dmso-S)(Him)],
(Him=imidazole; dmso=dimethyl sulfoxide),
to the DNA model base 9-methyladenine
(9-MeAde) was investigated in water.
NMR spectroscopy was first applied for
the study of the molecular stability
and hydrolysis of NAMI-A in aqueous
solution over a range of pH (3.0-7.4)
and chloride ion concentrations (0-1
M) at 37.0 degrees C. In physiological
conditions (phosphate buffer, pH 7.4)
NAMI-A disappears from the solution
in 15 min due to chloride and dmso hydrolysis,
leading to uncharacterised poly-oxo
Ru species. Conversely, at lower pH
(3.0-6.0) and in water (pH approximately
5.5), only a partial dmso hydrolysis
occurs, slowly forming the [trans-RuCl(4)(H(2)O)(Him)](-)
complex. This latter species coordinates
to 9-MeAde (via the N7 of 9-MeAde),
forming the [trans-RuCl(4)(9-MeAde)(Him)](-)
complex. NAMI-A and [trans-RuCl(4)(H(2)O)(Him)](-)
give comparable intracellular ruthenium
concentrations and accumulate in KB
cells (human mouth carcinoma) and accumulate
these at the G(2)/M phase, while poly-oxo
Ru species do not, and their cell uptake
is reduced to 50%. On the contrary,
G(2)/M arrest and protein content in
the murine metastatic cell line metGM,
are not influenced by NAMI-A hydrolysis.
Hydrolysed NAMI-A species apparently
are easier taken up by the metGM cells,
showing intracellular ruthenium concentrations
one order of magnitude greater than
those of intact NAMI-A. Therefore, it
is proposed that the selective antimetastatic
activity of NAMI-A during in vivo experiments
can be attributed to its hydrolysed
species. |
| Solution,
solid state and biological characterization
of ruthenium(III)-DMSO complexes with
purine base derivatives.
Turel I, Pecanac M, Golobic A, Alessio
E, Serli B, Bergamo A, Sava G.
Faculty of Chemistry and Chemical Technology,
University of Ljubljana, Askerceva 5,
1000, Ljubljana, Slovenia.
J Inorg Biochem.;98(2):393-401,
Feb 2004.
ABSTRACT
Two new complexes of
Ru(III) with purine base derivatives,
[mer-RuCl(3)(acv)(DMSO-S)(C(2)H(5)OH)].C(2)H(5)OH
(1) (acv=acyclovir, DMSO=dimethyl sulfoxide)
and [trans-RuCl(4)(guaH)(DMSO-S)].2H(2)O
(2) (guaH=protonated molecule of guanine),
were prepared from the same Ru(III)
precursor, [trans-RuCl(4)(DMSO-S)(2)](-),
by substitution of one DMSO-S. Coordination
of acv induced also replacement of one
chloride by an ethanol molecule. This
reactivity difference was explained
by striking contrasts in the hydrogen
bonding schemes of the two complexes,
evidenced in their X-ray crystal structures.
In 1 the guanine derivative acyclovir
is coordinated to ruthenium through
the N(7) atom, while in 2 the protonated
guanine molecule is bound through the
N(9) atom. Both complexes were also
characterized by various physico-chemical
methods in the solid state and in the
solution. In vitro, the biological
activity of 2 and of the previously
described complexes [mer-RuCl(3)(acv)(DMSO-S)(CH(3)OH)].0.5CH(3)OH
(3) and [mer-RuCl(3)(acv)(DMSO-S)(H(2)O)].H(2)O
(4) on tumour cells appear to be very
similar to that of NAMI-A (NAMI-A=[ImH][trans-RuCl(4)(DMSO-S)Im]).
All compounds are only weakly active
on tumour cell proliferation but show
an interesting proadhesive effect that
suggest possible activity on tumour
malignancy. |
|
Synthesis,
characterization and biological activity
of copper complexes with pyridoxal thiosemicarbazone
derivatives. X-ray crystal structure
of three dimeric complexes.
Ferrari MB, Bisceglie F, Pelosi G,
Tarasconi P, Albertini R, Dall'Aglio
PP, Pinelli S, Bergamo A, Sava G.
Dipartimento di Chimica Generale ed
Inorganica, Chimica Analitica e Chimica
Fisica, Parco Area delle Scienze 17
A, Universita di Parma, I-43100, Parma,
Italy
J Inorg Biochem.;98(2):301-312,
Feb 2004.
ABSTRACT
A dimeric copper complex
of the unsubstituted pyridoxal thiosemicarbazone
(H(2)L), [{Cu(HL)(OH(2))}(2)]Cl(2).2H(2)O,
previously tested on Friend murine cell
lines has been recently resynthesized
to evaluate its behavior on different
murine and human leukemic cell lines
and has been compared, in vitro
and in vivo, with its monomeric
counterpart [Cu(H(2)L)(OH(2))Cl]Cl.
On TS/A murine adenocarcinoma cell line
in vitro, both compounds significantly
inhibit cell proliferation at micromolar
concentrations, although the dimeric
compound is more active. Despite this
cytotoxicity they lack activity on TLX5
lymphoma. The unsubstituted dimeric
[{Cu(HL)(OH(2))}(2)]Cl(2).2H(2)O induces
apoptosis on CEM and U937 human cell
lines, with IC(50) concentrations of
1.2x10(-5) and 6.7x10(-6) M, respectively,
but it is inactive on K562. Moreover,
it alters significantly the cell cycle
of U937 and CEM lines and decreases
the telomerase activity of U937.To verify
if other dimeric copper complexes show
relevant biological activity new complexes
with N-substituted pyridoxal thiosemicarbazones
have been synthesized and characterized
using spectroscopic techniques. Three
of them, namely [Cu(Me(2)-HL)Cl](2).6H(2)O
(Me(2)-H(2)L=pyridoxal N1,N1-dimethylthiosemicarbazone)
(1), [Cu(MeMe-HL)Cl](2)Cl(2).4H(2)O
(MeMe-HL=pyridoxal N1,N2-dimethylthiosemicarbazone)
(2), [Cu(Et-H(2)L)Cl](2)Cl(2).2H(2)O
(Et-H(2)L=pyridoxal N1-ethylthiosemicarbazone)
(3), were also characterized by X-ray
diffractometry. These complexes are
dimeric and all three present a square
pyramidal coordinative geometry with
the ligand showing an SNO tridentate
behavior. Their biological activities
have been tested in vitro on
U937, CEM and K562 cell lines to ascertain
their effectiveness in comparison to
the corresponding unsubstituted complex
[{Cu(HL)(OH(2))}(2)]Cl(2).2H(2)O. Compound
1 shows weak proliferation inhibition
on all three cell lines, but it does
not induce apoptosis and it does not
inhibit telomerase activity, compound
2 is not effective at low concentration
and is toxic at higher doses; compound
3 inhibits CEM cell growth better than
complex 1 but it does not exert any
other biological effect.
|
| Reduction
of in vivo lung metastases
by dinuclear ruthenium complexes is
coupled to inhibition of in vitro tumour
invasion.
Bergamo A, Stocco G, Casarsa C, Cocchietto
M, Alessio E, Serli B, Zorzet S, Sava
G.
Foundation Callerio-Onlus, I-34127 Trieste,
Italy. a.bergamo@callerio.org
Int J Oncol.;24(2):373-9, Feb 2004.
ABSTRACT
Mononuclear ruthenium-dmso
compounds showed interesting antimetastatic
properties on experimental models of
solid tumours. In line with the interesting
results with multinuclear platinum complexes,
which proved to overcome cisplatin resistance,
we thought it worthwhile to test the
pharmacological properties of some dinuclear
ruthenium complexes to ascertain the
possible advantages due to the introduction
of a second metal centre over NAMI-A
and its mononuclear analogues. These
compounds belong to the general formula
X2[[RuCl4(dmso-S)]2(mu-L)] or [X][[RuCl4(dmso-S)](mu-L)[RuCl3(dmso-S)(dmso-O)]]
where L is a nitrogen donor ligand (pyrazine;
pyrimidine; 4,4'-bipyridine; 1,2-bis(4-pyridyl)ethane;
1,2-bis(4-pyridyl) ethylene; 1,3-bis(4-pyridyl)propane)
and X a counterion. We focused on parameters
related to metastatic ability such as
gelatinase activity, detected by zymography,
and invasive potential, measured by
means of a transwell chamber. These
activities were correlated to the ability
to inhibit tumour metastases in
vivo. All dinuclear complexes,
except compound D8 ([NH4]2[[RuCl4(dmso-S)]2(mu-pyz]),
decrease the number of tumour cells
that cross a matrigel barrier, and inhibit
MMP-9 gelatinolytic activity at concentrations
lower than that of NAMI-A and of other
mononuclear ruthenium complexes. In
vivo compounds D5 (Na2[[RuCl4(dmso-S)]2(mu-ethylbipy)])
and D7 ([NH4][[RuCl4(dmso-S)](mu-pyz)[RuCl3(dmso-S)
(dmso-O)]]) show anti-metastasis activity,
at two dose levels, with mild or null
effect on primary tumour growth; compound
D8 is the weakest active. All compounds
tend to accumulate in liver and kidneys,
rather than in tumour and lungs. However,
compound D5, the most active in
vitro on invasion and gelatinases
and active in vivo on metastasis,
is better concentrated in the lungs
than compound D8 which is less active
or inactive in vitro and in
vivo. Histological analysis show
liver, as well as kidney toxicities
that limit in vivo activity.
