Indian Patents. 212756:A PHARMACEUTICAL COMPOSITION COMPRISING A PHARMACEUTICAL CARRIER OR EXCIPIENT AND AS ACTIVE INGREDIENT AN ACRYLOYL DISTAMYCIN DERIVATIVE

Title of Invention	A PHARMACEUTICAL COMPOSITION COMPRISING A PHARMACEUTICAL CARRIER OR EXCIPIENT AND AS ACTIVE INGREDIENT AN ACRYLOYL DISTAMYCIN DERIVATIVE
Abstract	The present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and, as active ingredient, an acryloyl distamycin derivative of formula (I): wherein: Rl is a bromine or chlorine atom; R2 is a distamycin or distamycin-like framework or a pharmaceutically acceptable salt thereof; and - an antimicrotubule agent and/or an antimetabolite.

Full Text	COMBINED THERAPY AGAINST TUMORS COMPRISING SUBSTITUTED ACRYLOYL DISTAMYCXN DERIVATIVES, TAXANES AND/OR ANTIMETABOLITES The present invention relates to the field of cancer treatment and provides an antitumor composition comprising a substituted aciyloyl distaMycin derivative, more particularly an α-bromo- or α-chloro-acryloyl distamycin derivative, an antimicrotubule agent and/or an antimetabolite, 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. Distamycm. A is an antibiotic substance wilh antiviral and antiprotozoal activity, having a polypyrrole fiamework [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 from 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 distamycm. is optionally replaced by nitrogen-contaiuing eading groups such as, for instance, cyanamidino, N-methylamidino, guanidino, carbamoyl, amidoxime, cyano and the like, and/or wherein the polypyrrole framework of distamycin, or part of it, is replaced by varying carbocyclic or heterocyclic moieties. The present invention provides, in a first aspect, a pharmaceutical composition for use in antineoplastic therapy in mammals, including humans, comprising a pharmaceutically acceptable carrier or excipient; an acryloyi 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 antimicrotabule agent and/or an antimetabolite. 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-drags) of the compounds of formula (I). In the present description, unless olherwise 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 Antimicrotubule agents and antimetabolites are widely known in the art as antitumor agents; see, for a general reference. Cancer, Principles and Practice of Oncology, lippincott-Raven Ed. (1997), 432-452 and 467-483 According to a preferred embodiment of the invention, herewith provided are the above pharmaceutical compositions wherein the antimicrotubule agents are, for instance, taxanes, e.g. paclitaxel or docetaxel; vinca alkaloids, e.g. vincristine, vinblastine, vindesine, vinorelbine; and estramustine, optionally encapsulated within liposome. Preferred antimnetabolites are, for instance, antifolates, e.g. metotrexate, trimetrexate, tomudex; 5-fluoropyrimidines, e.g, 5-FU, floxuridine, florafur and capecitabine; cytidine analogs, e.g, cytarabine, azadtidine and gemdtabine. Particularly preferred antimicrotubule agents are paclitaxel and estramustine whereas preferred antimetabolites are 5-fluorouracil or gemcitabine. 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 (II) below: whereru m is an integer from 0 to 2; n is an integer from 2 to 5; r is O or l;. 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 (III) below: 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 In the present description, unless otherwise specified, with the term C1-C4 alkyl or alkoxy group we intend a straight or branched group selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobniyl, sec-butyl, tert-butyl, methoxy, ethoxy, nrpropoxy, isopropoxy, n-butoxy, isobutoxy, seobutoxy 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 (D) 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) wherem 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, X and Y are both CH groups and B is selected from: wherein R4 is cyano or hydroxy and R5, R6, and R7, the same or different, are hydrogen or CrC4 alkyl. Pharmaceutically acceptable salts of the compounds of formula (J) are those with pharmaceutically acceptable inorganic or organic adds such as, for instance, hydrochloric, hydrobromic, sulfuric, nitric, acetic, propionic, succinic, malonic, citric, tartaric, methanesulfonic, p-toluenesulfonic acid and the like. Examples of preferred acryloyl distamycin derivatives of formula (I), within the compositions object of the invention, optionally in the form of pharmaceutically acceptable salts, preferably with hydrochloric acid, are: 1. N-(5"{[(5-{{(5-{{(2-{[amino(iniino)methyl]amino}ethyl)amino]carbon methyl"lH-pyrrol-3-yl)amino]carbonyl}-l-methyl-ip-pyrrol-3- The above compounds of formula (T), 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, m the aforementioned international patent applications WO 90/11277, WO 98/04524, WO 98/21202, WO 99/50265 and WO 99/50266 and WO 01/40181. The present invention further provides a product comprising an acryloyl distamycin derivative of formula (I), as defined above, an antimicrotubule agent