Title of Invention

A PHARMACEUTICAL COMPRISING SUBSTITUTED ACRYLOYL DISTAMYCIN DERIVATIVES AND ALKYLATING AGENTS

Abstract The present invention to a composition comprising of acryloyl distamycin derivatives, in particular α -bromo- and α-chloro-acryloyl distamycin derivatives of formula (I), as set forth in the specification, and an alkylating agent, in the treatment of tumors. Also provided is the use of the said combination in the treatment or prevention of metastasis or in the treatment of tumors by inhibition of angiogenesis.
Full Text

COMBINED THERAPY AGAINST TUMORS COMPRISING SUBSTITUTED ACRYLOYL DISTAMYCIN DERIVATIVES AND ALKYLATING AGENTS
The present invention relates to the field of cancer treatment and provides an antitumor composition comprising a substituted acryloyl distamycin derivative, more particularly an a-bromo- or a-chloro-acryloyl distamycin derivative, and an alkylating agent, having a synergistic antineoplastic effect.
Distamycin A and analogues thereof, hereinafter referred to as distamycin and distamycin-like derivatives, are known in the art as cytotoxic agents useful in antitumor therapy.
Distamycin A is an antibiotic substance with antiviral and antiprotozoal activity, having a polypyrrole framework [Nature 203:1064 (1964); J. Med. Chem, 32: 774-778 (1989)]. The international patent applications WO 90/11277, WO 98/04524, WO 98/21202, WO 99/50265, WO 99/50266 and WO 01/40181 (claiming priority fi-om British patent application No. 9928703.9), all in the name of the applicant itself and herewith incorporated by reference, disclose acryloyl distamycin derivatives wherein the amidino moiety of distamycin is optionally replaced by nitrogen-containing ending groups such as, for instance, cyanamidino, N-methylamidino, guanidino, carbamoyl, amidoxime, cyano and the like, and/or wherein the polypyrrole firamework of distamycin, or part of it, is replaced by varying carbocyclic or heterocyclic moieties.


R2 is a distatnycm or distamycin-like framework; or a pharmaceutically acceptable salt thereof; and an alkylating agent.
The present invention includes, within its scope, the pharmaceutical compositions comprising any of the possible isomers covered by the compounds of formula (I), both considered separately or in admixture, as well as the metabolites and the pharmaceutically acceptable bio-precursors (otherwise known as pro-drugs) of the compounds of formula (I).
In the present description, unless otherwise specified, with the term distamycin or distamycin-like framework R2 we intend any moiety structurally closely related to distamycin itself for instance by optionally replacing the ending amidino moiety of distamycin and/or its polypyrrole framework, or part of it.
Alkylating agents are widely known in the art as described in various scientific
publications.
Representatives for this class of compounds are, for mstance, mustards such as
melphalan, chlorambucil, mechlorethamine, cyclophosphamide, ifosfamide and busulfan;
nitrosoureas such as caimustine, lormustine, semustine and fotemustine; tetrazines such
as dacarbazine and temozolomide; aziridiues such as thiotepa and mitomycin C and
platinum derivatives such as cisplatin, carboplatin, oxaliplatin, nedaplatin and lobaplatin
and the like.
See, for a general reference. Cancer Principles and Practice of Oncology, Lippincott-
Raven Ed. (1997), 405-432.
According to a preferred embodiment of the invention, herewith provided are the above pharmaceutical compositions wherein the alkylating agent is selected from mustards and platinum derivatives such as cisplatin, carboplatin and oxaliplatin.
According to another preferred embodiment of the invention, herewith provided are the above pharmaceutical compositions wherein, within the acryloyl distamycin derivative

of formula (I), R1 has the above reported meanings and R2 is a group of formula (IT) below:
wherein
m is an integer from 0 to 2;
n is an integer from 2 to 5;
r is O or 1;
X and Y are, the same or different and independently for each heterocyclic ring, a
nitrogen atom or a CH group;
G is phenylene, a 5 or 6 membered saturated or unsaturated heterocyclic ring with from 1
to 3 heteroatoms selected among N, O or S, or it is a group of formula (EI) below:


alkoxy group we intend a straight or branched group selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.
Even more preferred are the pharmaceutical compositions of the invention comprising the above acryloyl distamycin derivative of formula (I) wherein R1 is bromine or chlorine; R2 is the above group of formula (II) wherein r is 0, m is 0 or 1, n is 4 and B has the above reported meanings.
Still more preferred, within this class, are the pharmaceutical compositions comprising the compounds of formula (I) wherein R1 is bromine or chlorine; R2 is the above group of formula (H) wherein r is 0, m is 0 or 1, n is 4, X and Y are both CH groups and B is selected from;




methyl-1 H-pyrrol-3-yl} -4-[(2-bromoacryloyl)amino]-1 -methyl- lH-pyrrole-2-carboxamide.
The above compounds of fonnula (I), either specifically identified as such or by means of the general formula, are known or easily prepared according to known methods as reported, for instance, in the aforementioned international patent applications WO 90/11277, WO 98/04524, WO 98/21202, WO 99/50265 and WO 99/50266 as well as in WO 01/40181.
The present invention further provides a product comprising an acryloyl distamycin derivative of fonnula (I), as defined above, and an alkylating agent, as a combined preparation for simultaneous, separate or sequential use in antitumor therapy.
A fiarther aspect of the present invention is to provide a method of treatmg a mammal, including humans, suffering from a neoplastic disease state, which method comprises administering to said mammal the above acryloyl distamycin derivative of formula (I) and an alkylating agent, in amounts effective to produce a synergistic antineoplastic effect
The present invention also provides a method for lowering the side effects caused by antineoplastic therapy with an antineoplastic agent in a mammal in need thereof, including humans, the method comprising administering to said mammal a combined preparation comprising an alkylating agent and an acryloyl distamycin derivative of formula (I), as defined above, in amounts effective to produce a synergistic antineoplastic effect.
By the term "synergistic antineoplastic effect", as used herein, it is meant the inhibition of the growth tumor, preferably the complete regression of the tumor, by administering an effective amount of the combination comprising an acryloyl distamycin derivative of formula (I) and an alkylating agent to mammals, including humans.

By the term "administered " or "administering", as used herein, it is meant parenteral and/or oral administration; the term "parenteral" means intravenous, subcutaneous and intramuscular administration.
In the method of the present invention, the acryloyl distamycin derivative may be administered simultaneously with the alkylating agent or, alternatively, both compounds maybe administered sequentially in either order.
In this respect, it will be appreciated that the actual preferred method and order of administration will vary according to, inter alia, the particular formulation of the acryloyl distamycin of formula (I) being used, the particular formulation of the alkylating agent being used, the particular tumor model being treated as well as the particular host being treated.
To administer the acryloyl distamycin derivative of formula (T), according to the method of the invention, the course of therapy generally employed comprises doses varying from about 0.05 to about 100 mg/m2 of body surface area and, more preferably, from about 0.1 to about 50 mg/m2 of body surface area.
For the administration of the alkylating agent, according to the method of the
invention, the course of therapy generally employed comprises:
for the administration of mustard compounds doses varying from about 1 mg/m2 to
about 5000 mg/m2 of body surface area and, more preferably, from about 10 to about
1000 mg/m2 of body surface area.
for the administration of nitrosourea derivatives doses varying from about 1 mg/m to
about 1000 mg/m2 of body surface area and, more preferably, from about 10 to about
1000 mg/m2 of body surface area.
for the administration of tetrazine and aziridine compounds doses varying from about
1 mg/m2 to about 1000 mg/m2 of body surface area and, more preferably, from about
10 to about 1000 mg/m of body surface area.
for the administration of platinum derivatives doses varying from about 1 mg/m to
about 1000 mg/m2 of body surface area and, more preferably, from about 10 to about
500 mg/m of body surface area.