These data thus suggest dinuclear ruthenium
complexes as promising anti-invasive
agents for cancer treatment.
|
| Biological
role of adduct formation of the ruthenium(III)
complex NAMI-A with serum albumin and
serum transferrin.
Bergamo A, Messori L, Piccioli F, Cocchietto
M, Sava G.
Callerio Foundation Onlus, Via A. Fleming
22-31, 34127 Trieste, Italy. a.bergamo@callerio.org
Invest New Drugs.;21(4):401-115,
Nov 2003.
ABSTRACT
NAMI-A is an innovative
ruthenium(III) complex with a very encouraging
preclinical profile of metastasis inhibition,
which is undergoing initial phases of
clinical trials. To assess the pharmacological
relevance of the drug fraction associated
to plasma proteins, adducts of NAMI-A
with either serum albumin or serum transferrin
were prepared and their biological effects
tested in vitro and in
vivo. Specifically, adducts of
NAMI-A with either serum albumin or
serum transferrin, prepared and characterized
at a ruthenium-to-protein molar ratio
of 4:1, were evaluated in vitro
on the KB human tumor cell line and
in vivo on the MCa mammary
carcinoma tumor. The effects of NAMI-A/protein
adducts on cell viability and on cell
cycle progression were found to be far
smaller than those produced by free
NAMI-A. GFAAS measurements point out
that the amount of ruthenium that gets
into cells is drastically reduced when
NAMI-A is presented in its protein-bound
form. In vivo use of NAMI-A
adducts with albumin and transferrin
resulted markedly less effective on
lung metastasis reduction, than free
NAMI-A. Overall, the present results
suggest that binding to plasma proteins
causes a drastic decrease of NAMI-A
bioavailability and a subsequent reduction
of its biological activity, implying
that association to plasma proteins
essentially represents a mechanism of
drug inactivation.
|
| NAMI-A
inhibits the PMA-induced ODC gene expression
in ECV304 cells: involvement of PKC/Raf/Mek/ERK
signalling pathway.
Debidda M, Sanna B, Cossu A, Posadino
AM, Tadolini B, Ventura C, Pintus G.
Department of Biomedical Sciences, Division
of Biochemistry, Laboratory of Cardiovascular
Research, National Institute of Biostructures
and Biosystems, University of Sassari,
I-07100 Sassari, Italy
Int J Oncol.;23(2):477-82, Aug 2003.
ABSTRACT
Imidazolium trans-imidazole
dimethyl sulfoxide tetrachlororuthenate
(NAMI-A) is a new compound active against
lung metastasis of solid metastasizing
tumours. While its in vivo
effect has been studied, the molecular
insights that underlie its action are
largely unknown. Among the possible
pathways responsible for malignant transformation,
PKC arose as one of the most promising
targets for new antineoplastic drugs.
We demonstrated the capability of NAMI-A
of inhibiting PMA induced-PKC activity
in ECV304 in a dose-dependent fashion.
Furthermore, NAMI-A through modulation
of PKC activity has been proved capable
of reducing the phorbol ester induced
expression of ornithine decarboxilase
(ODC) gene and to abrogate the activation
of the Raf/MEK/ERK pathway. Taken together
these results suggest that many of the
in vivo outcomes of NAMI-A
treatment may be the result of a direct
action on PKC.
|
| Antiangiogenic
properties of selected ruthenium(III)
complexes that are nitric oxide scavengers.
Morbidelli L , Donnini S, Filippi S,
Messori L, Piccioli F, Orioli P, Sava
G, Ziche M.
British Journal of Cancer; 88: 1484
1491, 2003.
ABSTRACT
The nitric oxide synthase
(NOS) pathway has been clearly demonstrated
to regulate angiogenesis. Increased
levels of NO correlate with tumour growth
and spreading in different experimental
and human cancers. Drugs interfering
with the NOS pathway may be useful in
angiogenesis-dependent tumours. The
aim of this study was to pharmacologically
characterise certain ruthenium-based
compounds, namely NAMI-A, KP1339, and
RuEDTA, as potential NO scavengers to
be used as antiangiogenic/antitumour
agents. NAMI-A, KP1339 and RuEDTA were
able to bind tightly and inactivate
free NO in solution. Formation of ruthenium
NO adducts was documented by electronic
absorption, FT-IR spectroscopy and 1H-NMR.
Pretreatment of rabbit aorta rings with
NAMI-A, KP1339 or RuEDTA reduced endothelium-dependent
vasorelaxation elicited by acetylcholine.
This effect was reversed by 8-BrcGMP.
The key steps of angiogenesis, endothelial
cell proliferation and migration stimulated
by vascular endothelial growth factor
(VEGF) or NO donor drugs, were blocked
by NAMI-A, KP1339 and RuEDTA, these
compounds being devoid of any cytotoxic
activity. When tested in vivo,
NAMI-A inhibited angiogenesis induced
by VEGF. It is likely that the antitumour
properties previously observed for ruthenium-based
NO scavengers, such as NAMI-A, are related
to their NO-related antiangiogenic properties. |
| Development
of a LC method for pharmaceutical quality
control of the antimetastatic ruthenium
complex NAMI-A.
Bouma M, Nuijen B, Jansen MT, Sava
G, Picotti F, Flaibani A, Bult A, Beijnen
JH.
J Pharm Biomed Anal 26; 31(2):215-28,
Feb 2003.
ABSTRACT
Imidazolium trans-tetrachloro(dimethylsulfoxide)imidazoleruthenium(III)
(NAMI-A) is a novel ruthenium complex
with selective activity against metastases
currently in Phase I clinical trials
in the Netherlands. Pharmaceutical quality
control of NAMI-A drug substance and
lyophilized product warranted the development
of an assay for determination and quantification
of NAMI-A and degradation products.
A high performance liquid chromatography
(HPLC) method was developed, consisting
of a C18 column with 0.50 mM sodium
dodecylsulfate in 3% methanol at pH
2.5 (acidified using trifluoromethanesulfonic
acid) as the mobile phase and UV-detection
at 358 nm. The HPLC method was proven
to be linear, accurate and precise.
Stress testing showed that degradation
products were separated from the parent
compound. By combining results of nuclear
magnetic resonance (NMR) and HPLC experiments,
one degradation product was identified
as the mono-hydroxy species of NAMI-A.
HPLC analysis with off-line detection
of the eluate with flameless atomic
absorption spectrometry (F-AAS) showed
that under most conditions, all ruthenium-containing
compounds show a peak in the HPLC chromatogram
and that all ruthenium applied to the
column is recovered quantitatively.
For completely degraded solutions of
NAMI-A some ruthenium is retained on
the column. Suitability of the HPLC
method for the pharmaceutical quality
control of NAMI-A lyophilized product
was demonstrated. |
| Molecular
structure, solution chemistry and biological
properties of the novel [ImH][trans-IrCl(4)(Im)(DMSO)],
(I) and of the orange form of [(DMSO)(2)H][trans-IrCl(4)(DMSO)(2)],
(II), complexes.
Messori L, Marcon G, Orioli P, Fontani
M, Zanello P, Bergamo A, Sava G, Mura
P.
J Inorg Biochem 1; 95(1):37-46,
May 2003.
ABSTRACT
The new iridium(III)
complex, imidazolium[trans(DMSO,imidazole)tetrachloroiridate(III)],
(I) (DMSO=dimethyl sulfoxide), and the
orange form of [(DMSO)(2)H][trans(DMSO)(2)-tetrachloroiridate(III)],
(II) have been prepared and characterized,
both in the solid state and in solution,
by X-ray diffraction and by various
physicochemical techniques. Single crystal
X-ray diffraction studies point out
that complex (II) is isomorphous to
the ruthenium(III) analogue, [(DMSO)(2)H][trans-RuCl(4)(DMSO)(2)],
(III). Crystallographic data are the
following: a=16.028(2) A, b=24.699(3)
A, c=8.262(1) A, in space group Pbca
(Z=8) for (imidazolium) [trans(DMSO,imidazole)tetrachloroiridate(III)],
(I); and a=9.189(2) A, b=16.511(4) A,
c=14.028(3) A, beta=100.82(2) degrees
in space group P2/n (Z=4) for [(DMSO)(2)H][trans(DMSO)-(2)tetrachloroiridate(III)],
(II). Visible absorption spectra show
that both complexes are stable for several
days, at pH 7.4, at room temperature.