and/or an antimetabolite, as a combined preparation for simultaneous, separate or sequential use in antitumor therapy. A further aspect of the present invention is to provide a method of treating 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), an antimicrotubule agent and/or an antimetabolite, 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 compxising administering to said mammal a combined preparation comprising an acryloyl distamycin derivative of formula (I), an antimicrotubule agent and/or an antimetabolite, 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), an antimicrotubule agent and/or an antimetabolite 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 antimicrotubule agent or with the antimetabolite. Alternatively, the two drugs may be administered sequentially in either order. When the acryloyl distamycin derivative is administered with both the antimicrotubule agent and the antimetabolite, according to an embodiment of the invention, the drugs are preferably administered sequentially, in any order. In this respect, it will be appreciated that the actual preferred method and order of administration will vary according to, inter alias, the particular formulation of the acryloyl distamycin of formula Q) being used, the particular formulation of the antimicrotubule agent and/or the antimetabolite being used, theparticular tumor model being treated as well as the particular host being treated. To administer the acryloyl distamycin derivative of formula (I), 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 taxanes, according to the method of the invention, the course of therapy generally employed comprises doses varying from about 1 to about 1000 mg/m2 of body surface area and, more preferably, from about 10 to about 500 mg/m2 of body surface area. For the administration of the vinca alkaloids, according to the method of the invention, the course of therapy generally employed comprises doses varying from about 0,1 to about 1000 mg/m2 of body surface area and, more preferably, from about 0.5 to about 100 mg/m2 of body surfece area. For the administration of the antimetabolite according to the invention, the course of therapy generally employed comprises doses varying from about 0.1 to about 10 g/m2 of body surface area and, more preferably, from about 1 to about 5 g/m2 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 a composition comprising an effective amount of an acryloyl distamycin derivative of formula (I), as defined above, an antimicrotubule agent and/or an antimetabolite, 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 (1) with an antimicrotubule agent and/or an antimetabolite, 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 other drug, being either an antimicrotubule, an antimetabolite or a combination thereof and, hence, provides the most effective and least toxic treatment for tumors. The superadditive effects of the combined preparations of the invention are shown, for instance, by the following in vivo antitumor activity data which are intended to illustrate the present invention without posing any limitation to it. L » At the dose of 15 mg/kg of gemcitabine alone (day +1 after tumor injection, 2 h after PNU166196 administration) and at the dose of 0-7 mg/kg of PNU 166196 alone (days +1,6) were associated, without toxicity, ]LS% values of 50 and 58, respectively. Combining gemcitabine and PNU 166196 at the same doses with the same schedule, an increase of activity with ILS% values of 127 were observed, thus indicating a synergistic effect Table 1: Antileukemic activity against disseminated L1210^ murine leukemia of an acryloyl distamycin derivative (1) in combination with gemcitabine. 1) L1210 leukemia cells (105/mouse) are injected iv on day 0. 2) Treatment is given starting on day 1 after tumor transplantation (day 0), 3) Increase in life span: [(median survival time of treated micromedian survival time of controls)x 100]-100 4) Number of toxic deaths/number of mice. () treatment 2 h after PNU 166196 administration. We CLAIMS 1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and, as active ingredient, an acryloyl distamycin derivative of formula (I): wherein: R1i is a bromine or chlorine atom; R2 is a distamycin or distamycin-like framework; or a pharmaceutically acceptable salt thereof; and an antumicrotubule agent and/or an antimetabolite. 2. A pharmaceutical composition according to claim 1 wherein the antimicrotubule agent is selected from taxanes, including paclitaxel and docetaxel; vinca alkaloids, including vincristine, vinblastine, vindesine, vinorelbine; and estramustine, optionally encq)sulated within liposomes. 3. A pharmaceutical composition according to claim 2 wherein the antimicrotubule agent is paclitaxel or estramustine. 4. A pharmaceutical composition according to claim 1 wherein the antimetabolite is selected from antifolates, including methotrexate, tomudex and trimetrexate; 5-fluoropyrimidine derivatives, including 5-fluorouracil, floxuridine, ftorafur and capedtabine; and cytidine analogs, including cyterabine, azacitidine and gemcitabine. 