The antineoplastic therapy of the present invention is particularly suitable for treating breast, ovary, lung, colon, kidney, stomach, pancreas, liver, melanoma, leukemia and brain tumors in mammals, including humans.
In a further aspect, the present invention is directed to the preparation of a pharmaceutical composition comprising an effective amount of an acryloyl distamycin derivative of formula (I), as defined above, and an alkylatiug agent, in the preparation of a medicament for use in the prevention or treatment of metastasis or in the treatment of tumors by inhibition of angiogenesis.
As stated above, the effect of an acryloyl distamycin derivative of formula (I) and aa alkylating agent, for instance cisplatin and carboplatin, is significantly increased without a parallel increase of toxicity. In other words, the combined therapy of the present invention enhances the antitumoral effects of the acryloyl distamycin derivative and of the alkylating agent and, hence, provides the most effective and least toxic treatment for tumors.
The synergistic or super additive effect of the combined preparations of the invention is shown, for instance, by the following in vivo tests which are intended to illustrate the present invention without posing any limitation to it.
Table 1 shows the antileukemic activity on disseminated L1210 murine leukemia
obtained by combining the representative compound of formula (I) of the invention N-
(5-{[(5-{[(5-{[(2-{[amino(imno)methyl]amino}ethyI)amino]carbonyI}-l-methyI-lH-
pyrrol-3-yl)amino]carbonyl}-l-methyl4H-pyrrol-3-yl)amino]carbonyl}-l-methyl-lH--
pyrrol-3-yl)-4-[(2-bromoacryloyl)amino]-l-methyl--lH-pyrrole-2--carboxariiide
hydrochloride - internal code PNU 166196, with cisplatin.
At the dose of 5.9 mg/kg of cisplatin alone (day +3) and at the dose of 0.26 mg/kg of
PNU 166196 alone (days +1,2) were associated, without toxicity, with JLS% values of
67 and 33, respectively.

Combining cisplatin and PNU 166196 at the same doses with the same schedule, an increase of activity with E.S% values of 125 were observed, thus indicating a synergistic antitumor effect.
Table 2 shows the antileukemic activity on disseminated LI 210 murine leukemia
obtained by combining the above PNU 166196 derivative with carboplatin.
At the dose of 135 mg/kg of carboplatin alone (day +3) and at the dose of 0.26 mg/kg
of PNU 166196 alone (days +1,2) were associated, without toxicity, with ILS% values
of 50 and 33, respectively.
By combining carboplatin and PNU 166196 at the same doses and with the same
schedule, an increase of activity with ILS% values of 92 were observed, again
indicating a more than additive effect.
Table 3 shows the antitumor effect on subcutaneous implanted HCT-116 human colon
carcinoma obtained by combining PNU 166196 with cisplatin.
At the dose of 2 mg/kg of cisplatin alone (q7dx3) and at the dose of 0.4 mg/kg of PNU
166196 alone (q7dx3) were associated, without toxicity, T/C% values of 92 and 61,
respectively.
By combining cisplatin and PNU 166196, iostead, a significant increase in tumor
growth delay was observed, hence indicating a therapeutic advantage of the
combination (synergism) in comparison to the administration of the drugs alone.
For these experiments PNU 166196 was solubilized in water for injection, while standard pharmaceutical preparations were used for cisplatin and carboplatin.


a L1210 leukemia cells (10^/mouse CD2F1) are injected TV on Day 0.
b Treatment is given IV.
c Increase in life span: [(median survival time of treated mice/median survival time
of controls)x100]-100. ^ Number of toxic deaths/number of mice.

1 L1210 leukemia cells (105mouse CD2F1) are injected IV on Day 0.
2 Treatment is given IV,
3 Increase in life span: [(median survival time of treated mice/median survival time
of controls)x100]-100. 4 Number of toxic deaths/number of mice.


b Tumor regression (T/C %) on day 20 after treatment (according to NCI standards:
T/C The co-pending application no78/CHSNP/2003 relates to "combined therapy against tumors comprising substituted acrylcyl distamycin derivatives, taxanes and/or antimetabclites".