No significant chloride hydrolysis is
observed, even at high temperature (70
degrees C), over 24 h. The extreme stability
of these iridium(III) complexes within
a physiological buffer was further assessed
by (1)H NMR; in addition, cyclic voltammetry
measurements evidenced a high stability
of the oxidation state +3. Preliminary
biological studies show that both complexes
do not bind appreciably bovine serum
albumin nor inhibit significantly the
proliferation of representative human
tumor cell lines, suggesting that hydrolysis
of coordinated chlorides is a crucial
feature for the biological properties
and the antitumor activity of the parent
ruthenium(III) complexes. |
| Distinct
effects of dinuclear ruthenium(III)
complexes on cell proliferation and
on cell cycle regulation in human and
murine tumor cell lines.
Bergamo A, Stocco G, Gava B, Cocchietto
M, Alessio E, Serli B, Iengo E, Sava
G.
J Pharmacol Exp Ther; 305(2):725-32,
May 2003.
ABSTRACT
We have examined the
biological and antitumor activity of
a series of dinuclear ruthenium complexes.
The aim of this study was to compare
the in vitro effects of these
new compounds on cell proliferation,
cell distribution among cell cycle phases,
and the expression of some proteins
involved in cell cycle regulation. Results
obtained show a mild cytotoxic activity
against human and murine cell lines,
more evident after prolonged exposure
of cell challenge. Two of the eight
dinuclear complexes [namely, compounds
D3 (Na(2)[(RuCl(4)(dmso-S))(2)(mu-bipy)])
and D7 ([NH(4)][(RuCl(4)(dmso-S))(mu-pyz)(RuCl(3)(dmso-S)(dmso-O))])
modify cell cycle distribution similarly
to imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate
(NAMI-A), whereas the others have a
low or negligible effect on this parameter.
If we correlate the induction of cell
cycle modifications with ruthenium uptake
by tumor cells and with the modulation
of proteins regulating cell cycle, we
may stress that the induction of G(2)-M
cell cycle arrest is related to the
achievement of a threshold concentration
of ruthenium inside the cells, which
is dependent on the cell line being
used, and that only cyclin B, among
cell cycle regulating proteins examined
by immunoblotting assays, appears to
be significantly modified. This in
vitro study shows that dinuclear
ruthenium complexes may have a behavior
similar to that of the monomer NAMI-A.
These results encourage the future experimentation
of their pharmacological properties
in in vivo models. |
| Dual
Action of NAMI-A in Inhibition of Solid
Tumor Metastasis: Selective Targeting
of Metastatic Cells and Binding to Collagen.
Sava G, Zorzet S, Turrin C, Vita F,
Soranzo M, Zabucchi G, Cocchietto M,
Bergamo A, DiGiovine S, Pezzoni G, Sartor
L, Garbisa S.
Clin Cancer Res; 9(5):1898-1905,
May 2003.
ABSTRACT
NAMI-A is a ruthenium
complex endowed with a selective effect
on lung metastases of solid metastasizing
tumors. The aim of this study is to
provide evidence that NAMI-A's effect
is based on the selective sensitivity
of the metastasis cell, as compared
with other tumor cells, and to show
that lungs represent a privileged site
for the antimetastatic effects. The
transplantation of Lewis lung carcinoma
cells, harvested from the primary tumor
of mice treated with 35 mg/kg/day NAMI-A
for six consecutive days, a dose active
on metastases, shows no change in primary
tumor take and growth but a significant
reduction in formation of spontaneous
lung metastases. Transmission electron
microscopy examination of lungs and
kidney shows NAMI-A to selectively bind
collagen of the lung extracellular matrix
and also type IV collagen of the basement
membrane of kidney glomeruli. The half
lifetime of NAMI-A elimination from
the lungs is longer than for liver,
kidney, and primary tumor. NAMI-A bound
to collagen is active on tumor cells
as shown in vitro by an invasion test,
using a modified Boyden chamber and
Matrigel, and it inhibits the matrix
metallo-proteinases MMP-2 and MMP-9
at micromolar concentrations, as shown
in vitro by a zimography test. These
data show NAMI-A to significantly affect
tumor cells with metastatic ability.
Binding to collagen allows NAMI-A to
exert its selective activity on metastatic
cells during dissemination and particularly
in the lungs. These data also stress
the wide spectrum of daily doses and
treatment schedules at which NAMI-A
is active against metastases.
|
| Primary
tumor, lung and kidney retention and
antimetastasis effect of NAMI-A following
different routes of administration.
Cocchietto M., Zorzet S., Sorc A.,
Sava G.
Investigational New Drugs; 21: 55-62,
2003.
ABSTRACT
Imidazolium-trans-dimethylsulfoxideimidazoletetrachlororuthenate
(NAMI-A) is a ruthenium compound effective
on solid tumor metastases. In this study,
we evaluated the effects of different
routes of administration of NAMI-A on
the distribution to primary tumor, lungs
and kidneys in BD2F1 hybrids with Lewis
lung carcinoma or in CBA inbred mice
with MCa mammary carcinoma. NAMI-A concentration
and the percentage of cumulative dose
(%Dtot) retained in these
tissues is indipendent of the animal
strain and of the tumor model used.
Also the presence of the tumor does
not change the distribution of NAMI-A
in the lungs and in the kidneys. A dose-dependent
antimetastatic effect is evident with
intraperitoneal (i.p.) treatments at
three different doses. Treatment of
tumor bearing mice with NAMI-A administrered
i.p., per os or by aerosol
showed a similar effect on lung metastases,
although the concentration of ruthenium
reached in the lungs was markedly different.
On the basis of the data obtained, we
can conclude that the antimetastatic
effects are related to the amount of
NAMI-A administered, rather than to
the lung' s concentration of the compound. |
| Synthesis,
catalytic properties and biological
activity of new water soluble ruthenium
cyclopentadienyl PTA complexes [(C5R5)RuCl(PTA)2]
(R= H, Me; PTA= 1,3,5-triaza-7-phosphaadamantane).
Akbayeva DN, Gonsalvi L, Oberhauser
W, Peruzzini M, Vizza F, Brugeller P,
Romerosa A, Sava G, Bergamo A.
Chem Commun; 264-265, 2003.
ABSTRACT
The new water soluble
ruthenium complexes [(C5R5)RuCl(PTA)2]
(R = H, Me; PTA = 1,3,5-triaza-7- phosphaadamantane)
were synthesised and characterised.
Their evaluation as regioselective catalysts
for hydrogenation of unsaturated ketones
in aqueous biphasic conditions and as
cytotoxic agents towards the TS/A adenocarcinoma
cell line is briefly presented. |
| Inhibition
of the MEK/ERK signaling pathway by
the novel antimetastatic agent NAMI-A
down regulates c- myc gene
expression and endothelial cell proliferation.
Gianfranco Pintus, Bruna Tadolini,
Anna Maria Posadino, Bastiano Sanna,
Marcella Debidda, Federico Bennardini,
Gianni Sava and Carlo Ventura.
Eur.J Biochem; 269: 5861 5870,
2002.
ABSTRACT
Imidazolium trans-imidazoledimethyl
sulfoxide-tetrachlo-roruthenate (NAMI-A)
is a novel ruthenium-containing experimental
antimetastatic agent. Compelling evidence
ascribes a pivotal role to endothelial
cells in the orchestration of tumor
angiogenesis and metastatic growth,
suggesting antiangiogenic therapy as
an attractive approach for anticancer
treatment. In this context, activation
of the mitogen-activated protein kinase
(MAPK)/extracellular signal-regulated
kinase (ERK) signaling pathway has been
found fundamental in transducing extracellular
stimuli that modulate a number of cellular
process including cell proliferation,
migration and invasion. Here we show
that exposure of the transformed endothelial
cell line ECV304 to NAMI-A significantly
inhibited DNA synthesis, as well as
the expression of the proliferating
cell nuclear antigene (PCNA). These
responses were associated with a marked
down-regulation of ERK phosphorylation
in serum-cultured cells. In addition,
NAMI-A markedly reduced
serum stimulated- and completely suppressed
phorbol 12-myristate 13-acetate (PMA)-triggered
MAPK/ERK kinase activity. NAMI-A was
also able to inhibit the phosphorylation
of MEK, the upstream activator of ERK,
and, similar to both the protein kinase
C (PKC) inhibitor GF109203X and the
MAPK/ERK (MEK) inhibitor PD98059, it
completely counteracted PMA-induced
ERK phosphorylation. Finally,NAMI-A
and PD98059 down regulated c-myc
gene expression to the same extent in
serum-cultured cells and dose-dependently
counteracted, and ultimately abolished,
the increase in c-myc gene
expression elicited by PMA in serum-free
cells.These results suggest that inhibition
of MEK/ERK signaling by NAMI-A may have
an important role in modulating c-myc
gene expression and ECV304 proliferation. |
| Tumour
cell uptake of the metastasis inhibitor
ruthenium complex NAMI-A and its in
vitro effects on KB cells.