5. A pharmaceutical composition according to claim 4 wherein the antimetabolite is selected from 5-fluorouracil or gemcitabine. 6. A pharmaceutical composition according to claim 1 comprising an acryloyl distamycin derivative of formula (I) wherein. m is an integer from 0 to 2; n is an integer from 2 to 5; risOorl; 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 (ED) below: wherein Q is anitrogen 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 wherein R4 is cyano, amino, hydroxy or C1-C4 alkoxy; R5, R6 and R7, the same or different, are hydrogen or C1-C4 alkyl 7. A pharmaceutical composition according to claim 6 comprising an acryloyl distamycin derivative of formula (I) wherein R1, R2 and B are as defined in claim 6, r is 0,m is O or 1 andn is 4 8. A pharmaceutical composition according to claim 7 comprising an acryloyl distamycin derivative of formula (I) wherein R1 and R2 are as defored in claim 6, r is 0, mis0orl,n is 4,X and Y are both CH groups and B is selected from: wherein R4 is cyano or hydroxy and R5, R6 and R7, the same or different, are hydrogen or C1-C4 alkyl, 9. A pharmaceutical composition according to claim 1 comprising an acryloyl distamydn derivative, optionally in the form of a pharmaceutically acceptable salt, selected from the group consisting of: wherein: R1 is a bromine or chlorine atom; R2 is a distaznycin or distamycin-like framework; or a pharmaceutically acceptable salt thereof an antimicrotubule ageat and/or an antimetabolite, as a combined preparation for simultaneons, separate or sequential use in the treatment of tumors. 11. Products according to claim 10 wherein the antimicrotubule agent is selected from taxanes, including paclitaxel and docetaxel; vinca alkaloids, including vincristine, vinblastrue, vindesine, vinorelbine; and estramustine, optionally encapsulated within liposomes. 12. Products according to claim 11 wherein the antimicrotubule agent is paclitaxel or estramustine. 13. Products according to claim 10 wherein the antimetabolite is selected from antifolates, including methotrexate, tomudex and trimetrexate; 5-fluoropyrimidine derivatives, including 5-fluorouracil, floxuridine, ftorafur and capecitabine; and cytidine analogs, including cytarabine, azacitidine and gemdtabine, 14. Products according to claim 13 wherein the antimetabolite is selected from 5- fluorouracil or gemdtabine. 15. Products according to claim 10 comprising an acryloyl distamycin derivative of formula (I) wherein m is an integer from 0 to 2; n is an integer from 2 to 5; r is O or l; 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 fiom 1 to 3 heteroatoms selected among N, O or S, or it is a group of formula (III) below: 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 16. Products according to claim 10 wherein the acryloyl distamycin derivative is selected from the group as defined in claim 9. 17. Use of an acryloyl distamycin derivative of formula (I) , as defined in claim 1 or in any one of claims from 6 to 9 in the preparation of a medicament for use in combination therapy with an and microtubule agent and/or an antimetabolite in the treatment of tumors. 18. Use according to claim 17 wherein the medicament further comprises the said antimicrotubule agent and/or antimetabolite. 19. Use according to claim 17 or 18 wherein the acryloyl distamydn derivative is selected Scorn the group as defined in claim 9. 20. Use according to any one of claims 17 to 19 wherein the tumor is selected from breast, ovary, lung, colon, kidney, stomach, pancreas, liver, melanoma, leukemia and brain tumors. 21. Use of an acryloyl distamycin derivative of formula (I), as defined in claim 1 or in any one of claims form 6 to 9 in the preparation, of a medicament for use in combination therapy with an antimicrotubule agent and/or an antimetabolite in the prevention or treatment of metastasis or in the treatment of tumors by inhibition of angiogenesis. 22. Use according to claim 21 wherein the medicament further comprises the said antimicrotabule agent and/or antimetabolite. 23. A method of treating a mammal, including humans, suffering from a neoplastic disease state, which method compiises administering to said mammal the acryloyl distamycin derivative of formula (I), as defined in claim 1 or any one of claims 6 to 9, an antimicrotubule agent and/or.an antimetabolite, in amounts effective to produce a synergistic antineoplastic effect 24. A method for lowering the side effects caused by antineoplastic therapy with an antineoplastic agent, in a mammal in need thereof including hmnans, the method comprising administering to said mammal a combined preparation comprising an antimicrotubule agent and/or an antimetabolite with an acryloyl distamycin derivative of formula (I), as defined in claim 1 or any one of claims from 6 to 7, in amounts effective to produce a synergistic antineoplastic effect 26, A pharmaceutical composition, substantially as hereinabove described and exemplified.