WE CLAIM:
1, A phannaceutical composition comprising a pharmaceutically acceptable currier or excipient and as active ingredient,
an acryloyl distamycin derivative of formula (I);
wherein:
R1 is a bromine or chlorine atom;
R2 is a distamycin or distamycin-like framework; or a pharmaceutically acceptable salt
thereof; and
an alkylating agent wherein the alkylating
agent is selected from the group consisting of mustards such as melphalan, chlorambucil, mechlorethamine, cyclophosphamide, ifosfamide and busulfan; nitrosoureas such as carmustine, lormustinc, semustine and fotemustine; tetrazines such as dacarbazine and temozolomide; aziridines such as thiotepa and mitomycin C and platinum derivatives such as cisplatin, carboplatin, oxaliplatin, nedaplatin and lob^latin.
2. The pharmaceutical composition according to claim 1 comprising an acryloyl distamycin derivative of formula (I)

wherein:
R1 is a bromine or chlorine atom; R2 is a group of formula (II)


nitrogen atom or a CH group;
G is phenylene, a 5 or 6 membered saturated or unsaturated heterocyclic ring with from 1
to 3 heteroatoms selected among N, O or S, or it is a group of formula (HI) below;

3. The pharmaceutical composition according to claim 2 comprising an acryioyl distamycin derivative of formula (I) wherein R1, R2 and B are as defined in claim 3, r is





7. The composition according to claim 6, wherein the alkylating agent is selected from mustards such as melphalan, chlorambucil, mechlorethamine, cyclophosphamide,
ifosfamide and busulfan; mtrosoureas such as carmustine, lonnustine, semustine and
fotemustine; tetrazines such as dacarbazine and temozolomide; aziridines such as
thiotepa and mitomycin C and platinum derivatives such as cisplatin, carboplatin,
oxaliplatin, ncdaplatin and lobaplatin.


wherein Q is a nitrogen atom or a CH group and W is an oxygen or sulfur atom or it is a group NR3 wherein R3 is hydrogen or C1-C4 alkyl; B is selected from the group consisting of

9. The composition according to claim 6, wherein the acryloyl distamycin derivative is selected from the group as claimed in claim 5.


Documents:

076-chenp-2003-abstract.pdf

076-chenp-2003-claims filed.pdf

076-chenp-2003-claims granted.pdf

076-chenp-2003-correspondnece-others.pdf

076-chenp-2003-correspondnece-po.pdf

076-chenp-2003-description(complete) filed.pdf

076-chenp-2003-description(complete) granted.pdf

076-chenp-2003-form 1.pdf

076-chenp-2003-form 26.pdf

076-chenp-2003-form 3.pdf

076-chenp-2003-form 5.pdf

076-chenp-2003-other documents.pdf

076-chenp-2003-pct.pdf


Patent Number 209628
Indian Patent Application Number 76/CHENP/2003
PG Journal Number 50/2007
Publication Date 14-Dec-2007
Grant Date 05-Sep-2007
Date of Filing 13-Jan-2003
Name of Patentee M/S. NERVIANO MEDICAL SCIENCES S R L
Applicant Address VIALE PASTEUR 10, NERVIANO (MI) 20014,
Inventors:
# Inventor's Name Inventor's Address
1 GERONI, Maria, Cristina, Rosa Via Correggio, 48 I-20149 Milano
2 COZZI, Paolo Via Zanella 48/5 I-20133 Milano
3 BERIA, Italo Via S Anna, 16 I-20014 Nerviano
PCT International Classification Number A61K 45/06
PCT International Application Number PCT/EP2001/007064
PCT International Filing date 2001-06-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0015447.6 2000-06-23 U.K.