Fabiana Frausin, Moreno Cocchietto,
Alberta Bergamo, Vito Scarcia, Ariella
Furlani, Gianni Sava
Cancer Chemother Pharmacol; 50:
405-411, 2002.
ABSTRACT
Purpose:The uptake
of NAMI-A (imidazolium trans
-imidazoledimethylsulphoxidetetrachlororuthenate)
by KB cells in vitro was compared
with the effects of this compound on
the cell cycle phase distribution of
the cells. Methods:NAMI-A
uptake was determined by flameless atomic
absorption spectroscopy, and the cell
cycle phase distribution was determined
by flow cytometry.Results:NAMI-A
uptake was proportional to its concentration
in the incubation medium.The use of
a number of incubation conditions showed
that NAMI-A uptake from MEM was independent
of the presence of serum and dependent
on the presence of amino acids in the
incubation medium,and that NAMI- A uptake
was markedly higher when the cells were
incubated in PBS.The uptake increase
observed in PBS did not occur when the
cells were kept at 0 4° C, suggesting
the presence of active transportation
of NAMI-A into cells.In addition,the
presence of divalent cations such as
Ca2+ and Mg2+
,appeared to facilitate NAMI-A uptake.The
anionic substance transport inhibitor
probenecid significantly reduced the
active transportation of NAMI-A into
cells.The effects of NAMI-A on cell
cycle distribution were strictly dependent
on its uptake by tumour cells and not
on its extracellular concentration.
Conclusions:These findings
suggest the interaction of NAMI-A with
biological components resulting in possible
consequences for the distribution of
the compound itself. Furthermore, NAMI-A
enters tumour cells both by passive
diffusion and by active transportation. |
| Ruthenium-based
NAMI-A type complexes with in vivo
selective metastasis reduction and in
vitro invasion inhibition unrelated
to cell cytotoxicity.
Bergamo A, Gava B, Alessio E, Mestroni
G, Serli B, Cocchietto M, Zorzet S,
Sava G
Int J Oncol ;21(6):1331-8, Dec 2002.
ABSTRACT
A series of analogues
of NAMI-A, a reference compound active
on solid tumor metastases, were synthesized
(NAMI-A type complexes). They share
the same chemical structure of NAMI-A,
and differ from it in the nature of
the coordinated nitrogen ligand, such
as pyrazole, thiazole and pyrazine,
which are less basic than imidazole.
This modification confers to the new
NAMI-A type complexes a better stability
in aqueous solution compared to the
parent compound, a very important characteristic
for a class of compounds that, with
NAMI-A, is currently completing a phase
I clinical trial at the Netherlands
Cancer Institute of Amsterdam. Cytotoxicity
and the effects on cell cycle and invasion
were investigated on TS/A, B16-F10 and
MCF-7 tumor cell lines, while the inhibition
of lung metastases was determined on
the mouse experimental tumors Lewis
lung carcinoma and MCa mammary carcinoma.
The new complexes show a pharmacological
activity very similar to that of the
parental compound NAMI-A: in vitro
they are devoid of meaningful cytotoxicity
against tumor cells, and in vivo
they inhibit metastasis formation and
growth approximately to the same extent
as NAMI-A. Thus the new NAMI-A type
complexes retain the same potent characteristic
of NAMI-A to selectively interact with
solid tumor metastases.
However, compared to NAMI-A they do
not stop cell cycle progression at G2-M
level and are more active in preventing
the spontaneous invasion of Matrigel
by tumor cells exposed for 1 h to 10(-4)
M concentration. Globally, these complexes
take advantage of the knowledge on NAMI-A
and appear particularly interesting
for future clinical handling and applications. |
| A
kinetic study of the chemical stability
of the antimetastatic ruthenium complex
NAMI-A.
Bouma M, Nuijen B, Jansen MT, Sava
G, Flaibani A, Bult A, Beijnen JH
Department of Pharmacy and Pharmacology,
Slotervaart Hospital/The Netherlands
Cancer Institute, Louwesweg 6, 1066
EC, Amsterdam, The Netherlands.
Int J Pharm 6;248(1-2):239-46, Nov
2002.
ABSTRACT
NAMI-A is a novel ruthenium
complex with selective activity against
cancer metastases currently in Phase
I clinical trials in The Netherlands.
The chemical stability of this new agent
was investigated utilizing a stability-indicating
reversed-phase high performance liquid
chromatographic assay with ultraviolet
detection and ultraviolet/visible light
spectrophotometry. The degradation kinetics
of NAMI-A were studied as a function
of pH, buffer composition, and temperature.
Degradation of NAMI-A follows first-order
kinetics at pH<6 and zero-order kinetics
at pH >/=6. A pH-rate profile, employing
rate constants extrapolated to zero
buffer concentration, was constructed,
demonstrating that NAMI-A is most stable
in pH region 3-4. The degradation rate
is not significantly affected by specific
buffer components. Storage temperature
strongly influences the degradation
rate. |
| Pharmaceutical
development of a parenteral lyophilized
formulation of the antimetastatic ruthenium
complex NAMI-A.
Bouma M, Nuijen B, Sava G, Perbellini
A, Flaibani A, van Steenbergen MJ, Talsma
H, Kettenes-van den Bosch JJ, Bult A,
Beijnen JH
Department of Pharmacy and Pharmacology,
Slotervaart Hospital/The Netherlands
Cancer Institute, Louwesweg 6, 1066
EC, Amsterdam, The Netherlands.
Int J Pharm 6;248(1-2):247-59, Nov 2002.
ABSTRACT
This paper describes
the development of a stable pharmaceutical
dosage form for NAMI-A, a novel antimetastatic
ruthenium complex, for Phase I testing.
NAMI-A drug substance was characterized
using several spectrometric and chromatographic
techniques. In preformulation studies,
it was found that NAMI-A in aqueous
solution was not stable enough to allow
sterilization by moist heat. The effect
of several excipients on the stability
of the formulation solution was investigated.
None of them provided sufficient stability
to allow long-term storage of an aqueous
solution of NAMI-A. Therefore, a lyophilized
product was
developed. Five different formulations
were prepared and subjected to thermogravimetric
(TG) analysis and stability studies
at various conditions for 1 year. Minimal
degradation during the production process
is achieved with a formulation solution
of pH 3-4. Of the acids tested, only
hydrochloric acid (HCl 0.1 mM) both
stabilized the formulation solution
and was compatible with the
lyophilized product. This product was
stable for at least 1 year when stored
at -20 degrees C, 25 degrees C/60% relative
humidity (RH) and 40 degrees C/75% RH,
and was also photostable. |
| Photostability
profiles of the experimental antimetastatic
ruthenium complex NAMI-A.
Bouma M, Nuijen B, Jansen MT, Sava
G, Bult A, Beijnen JH.
Slotervaart Hospital/The Netherlands
Cancer Institute, Department of Pharmacy
and Pharmacology, Louwesweg 6, 1066
EC, Amsterdam, The Netherlands.
J Pharm Biomed Anal 7;30(4):1287-96,
Nov 2002.
ABSTRACT
NAMI-A is a novel ruthenium
complex with selective activity against
metastases currently in Phase I clinical
trials in The Netherlands. The photostability
of this new agent in solid state and
in solution has been investigated utilizing
a stability-indicating reversed-phase
high performance liquid chromatographic
(HPLC) assay and ultraviolet/visible
(UV/VIS) light spectrophotometry. In
solid state, NAMI-A proved to be photostable.
In solution, however, the compound degraded
rapidly, in a pH-independent manner
in the pH range of 2-5. At alkaline
pH, the degradation rate was higher
than at acidic pH. The type of buffer
species had little influence. NAMI-A
concentration was inversely related
to the photostability. Addition of photostabilizers
(5% DMSO, 2% benzyl alcohol, 0.001%
curcumin) marginally increased the half-life.
NAMI-A's photostability in solution
was influenced to the greatest extent
by addition of an alcohol, with the
least polar solvent system (50% propylene
glycol) providing the most stable medium.
Based on the presented results, it is
recommended to store NAMI-A solutions
in the dark. |
| The
anti-metastatic agent imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate
induces endothelial cell apoptosis by
inhibiting the mitogen-activated protein
kinase/extracellular signal-egulated
kinase signaling pathway.
Bastiano Sanna, Marcella Debidda, Gianfranco
Pintus, Bruna Tadolini, Anna M. Posadino,
Federico Bennardini, Gianni Sava, Carlo
Ventura
Archives of Biochemistry and Biophysics;
403: 209 218, 2002.