Full Text

COMBINED THERAPY AGAINST TUMORS COMPRISING SUBSTITUTED ACRYLOYL DISTAMYCXN DERIVATIVES, TAXANES AND/OR ANTIMETABOLITES
The present invention relates to the field of cancer treatment and provides an antitumor composition comprising a substituted aciyloyl distaMycin derivative, more particularly an α-bromo- or α-chloro-acryloyl distamycin derivative, an antimicrotubule agent and/or an antimetabolite, 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.
Distamycm. A is an antibiotic substance wilh antiviral and antiprotozoal activity, having a polypyrrole fiamework [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 from 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 distamycm. is optionally replaced by nitrogen-contaiuing eading groups such as, for instance, cyanamidino, N-methylamidino, guanidino, carbamoyl, amidoxime, cyano and the like, and/or wherein the polypyrrole framework of distamycin, or part of it, is replaced by varying carbocyclic or heterocyclic moieties.
The present invention provides, in a first aspect, a pharmaceutical composition for use in antineoplastic therapy in mammals, including humans, comprising a pharmaceutically acceptable carrier or excipient;
an acryloyi 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 antimicrotabule agent and/or an antimetabolite.
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-drags) of the compounds of formula (I).
In the present description, unless olherwise 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
Antimicrotubule agents and antimetabolites are widely known in the art as antitumor agents; see, for a general reference. Cancer, Principles and Practice of Oncology, lippincott-Raven Ed. (1997), 432-452 and 467-483
According to a preferred embodiment of the invention, herewith provided are the
above pharmaceutical compositions wherein the antimicrotubule agents are, for
instance, taxanes, e.g. paclitaxel or docetaxel; vinca alkaloids, e.g. vincristine,
vinblastine, vindesine, vinorelbine; and estramustine, optionally encapsulated within
liposome.
Preferred antimnetabolites are, for instance, antifolates, e.g. metotrexate, trimetrexate,
tomudex; 5-fluoropyrimidines, e.g, 5-FU, floxuridine, florafur and capecitabine;
cytidine analogs, e.g, cytarabine, azadtidine and gemdtabine.
Particularly preferred antimicrotubule agents are paclitaxel and estramustine whereas
preferred antimetabolites are 5-fluorouracil or gemcitabine.
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 (II) below:

whereru
m is an integer from 0 to 2;
n is an integer from 2 to 5;
r is O or l;.
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 (III) below:

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

In the present description, unless otherwise specified, with the term C1-C4 alkyl or alkoxy group we intend a straight or branched group selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobniyl, sec-butyl, tert-butyl, methoxy, ethoxy, nrpropoxy, isopropoxy, n-butoxy, isobutoxy, seobutoxy 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 (D) 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) wherem 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, X and Y are both CH groups and B is selected from:

wherein R4 is cyano or hydroxy and R5, R6, and R7, the same or different, are hydrogen or CrC4 alkyl.
Pharmaceutically acceptable salts of the compounds of formula (J) are those with pharmaceutically acceptable inorganic or organic adds such as, for instance, hydrochloric, hydrobromic, sulfuric, nitric, acetic, propionic, succinic, malonic, citric, tartaric, methanesulfonic, p-toluenesulfonic acid and the like.
Examples of preferred acryloyl distamycin derivatives of formula (I), within the compositions object of the invention, optionally in the form of pharmaceutically acceptable salts, preferably with hydrochloric acid, are: 1. N-(5"{[(5-{{(5-{{(2-{[amino(iniino)methyl]amino}ethyl)amino]carbon methyl"lH-pyrrol-3-yl)amino]carbonyl}-l-methyl-ip-pyrrol-3-

The above compounds of formula (T), 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, m the aforementioned international patent applications WO 90/11277, WO 98/04524, WO 98/21202, WO 99/50265 and WO 99/50266 and WO 01/40181.
The present invention further provides a product comprising an acryloyl distamycin derivative of formula (I), as defined above, an antimicrotubule agent and/or an antimetabolite, as a combined preparation for simultaneous, separate or sequential use in antitumor therapy.

A further aspect of the present invention is to provide a method of treating 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), an antimicrotubule agent and/or an antimetabolite, 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 compxising administering to said mammal a combined preparation comprising an acryloyl distamycin derivative of formula (I), an antimicrotubule agent and/or an antimetabolite, 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), an antimicrotubule agent and/or an antimetabolite 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 antimicrotubule agent or with the antimetabolite.
Alternatively, the two drugs may be administered sequentially in either order.
When the acryloyl distamycin derivative is administered with both the antimicrotubule
agent and the antimetabolite, according to an embodiment of the invention, the drugs
are preferably administered sequentially, in any order.
In this respect, it will be appreciated that the actual preferred method and order of
administration will vary according to, inter alias, the particular formulation of the
acryloyl distamycin of formula Q) being used, the particular formulation of the
antimicrotubule agent and/or the antimetabolite being used, theparticular tumor model
being treated as well as the particular host being treated.
To administer the acryloyl distamycin derivative of formula (I), 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 taxanes, according to the method of the invention, the
course of therapy generally employed comprises doses varying from about 1 to about

1000 mg/m2 of body surface area and, more preferably, from about 10 to about 500
mg/m2 of body surface area.
For the administration of the vinca alkaloids, according to the method of the invention,
the course of therapy generally employed comprises doses varying from about 0,1 to
about 1000 mg/m2 of body surface area and, more preferably, from about 0.5 to about
100 mg/m2 of body surfece area.
For the administration of the antimetabolite according to the invention, the course of
therapy generally employed comprises doses varying from about 0.1 to about 10 g/m2
of body surface area and, more preferably, from about 1 to about 5 g/m2 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 a composition comprising an effective amount of an acryloyl distamycin derivative of formula (I), as defined above, an antimicrotubule agent and/or an antimetabolite, 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 (1) with an antimicrotubule agent and/or an antimetabolite, 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 other drug, being either an antimicrotubule, an antimetabolite or a combination thereof and, hence, provides the most effective and least toxic treatment for tumors.
The superadditive effects of the combined preparations of the invention are shown, for instance, by the following in vivo antitumor activity data which are intended to illustrate the present invention without posing any limitation to it.