ABSTRACT
Imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate
(NAMI-A) is a new ruthenium compound
active against lung metastasis in vivo
and tumor cell invasion in vitro. Since
angiogenesis was recognized as a key
event in the metastasizing process,
the manipulation of neo-vessel formation
has been developed as a new therapeutic
approach. Within this context, a pivotal
role for apoptosis in regulating cellular
growth has been proposed. In the present
study, we exposed to NAMI-A the spontaneously
transformed human endothelial cell line
ECV304 and assessed a number of apoptosis-related
features, including the DNA degradation
rate, the activation of caspase-3 protease,
the expression of Hsp27, and the release
of cytochrome c. Cell treatment with
NAMI-A elicited a signi?cant increment
in the apoptotic response, as indicated
by DNA fragmentation and caspase-3 activation,
two classical hallmarks of cellular
suicide. Furthermore, NAMI-A was able
to down-regulate Hsp27 protein expression
and provoke the release of mitochondrial
cytochrome c in the cytosol. Here, we
analyze the involvement of the mitogen-activated
protein kinase (MAPK)/extracellular
signal-regulated kinase (ERK) signal
transduction pathway in the induction
of apoptosis elicited by NAMIA.
Such a response was associated with
a marked inhibition of MAPK/ERK kinase
(MEK) and ERK phosphorylation with a
time course and dose dependency overlapping
those observed throughout NAMI-A-induced
apoptosis. In addition, we report that
PD98059, a selective MEK inhibitor,
is able to induce apoptosis by itself
in the ECV304 cell line. These results
suggest that inhibition of MEK/ERK signaling
by NAMI-A may have an important role
in modulating an apoptotic event in
ECV304. |
| In
vivo
Biodistribution Studies on Hyaluronan
Butyrate by Means of 99mTc
Direct Labelling and YAP Camera.
R. Rossin, S. Zorzet, A. Zanella, C.
Turrin, G. Sava, G. Moschini, A. Perbellini,
U. Mazzi
In: Technetium, Rhenium and Other
Metals in Chemistry and Nuclear Medicine
(M. Nicolini, U. Mazzi Eds.), pp 689-693,
SGEditoriali, Padova, 2002.
ABSTRACT
The glycosaminoglican
hyaluronan is one of the main components
of the extracellular matrix. The primary
receptor for HA (CD44) is overexpressed
on the surface of most malignant tumour
cells, suggesting the use of HA for
the targeted delivery of antitumour
drugs such as butyrate. In this study
the potential anti-tumour prodrug HA-But,
directly labelled with 99mTc,
has been injected in mice intravenous,
intraperitoneum and subcutaneous, and
its biodistribution has been studied
by means of a YAP camera. The collected
scintigrafic images showed a rapid blood
clearance and liver uptake after i.v.
injection and a slow diffusion after
i.p. and s.c. injection. These results
confirm for HA-But the biodistribution
of native HA and suggest its use for
local therapy of solid tumours. |
| Analysis
of the cytotoxicity of synthetic antimicrobial
peptides on mouse leucocytes: implications
for systemic use.
Sabrina Pacor, Anna Giangaspero, Marina
Bacac, Gianni Sava, Alessandro Tossi
Journal of Antimicrobial Chemotherapy;
50: 339-348, 2002.
ABSTRACT
We have analysed the
toxicity of highly cationic, artificial
alpha-helical antimicrobial peptides
on blood cells to assess their suitability
for systemic application. Flow cytometric
methods, based on the uptake of propidium
iodide, were used to obtain a rapid
and quantitative estimate of membrane
damage to resting and concanavalin A-activated
mouse lymphocytes, which was further
confirmed by morphological changes as
observed by scanning electron microscopy.
Membrane permeabilization appeared to
correlate with structural characteristics,
so that the peptide L-19(9/B), which
contains helix-stabilizing aminoisobutyric
acid (Aib) residues and is a potent
antimicrobial, was also the most lytic
towards both mouse lymphocytes and human
erythrocytes. Reducing the propensity
for helix formation in P19(9/B) resulted
in a marked reduction in in vitro
cytotoxicity. Changing the helical sense
in D-P19(9/B) also resulted in a significant
decrease in cytolytic activity towards
both erythrocytes and leucocytes. A
limited assessment in BALB/c mice confirmed
a lower in vivo toxicity of
P19(8) than L-19(9/B). In a study of
the systemic antimicotic activity of
P19(8) in a mouse protection model,
a modest prolongation in survival of
Candida albicans-infected animals
after intravenous administration was
observed at 5 mg/kg peptide but not
at higher doses. The implications of
these observations for the systemic
use of this type of peptide are discussed. |
| A
review on usnic acid, an interesting
natural compound.
Moreno Cocchietto, Nicola Skert, Pier
Luigi Nimis e Gianni Sava
NATURWISSENSCHAFTEN; 89:137-146,
2002.
ABSTRACT
Lichens are a world-widespread
consortium of fungal and photosynthetic
partners. Usnic acid is one of the most
common and abundant lichen metabolites,
well known as an antibiotioc, but also
endowed with several other interesting
properties. This review summarises the
most relevuant studies on usnic acid,
focusing on a number of biological activities
in different flield. On the basis of
the existing literature, usnic acid
seems to be an exclusive lichen product.
No synthetic derivatives more effective
than the natural form are known. Both
the (+) and (-) enantiomers of usnic
acid are effective against a large variety
of Gram-positive (G+) bacterial strains,
including strains from clinical isolates,
irrespective of their resistant phenotype.
Of particular relevance is the inhibition
of growth of multi-resistant strains
of Streptococcus aureus, enterococci
and mycobacteria. The (+)-usnic acid
enantiomer appears to be selective against
Streptococcus mutans without
inducing perturbing side effects on
the oral saprophyte flora. On the other
hand, the (-)-usnic acid enantiomer
is a selective natural herbicide because
of its blocking action against a specific
key plant enzyme. Other recognised characteristics
of usnic acid are ultraviolet absorption
and preserving properties. The toxicology,
the in vitro anti-inflammatory effects
and the mechanism of action of usnic
acid need to be investigated in greater
detail in order to reach clinical trials
and to allow further applications. Furthermore,
more research is needed to make possible
intensive lichen culture, in order to
produce large quantities of lichen substances
for pharmaceutical, cosmetic and agricultural
purposes. Some biological aspects, i.e.
the possible biological roles of usnic
acid, are discussed. |
| Inhibition
of endothelial cell functions and of
angiogenesis by the metastasis inhibitor
NAMI-A.
Angelo Vacca, Michele Bruno, Angela
Boccarelli, Mauro Coluccia, Domenico
Ribatti, Alberta Bergamo, Spiridione
Garbisa, Luigi Sartor, Gianni Sava
BRITISH JOURNAL OF CANCER; 86: 993-998,
2002 www.bjcancer.com
ABSTRACT
NAMI-A is a ruthenium-based
compound with selective anti-metastasis
activitvy in experimentaI models of
solid tumours. We studied whether this
activity was dependent on anti-angiogenic
ability of NAMI-A. We thus investigated
its in vitro effects on endothelial
cell functions necessary for angiogenesis
to develop, as weIl as its in vivo
effects in the chick embryo chorioallantoic
membrane model. Endothelial cell proliferation,
chemotaxis, and secretion of the matrix-degrading
enzyme rnetalloproteinase-2 were inhibited
by NAMI-A in a dose-dependent manner,
and without morphologic signs of cell
apoptosis or necrosis. LastIy, NAMI-A
displayed a dose-dependent in vivo
anti-angiogenic activity in the chorioallantoic
membrane model. These data suggest that
the anti-angiogenic activity of NAMI-A
can contribute to its anti-metastatic
efficacy in mice bearing malignant solid
tumours. |
| Influence
of chemical stability on the activity
of the antimetastasis ruthenium compound
NAMI-A.
G. Sava, A.Bergamo, S. Zorzet, B. Gava,
C. Casarsa, M. Cocchietto, A. Furlani,
V. Scarcia, B. Serli, E. Iengo, E. Alessio,
G. Mestroni
EUROPEAN JOURNAL OF CANCER; 38:
427-435, 2002
www.ejconline.com
ABSTRACT
The influence of chemical
stability on the antimetastatic ruthenium(III)
compound imidazolium trans-imidazoletetrachlorodimethylsulphoxideruthenium(III)
(NAMI-A) in aqueous solution was studied
both in vitro and in vivo.
The loss of dimethyl-sulphoxide (DMSO)
ligand from the compound was tested
by using a NAMI-A solution acidified
with HCl at pH 3.0 and aged for 0, 4,
8 and 24 h prior to intraperitoneal
(i.p.) injection into CBA mice bearing
advanced MCa mammary carcinoma. The
activity of NAMI-A on lung metastases
showed no change even after the loss
of DMSO ligand from up to 50% of the
molecules. The reduction of NAMI-A did
not modify the number of KB cells blocked
in the S+G2M phases, independent
of whether the reduction occurred outside
the cells or after loading the cells
with the compound prior to treatment
with the reductants (ascorbic acid,
glutathione or cysteine). In vivo,
the complete reduction of NAMI-A with
equivalent amounts of ascorbic acid,
glutathione or cysteine prior to administration
to mice bearing advanced MCa mammary
carcinoma was more active than NAMI-A
alone. The data show that NAMI-A, although
undergoing a series of chemical modifications,
maintains its antimetastatic activity
in a broad range of experimental conditions. |
| Isolation
of a murine metastatic cell line and
preliminary test of sensitivity to the
anti-metastasis agent NAMI-A.
Sabrina Pacor1, Marta Vadori1,
Francesca Vita2, Marina Bacac2,
Maria Rosa Soranzo3, Giuliano
Zabucchi3, Gianni Sava1,2
1Department of Biomedical
Sciences, University of Trieste. via
L. Giorgieri 7;
2Callerio Foundation-Onlus,
via A. Fleming 22-31;
3Department of Physiology
and Pathology, University of Trieste,
via A. Fleming 22, 34127-Trieste, Italy
Anticancer Research; 21:2523-2530, 2001
ABSTRACT
We have isolated a
new cell line (metGM) obtained from
the spontaneous lung metastases of the
mouse MCa mammary carcinoma. MetGM is
a stable cell line which, after one
year from its isolation, grows in vitro
in suspension, forming cell aggregates,
with cells that show irregular blabbing
borders, active protein synthesis and
convoluted nuclei, and which have the
capacity of invading matrigel membranes
on which they give rise to a network
of branching colonies. The preliminary
study of the effects of the anti-metastasis
ruthenium complex NAMI-A on metGM showed
no direct cytotoxicity, with a mild
reduction of cell proliferation, independent
of the concentration of the ruthenium
complex and not evident before 24 hours
from treatment. A 10% DNA fragmentation
was also measured on metGM cells 24
hours after challenge for 1 hour with
10-5M NAMI-A, suggesting that this compound
is probably capable of apoptosis in
a metastasis-derived cell line. Besides
these effects on a limited percent of
the cell population, NAMI-A changed
the shape of the metGM cells and these
alterations might account for the non-cytotoxic
anti-metastatic properties of this innovative
ruthenium complex. Thus MetGM appears
to be a novel cell line suitable for
the in vitro study of compounds endowed
with anti-metastatic properties and
for the development of new drugs with
this activity. |
| Pharmacological
effects of the ruthenium complex NAMI-A
given orally to CBA mice with MCa mammary
carcinoma.
Sonia Zorzet1, Alenka Sorc2,
Claudia Casarsa2, Moreno
Cocchietto2, Gianni Sava1,2
1Department of Biomedical
Sciences, University of Trieste, via
L. Giorgieri 7,
2Fondazione Callerio Onlus,
via A. Fleming 22-31, 34127 Trieste,
Italy.
Metal Based Drugs; 8:1-7, 2001.
ABSTRACT
NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate,
is a ruthenium based compounds capable
of inhibiting the growth of lung metastases
of solid tumours in a number of experimental
conditions. The aim of this study was
to investigate the potential use of
NAMI-A by the oral route to treat lung
metastases of MCa mammary carcinoma
in the CBA mouse. Treatment of mice,
carrying intramuscular tumours in advanced
stage of growth, for 11 consecutive
days caused a significant reduction
of the weight of lung metastases over
the range of doses from 150 to 600 mg/kg/day.
No sign of toxicity was observed at
the histological analysis in the gut
epithelium or in the kidney parenchyma,
and NAMI-A concentration in the kidney
was more than 10-fold lower than after
intraperitoneal treatments. NAMI-A is
thus active against metastases also
by the oral route, suggesting the use
of this way to treat tumour bearing
hosts for long periods. |
| Tumour
cell uptake G2-M accumulationand cytotoxicity
of NAMI-A on TS/A adenocarcinoma cells.
Alberta Bergamo1, Sonia
Zorzet2, Moreno Cocchietto1,
Maria Elena Carotenuto1,
Monica Magnarin1, Gianni
Sava1,2
1Fondazione Callerio Onlus
via A. Fleming 22-31,
2Department of Biomedical
Sciences, University of Trieste, via
L. Giorgieri 7, 34127 Trieste, Italy.
Anticancer Research; 21:1893-1898,
2001.
ABSTRACT
The ruthenium(lll) complex imidazolium
trans-imidazoledimethylsulfoxide-tetrachlororuthenate
(NAMI-A) was tested on TS/A adenocarcinoma
cells to evaluate the relationship between
cell uptake, cell cycle arrest and cytotoxicity.
The in vitro challenge of TS/A
cells with 10-4M NAMI-A in
multiwell plates for 15 min to 4 hrs
shows a partial reduction of cell growth,
evaluated by the MTT or the sulforhodamine-B
test following a further 24 hrs cultivation
of the treated cells in the absence
of NAMI-A, only afler 4 hrs exposure.
NAMI-A causes the increase of cells
in G2-M, measured by flow
cytometry following PI staining, depending
on the duration of in vitro treatment,
whereas, ruthenium uptake by tumour
cells, determined by flameless atomic
absorption spectroscopy, increases up
to 2 hrs of cell incubation. The arrest
of cell cycle in the pre-mitotic G2-M
phase is transient and completely reversed
by 48 hrs after treatment. This study
shows that the effect of NAMI-A on cell
cycle of TS/A cells is not strictly
related to NAMI-A uptake as is the effect
on tumour cell proliferation. |
| Immunostimulating
effects of oral lysozyme on a vaccination
treatment with Vibrio anguillarum.
Pacor S, Bacac M, Vadori M, Vismara
D, Manfrin A, Sava G.
Far Ter; XVIII: 51-55, 2001.
ABSTRACT
The aim of the present
work has been of evaluating the effects
of an oral treatment with 100 mg/kg
hen egg-white lysozyme (HEL) for 6 days
followed by 3x105 cells/day Vibrio
anguillarum serotype ol (VA-o1),
heat inactivated, for 3 days, on host
immunity, in CBA mice. The oral treatment
of HEL increases lymphocytes (+47%)
in Peyer's patches as compared to untreated
controls. A small subset of these lymphocytes
is increased from 11% to 38% in the
group treated with HEL; these cells
are CD4+ (66%), CD54+ (96%) and have
significantly increased the protein
content. VA-o1 is devoid of such effects,
whereas HEL+VA-o1 shows the same effects
as HEL alone. Repetition of this schedule,
after 3-weeks, causes a statistically
significant increase in CD3+ cells into
mesenteric lymph nodes. These results
suggest the possibility of using the
combination HEL+VA-o1 to add aspecific
immune responses to the vaccination
response with a VA-o1. |
|
Pharmacological
effects of the ruthenium complex NAMI-A
given orally to CBA mice with MCa mammary
carcinoma.
Zorzet S, Sorc A, Casarsa C, Cocchietto
M, Sava G.
Metal Based Drugs, 8: 1-7, 2001.
ABSTRACT
NAMI-A imidazolium
trans-imdazoledimethylsulfoxidetrachlororuthenate
is a ruthenium based compounds capable
of inhibiting the growth of lung metastases
of solid tumours in a number of experimental
conditions. The aim of this study was
to investigate the potential use of
NAMI-A by the oral route to treat lung
metastases of MCa mammary carcinoma
in the CBA mouse. Treatment of mice,
carrying intramuscular tumours in advanced
stage of growth, for 11 consecutive
days caused a significant reduction
of the weight of lung metastases over
the range of doses from 150 to 600mg/kg/day.
No sign of toxicity was observed at
the histological analysis in the gut
epithelium or in the kidney parenchyma,
and NAMI-A concentration in the kidney
was more than 10-fold lower than after
intraperitoneal treatments. NAMI-A is
thus active against metastases also
by the oral route, suggesting the use
of this way to treat tumour bearing
hosts for long periods. |
| Effects
of NAMI-A and some related ruthenium
complexes on cell viability after short
exposure of tumor cells.
Bergamo A, Zorzet S, Gava B, Sorc A,
Alessio E, Iengo E, Sava G.
Anti-Cancer Drugs, 11: 667-672,
2000.
ABSTRACT
A series of three ruthenium
complexes, i.e. trans-dichlorote-trakisdimethyl-sulfoxide
ruthenium(ll) (trans-Ru), imidazolium
trans-imidazoletetra-chlororuthenate
(ICR) and sodium trans-tetramethylensulfoxideisoquinoline-tetrachlororuthenate
(TEQU), were studied in vitro
in comparison to NAMI-A, a potent ruthenium-based
antimetastasis agent. In vitro
challenge of TS/A adenocarcinoma or
KB oral carcinoma tumor cells with 10(-4)
M concentration for 1 h evidenced the
lack of cytotoxicity of NAMI-A, ICR
and trans-Ru, the accumulation of cells
in the G2/M pre-mitotic cell phase by
NAMI-A and the attachment of tumor cells
to the plastic substrate was significantly
greater for NAMI-A than for ICR. These
data stress that in vitro cytotoxicity
is not necessary for in vivo
activity of ruthenium antitumor complexes:
NAMI-A, ICR and trans-Ru, are in fact
known to be active against murine tumors
in the mouse system. Rather, TEQU, the
compound free of in vivo activity,
was the only one to reduce cell growth
of in vitro cultured cells. In
conclusion, the data on the effects
of NAMI-A on in vitro cultured
cells show that the increase of cell
adhesion properties and the transient
cell cycle arrest in the G2/M phase
are much more relevant than the effects
on cell properties relevant to cell
growth (i.e. on CD44, CD54 or CD71 antigens)
for determining in vivo antimetastasis
activity. |
| Ruthenium-based
compounds and tumour growth control
(review).