L »
At the dose of 15 mg/kg of gemcitabine alone (day +1 after tumor injection, 2 h after PNU166196 administration) and at the dose of 0-7 mg/kg of PNU 166196 alone (days +1,6) were associated, without toxicity, ]LS% values of 50 and 58, respectively. Combining gemcitabine and PNU 166196 at the same doses with the same schedule, an increase of activity with ILS% values of 127 were observed, thus indicating a synergistic effect
Table 1: Antileukemic activity against disseminated L1210^ murine leukemia of an acryloyl distamycin derivative (1) in combination with gemcitabine.

1) L1210 leukemia cells (105/mouse) are injected iv on day 0.
2) Treatment is given starting on day 1 after tumor transplantation (day 0),
3) Increase in life span: [(median survival time of treated micromedian survival time of controls)x 100]-100
4) Number of toxic deaths/number of mice.
(*) treatment 2 h after PNU 166196 administration.

We CLAIMS
1. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier or excipient and, as active ingredient,
an acryloyl distamycin derivative of formula (I):
wherein:
R1i is a bromine or chlorine atom;
R2 is a distamycin or distamycin-like framework; or a pharmaceutically acceptable salt
thereof; and
an antumicrotubule agent and/or an antimetabolite.
2. A pharmaceutical composition according to claim 1 wherein the antimicrotubule agent is selected from taxanes, including paclitaxel and docetaxel; vinca alkaloids, including vincristine, vinblastine, vindesine, vinorelbine; and estramustine, optionally encq)sulated within liposomes.
3. A pharmaceutical composition according to claim 2 wherein the antimicrotubule agent is paclitaxel or estramustine.
4. A pharmaceutical composition according to claim 1 wherein the antimetabolite is selected from antifolates, including methotrexate, tomudex and trimetrexate; 5-fluoropyrimidine derivatives, including 5-fluorouracil, floxuridine, ftorafur and capedtabine; and cytidine analogs, including cyterabine, azacitidine and gemcitabine.
5. A pharmaceutical composition according to claim 4 wherein the antimetabolite
is selected from 5-fluorouracil or gemcitabine.

6. A pharmaceutical composition according to claim 1 comprising an acryloyl distamycin derivative of formula (I)

wherein.
m is an integer from 0 to 2;
n is an integer from 2 to 5;
risOorl;
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 (ED) below:

wherein Q is anitrogen 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

wherein R4 is cyano, amino, hydroxy or C1-C4 alkoxy; R5, R6 and R7, the same or different, are hydrogen or C1-C4 alkyl
7. A pharmaceutical composition according to claim 6 comprising an acryloyl
distamycin derivative of formula (I) wherein R1, R2 and B are as defined in claim 6, r is
0,m is O or 1 andn is 4
8. A pharmaceutical composition according to claim 7 comprising an acryloyl
distamycin derivative of formula (I) wherein R1 and R2 are as defored in claim 6, r is 0,
mis0orl,n is 4,X and Y are both CH groups and B is selected from:

wherein R4 is cyano or hydroxy and R5, R6 and R7, the same or different, are hydrogen or C1-C4 alkyl,
9. A pharmaceutical composition according to claim 1 comprising an acryloyl
distamydn derivative, optionally in the form of a pharmaceutically acceptable salt,
selected from the group consisting of:

wherein:
R1 is a bromine or chlorine atom;
R2 is a distaznycin or distamycin-like framework; or a pharmaceutically acceptable salt
thereof an antimicrotubule ageat and/or an antimetabolite, as a combined preparation
for simultaneons, separate or sequential use in the treatment of tumors.
11. Products according to claim 10 wherein the antimicrotubule agent is selected
from taxanes, including paclitaxel and docetaxel; vinca alkaloids, including vincristine,
vinblastrue, vindesine, vinorelbine; and estramustine, optionally encapsulated within
liposomes.
12. Products according to claim 11 wherein the antimicrotubule agent is paclitaxel
or estramustine.
*
13. Products according to claim 10 wherein the antimetabolite is selected from
antifolates, including methotrexate, tomudex and trimetrexate; 5-fluoropyrimidine
derivatives, including 5-fluorouracil, floxuridine, ftorafur and capecitabine; and
cytidine analogs, including cytarabine, azacitidine and gemdtabine,
14. Products according to claim 13 wherein the antimetabolite is selected from 5-
fluorouracil or gemdtabine.