Sava G, Bergamo A.
Int J Oncol; 17: 353-365, 2000.
ABSTRACT
Heavy metals have often
been represented as an uncertain entity
related to renal and other risks of
toxicity. In favour of this thought
there are several lines of evidence,
first of all traffic pollution, other
evidence that metals such as arsenite,
mercury, cadmium or even iron or radioactive
heavy metals, that may be introduced
into the body by accident, have been
responsible of well known pathologies
(for example saturnism with lead) or
acute toxicity. Therefore, the biological
and medical literature have debated
on this subject, mainly from the toxicological
point of view, rather than studying
possible advantages that might come
from compounds based on these metals.
Exceptions are represented by studies
on the role of metal ions in the biochemistry
of enzymes and energy production and,
although with less emphasis, on their
possible use for correcting metabolic
malfunctions. Ruthenium, as a metal,
has received an even poorer interest
and besides the use in histology, neither
ruthenium ions nor ruthenium compounds
have a clear place in medicine and biology.
Nevertheless, since the middle seventies,
many studies have been published, showing
in a convincible and repetitive manner,
the possible advantages of ruthenium
as a base for new competitive drugs.
The aim of this review is therefore
that of critically examining the past
and the actual work on ruthenium compounds
with emphasis on their proposed role
in cancer therapy. |
| Increase
of tumour infiltrating lymphocytes in
mice treated with antimetastatic doses
of NAMI-A.
Magnarin M, Bergamo A, Carotenuto ME,
Zorzet S, Sava G.
Anticancer Res; 20: 2939-2944, 2000.
ABSTRACT
NAMI-A is a novel
antitumour agent, based on ruthenium,
which has proved effectiveness against
lung metastases of solid mouse tumours.
The study focuses on the effects of
NAMI-A on leukocyte infiltration into
the primary tumour of MCa mammary
carcinoma, implanted subcutaneously
(s.c.) or intramuscularly (i.m.) into
CBA mice. NAMI-A, given with a cycle
of daily treatments for six consecutive
days on advanced tumours at 35 mg/kg/day,
markedly reduces lung metastasis independently
of the tumour type (Lewis lung carcinoma,
MCa mammary carcinoma or TS/A adenocarcinoma)
being treated and of the site of tumour
implantation (s.c. or i.m.). The analysis
of leukocyte infiltration of the primary
tumour, performed on a single cell
suspension of cells isolated from
a Ficoll gradient on which a raw suspension
of primary tumour cells was layered,
showed NAMI-A to significantly increase
tumour infiltrating lymphocytes. These
lymphocytes are almost all CD3+ cells
with a significant increase of the
CD8+ over the CD4+ subpopulation that
reduces the helper/suppressor ratio
from 2.8 to 2.1. These data indicated
the absence of toxicity of NAMI-A
for tumour infiltrating lymphocytes
and suggested that this compound might
even synergize in combined treatments
with cancer immunotherapy.
|
| Antimetastatic
properties and DNA interactions of the
novel class of dimeric Ru(III) compounds
Na2[[trans-RuCl4(Me2SO)]2(mu-L)] (L
= ditopic, non-chelating aromatic N-ligand).
A preliminary investigation.
Alessio E, Iengo E, Zorzet S, Bergamo
A, Coluccia M, Boccarelli A, Sava G.
J Inorg Biochem; 79: 173-177, 2000.
ABSTRACT
A novel class of dianionic
Ru(III) dimers of formula Na2[[trans-RuCl4(Me2SO)]2(mu-L)],
with L = pyrazine (pyz, 1), pyrimidine
(pym, 2), 4,4'-bipyridine (bipy, 3),
and 1,2-bis(4-pyridine) ethane (etbipy,
4), was developed by us with the specific
aim of assessing their antitumor properties.
The dimers are in fact structurally
related to the antimetastatic mononuclear
compound (ImH) [trans-RuCl4(Me2SO)(Im)]
(NAMI-A, Im = imidazole). Preliminary
results concerning the antineoplastic
activity of 1-4 against the murine MCa
carcinoma model, a tumor which spontaneously
metastasizes in the lungs, are reported.
Similarly to what is normally observed
with NAMI-A, the treatment with the
dimeric complexes was scarcely effective
against the growth of the primary tumor.
However, dimers 1, 2, and 4 reduced
very effectively the number and, in
particular, the weight of lung metastases
(to about 5% with respect to controls);
in particular, Na2[[trans-RuCl4(Me2SO)]2(mu-etbipy)]
(4) was as effective as NAMI-A in reducing
the spontaneous metastases at a dosage
which, in terms of moles of ruthenium,
is about 3.5 times lower compared to
that normally used for NAMI-A. Furthermore,
in vitro tests showed that dimers
1-4 are capable of forming interstrand
cross-links with linearized plasmidic
DNA in a time-dependent manner. All
the dimeric species are more active
in inducing cross-links compared to
NAMI-A, and the dimer bridged by the
etbipy ligand (4) is the most effective
among those tested. |
| Lack
of In vitro cytotoxicity, associated
to increased G(2)-M cell fraction and
inhibition of matrigel invasion, may
predict In vivo-selective antimetastasis
activity of ruthenium complexes.
Zorzet S, Bergamo A, Cocchietto M, Sorc
A, Gava B, Alessio E, Iengo E, Sava
G.
J Pharmacol Exp Ther; 295: 927-933,
2000.
ABSTRACT
The ruthenium complexes
trans-dichlorotetrakisdimethylsulfoxide
ruthenium(II) (trans-Ru), imidazolium
trans-imidazoletetrachlororuthenate
(ICR), sodium trans-tetramethylensulfoxideisoquinolinetetrachlororuthenate
(TEQU), and imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate
(NAMI-A) are tested in vitro
by short exposure of MCF-7, LoVo, KB,
and TS/A tumor cells to 10(-4) M concentration,
and in vivo on Lewis lung carcinoma
by a daily i.p. treatment for 6 consecutive
days using equitoxic and maximum tolerated
doses. NAMI-A 1) inhibited tumor cell
invasion of matrigel, 2) induced a transient
accumulation of cells in the G(2)-M
phase, 3) did not modify in vitro
cell growth, and 4) markedly reduced
lung metastasis formation. TEQU showed
significant cytotoxicity in vitro
and was not antimetastatic in vivo.
ICR and trans-Ru did not modify cell
cycle distribution of in vitro
tumor cells nor did they inhibit matrigel
invasion; ICR was also devoid of antimetastasis
effects in vivo. Ruthenium uptake
by tumor cells did account for in
vitro cytotoxicity but not for other
in vitro actions or for in
vivo antimetastasis activity. The
contemporary absence of cytotoxicity,
associated to inhibition of matrigel
crossing and to transient block in the
premitotic G(2)-M phase, appears to
be prerequisites for a ruthenium compound
to show in vivo-selective antimetastasis
effect. The validation of this model
for other classes of compounds will
allow an understanding of the combined
weight of the above-mentioned phenomena
for tumor metastasis growth and control.
|
| Blood
concentration and toxicity of the antimetastasis
agent NAMI-A following repeated intravenous
treatment in mice.
Cocchietto M, Sava G.
Pharmacol Toxicol; 87: 193-197,
2000.
ABSTRACT
NAMI-A is a new generation
antitumour ruthenium-based agent and
characterised by strong efficacy against
lung metastases of experimental solid
tumours in mice. The effects of intravenous
administration of 15, 35 and 50 mg/kg/day
of NAMI-A for 5 consecutive days on
blood concentration and host toxicity
were tested on Swiss CD1 male and female
mice. The blood concentration of NAMI-A,
both after the first injection and at
the end of the 5-day treatment fell
rapidly and 5 min. after the last injection
it was always below 10% of the administered
dose. Kinetic parameters, calculated
at the end of the 5-day treatment cycle
according to a mono-compartment model
(fitting with R2=0.9), indicate a t
1/2 of about 18 hr. Toxicity i) was
observed only at the highest dose used
(50 mg/kg/day), ii) was greater in females
than in males, iii) in mice which survived
treatment was completely reversed within
3-weeks of the end of the treatment.