15. Products according to claim 10 comprising an acryloyl distamycin derivative of formula (I)

wherein
m is an integer from 0 to 2;
n is an integer from 2 to 5;
r is O or l;
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 fiom 1
to 3 heteroatoms selected among N, O or S, or it is a group of formula (III) below:

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

16. Products according to claim 10 wherein the acryloyl distamycin derivative is
selected from the group as defined in claim 9.
17. Use of an acryloyl distamycin derivative of formula (I) , as defined in claim 1 or
in any one of claims from 6 to 9 in the preparation of a medicament for use in
combination therapy with an and microtubule agent and/or an antimetabolite in the
treatment of tumors.
18. Use according to claim 17 wherein the medicament further comprises the said
antimicrotubule agent and/or antimetabolite.
19. Use according to claim 17 or 18 wherein the acryloyl distamydn derivative is
selected Scorn the group as defined in claim 9.
20. Use according to any one of claims 17 to 19 wherein the tumor is selected from
breast, ovary, lung, colon, kidney, stomach, pancreas, liver, melanoma, leukemia and
brain tumors.
21. Use of an acryloyl distamycin derivative of formula (I), as defined in claim 1 or
in any one of claims form 6 to 9 in the preparation, of a medicament for use in
combination therapy with an antimicrotubule agent and/or an antimetabolite in the

prevention or treatment of metastasis or in the treatment of tumors by inhibition of angiogenesis.
22. Use according to claim 21 wherein the medicament further comprises the said
antimicrotabule agent and/or antimetabolite.
23. A method of treating a mammal, including humans, suffering from a neoplastic
disease state, which method compiises administering to said mammal the acryloyl
distamycin derivative of formula (I), as defined in claim 1 or any one of claims 6 to 9,
an antimicrotubule agent and/or.an antimetabolite, in amounts effective to produce a
synergistic antineoplastic effect
24. A method for lowering the side effects caused by antineoplastic therapy with an
antineoplastic agent, in a mammal in need thereof including hmnans, the method
comprising administering to said mammal a combined preparation comprising an
antimicrotubule agent and/or an antimetabolite with an acryloyl distamycin derivative
of formula (I), as defined in claim 1 or any one of claims from 6 to 7, in amounts
effective to produce a synergistic antineoplastic effect

26, A pharmaceutical composition, substantially as hereinabove described and exemplified.

Documents:

078-chenp-2003-abstract.pdf

078-chenp-2003-assignement.pdf

078-chenp-2003-claims filed.pdf

078-chenp-2003-claims granted.pdf

078-chenp-2003-correspondnece-others.pdf

078-chenp-2003-correspondnece-po.pdf

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

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

078-chenp-2003-form 1.pdf

078-chenp-2003-form 26.pdf

078-chenp-2003-form 3.pdf

078-chenp-2003-form 5.pdf

078-chenp-2003-other documents.pdf

078-chenp-2003-pct.pdf

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Patent Number

212756

Indian Patent Application Number

78/CHENP/2003

PG Journal Number

07/2008

Publication Date

15-Feb-2008

Grant Date

14-Dec-2007

Date of Filing

13-Jan-2003

Name of Patentee

NERVIANO MEDICAL SCIENCES SRL

Applicant Address

Viale Pasteur 10, Nerviano (MI) Italy 20014

Inventors:

#	Inventor's Name	Inventor's Address
1	BERIA, Italo	Via S. Anna, 16 I-20014 Nerviano
2	GERONI, Maria, Cristina, Rosa	Via Correggio 48 I-20149 Milano
3	COZZI, Paolo	Via Zanella 48/5 I-20133 Milano

PCT International Classification Number

A16K 38/00

PCT International Application Number

PCT/EP2001/007060

PCT International Filing date

2001-06-20

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	0015446.8	2000-06-23	U.K.