Haematological examinations, clinical
chemistry data and histopathologic studies
were consistent in terms of the effect
on host lymphoid tissues, consisting
in spleen and lymph node depletion and
in a general increase of circulating
leukocytes. Data on ruthenium organ
retention confirm lack of brain penetration
and a relatively high lung concentration
which might account for the remarkable
effect on lung metastases. |
| Blood
levels of ruthenium following repeated
treatments with the antimetastatic compound
NAMI-A in healthy beagle dogs.
Sava G, Cocchietto M.
In Vivo; 14: 741-744, 2000.
ABSTRACT
NAMI-A is a new generation ruthenium
compound which is entering phase-I clinical
trials anti-metastatic agent. This study
analyses the effects of the i.v. injection
of NAMI-A to healthy Beagle dogs at
increasing doses from 0.4 (low) 4 (mid)
and 8 (high) mg/kg/day, given for 5
consecutive days. Only mild signs of
toxicity, consisting of emesis and mucoid
faeces, from which animals completely
recovered, occurred during treatment
at the high dose. Decay of ruthenium
concentration from the whole blood,
24 hr after 5-days treatment, was lower
than that observed after 1-day treatment.
T1/2 was about 20-23 hr, or slightly
longer when the animals were hydrated
with tap water prior to treatment; Cltot
was 21-22 ml*hr-1, decreasing to 13
ml*hr-1 after hydration and increasing
to 34 ml*hr-1 with the high dose. AUC
was proportional to the dose used. Thus
NAMI-A is well tolerated by healthy
dogs with blood levels comparable to
those obtained in mice treated with
an about 10-times higher daily dose.
|
| Pharmacological
control of lung metastases of solid
tumours by a novel ruthenium complex.
Sava G, Capozzi I, Clerici K, Gagliardi
R, Alessio E, Mestroni G.
Clinical Experimental Metastasis;
16: 371-379, 1998.
ABSTRACT
Imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate
ImH[trans-RuCl4(DMSO)Im]
(NAMI-A), a ruthenium compound that
replaces Na+ with ImH+
in the molecule of Na[trans-RuCl4(DMSO)Im]
(NAMI), was studied for the anti-metastasis
effects in models of solid metastasising
tumours of the mouse. NAMI-A, given
i.p. at 35 mg/kg/day for six consecutive
days, a dose equimolar to that of NAMI,
to mice bearing Lewis lung carcinoma
and MCa mammary carcinoma, markedly
reduces lung metastasis weight by 80-90%,
with an effect equal or even superior
to that of NAMI, depending on the experimental
system adopted. Correspondingly, NAMI-A
increases the content of connective
tissue in the tumour matrix, around
blood vessels, and in the tumour capsule,
augments the percentage of tumour cells
in G2/M phase and reduces
the amount of CD45+ cells
infiltrating the tumour parenchyma.
The effects of the same doses on spleen
lymphocytes corresponds to an increase
of CD8+ subset without any
change of the distribution of cells
in G0/G1, S and
G2/M phases. The study shows
that NAMI-A behaves similarly to NAMI
on the several parameters examined in
comparison experiments and therefore
we suggest to credit NAMI-A with all
the biological actions already described
for NAMI during the last 3 years. The
replacement of Na+ with ImH+
therefore, besides the better chemical
stability of the molecule, confers to
[trans-RuCl4(DMSO)Im]-
a closer similarity with a true drug
to be used in humans, and suggests this
molecule for future studies of preclinical
toxicology and phase I and II clinical
trials. |
| In
vitro
cell cycle arrest, in vivo action
on solid metastasizing tumors, and host
toxicity of the antimetastatic drug
NAMI-A and cisplatin.
Bergamo A, Gagliardi R, Scarcia V,
Furlani A, Alessio E, Mestroni G, Sava
G.
Journal of Pharmacology and Experimental
Therapeutics; 289: 559-564, 1999.
ABSTRACT
The effects of NAMI-A
(imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate)
are compared with cisplatin on tumour
cells cultured in vitro at doses
of 1 to 100 mM and on tumour metastases
in vivo at maximum tolerated
doses. Using mouse tumours that metastasise
to the lungs, NAMI-A given i.p. for
6 consecutive days at 35 mg/kg/day,
was effective independently of the tumour
line being treated and of the stage
of metastasis growth. Conversely, cisplatin
(2 mg/kg/day for 6 days) was as effective
as NAMI-A on MCa mammary carcinoma and
TS/A adenocarcinoma and less effective
than NAMI-A on Lewis lung carcinoma.
Cisplatin reduced body weight gain and
spleen weight during treatment and was
much more toxic than NAMI-A on liver
sinusoids, kidney tubules, and lung
epithelium. In vitro NAMI-A caused
a transient cell cycle arrest of tumour
cells in the premitotic G2/M
phase, whereas cisplatin caused a progressive
dose-dependent disruption of cell cycle
phases. Correspondingly NAMI-A did not
modify cell growth, whereas cisplatin
caused a dose-dependent reduction of
cell proliferation, as determined by
sulphorhodamine B test. Thus, NAMI-A,
unlike cisplatin is a potent agent for
the treatment of solid tumour metastases
as well as when these tumour lesions
are in an advanced stage of growth.
NAMI-A is endowed with a mechanism of
action unrelated to direct tumour cell
cytotoxicity, and such mechanism of
action is responsible for a reduced
host toxicity. |
| Drug
control of solid tumour metastases:
a critical view.
Sava G, Bergamo A.
Anticancer Research; 19: 1117-1124,
1999.
ABSTRACT
Metastasis of solid
tumours represent the target of election
for the pharmacological treatment of
cancer. Nevertheless, commonly used
treatments do not represent any selective
approach, provided that drugs are mostly
unspecific cytotoxics. Today many strategies
adopted to interfere with metastasis
growth concern the interaction with
biological signals of the metastatic
cells or of the host. One difference
should be made between anti-metastatic
and anti-metastasis drugs, in that only
the latter realise the goal of selectively
destroying metastases whereever they
are. In this context many agents active
on newly identified molecular targets
are more effective in preventing metastasis
formation than in inhibiting their growth.
NAMI-A, an innovative ruthenium compound,
seems to provide optimism for the future
and, in laboratory models, it is very
active on lung metastases independently
of the stage of their growth. The success
of NAMI-A against metastasis shpould
stimulate laboratory studies with appropriate
experimental models to predict clinical
activity, since the use of experimental
conditions closely similar to those
of human tumours should help the identification
of more active compounds. |
| Paracrine
effects of IL-4 transfection on TS/A
adenocarcinoma cells mediate reduced
in vivo growth.
Pacor S, Gagliardi R, Spessotto P,
Zabucchi G, Sava G.
Pathology and Oncology Research;
5: 110-116, 1999.
ABSTRACT
The in vitro / in vivo
growth capacity and phenotype of TS/A
and the IL4-transfected TS/A-IL4 cell
lines were studied by cell cycle analysis,
expression of ICAM-1/CD54, ransferrin
receptor/CD71 and E-cadherin and by
histology of the primary tumors. TS/A-IL4,
unlike the TS/A line, shows in vitro
a marked increase in the fibroblastoid
cell type and a decreased E-cadherin
expression. Administration of conditioned
medium containing IL4 obtained from
the TS/A-IL4 cell line, stimulates CD54
expression in the TS/A CELL LINE. TS/A-IL4
tumors grow more slowly in vivo
and are ultimately rejected. These processes
are accompanied by a marked increase
in collagen and extracellular matrix
proteins and increased recruitment and
degranulation of mast cells. The paracrine
effect of IL4, released by the transfected
tumor cells, might be responsible for
the reduced in vivo growth of
the TS/A cell line in the presence of
TS/A-IL4 cells. |
| In
vitro
down regulation of ICAM-1 and E-cadherin
and in vivo reduction of lung
metastases of TS/A adenocarcinoma by
a lysozyme derivative.
Pacor S, Gagliardi R, Di Daniel E,
Vadori M, Sava G.
International Journal of Molecular
Medicine; 4: 369-375, 1999.
ABSTRACT
The aim of the present
investigation was to examine the effects
of the lysozyme derivative mPEG-lyso
(hen egg-white lysozyme coupled with
polyoxyethylenglycol), on TS/A adenocarcinoma
cell line in vivo and in vitro.
mPEG-lyso reduces the number of ICAM-1+
and E-cadherin+ cells of
TS/A adenocarcinoma cell line in
vitro, and causes a marked decrease
of spontaneous lung metastases in
vivo. In both cases, mPEG-lyso reduces
the number of tumour cells in synthesis
and pre-mitotic phases. In connection
with the reduction of cells expressing
adhesion molecules, mPEG-lyso reduces
the number of infiltrating leukocytes
in the primary tumour in vivo
and reduces the binding capacity of
splenocytes to tumour cells in vitro.
These data stress, for the first time,
that the in vivo control of mPEG-lyso
on lung metastasis formation of solid
metastasising tumours may be due to
a combination of effects on tumour cells
in addition to those on host’s immune
system. |
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