WO2004035045A1

WO2004035045A1 - Use of substituted acrylolyl distamycin derivatives in combination with demethylating agents, in the treatment of cancer

Info

Publication number: WO2004035045A1
Application number: PCT/EP2003/050700
Authority: WO
Inventors: Maria Cristina Geroni; Camilla Fowst; Massimo Broggini
Original assignee: Pharmacia Italia SpA
Current assignee: Pfizer Italia SRL
Priority date: 2002-10-16
Filing date: 2003-10-07
Publication date: 2004-04-29
Anticipated expiration: 2005-04-16
Also published as: AU2003288263A1

Abstract

Pharmaceutical compositions comprising α-bromo- or α-chloro-acryloyl distamycin derivatives, as defined in the specification, or a Glutathione S-transferase (GST)-activated drug, and a demethylating agent are described; the above formulations may be used for treating tumors having aberrant GSTπ gene expression such as prostate, liver, renal, gastric and breast carcinomas.

Description

USE OF SUBSTITUTED ACRYLOLYL DISTAMYCIN DERIVATIVES IN COMBINATION WITH DEMETHYL ATING AGENTS , IN THE TREATMENT OF CANCER

The present invention provides an antitumor composition comprising a substituted acryloyl distamycin derivative, more particularly an α-bromo- or α-chloro-acryloyl distamycin derivative, or a Glutathione S-transferase (GST)-activated drug, and a pharmacologically active demethylating agent. Distamycin A is an antibiotic substance with antiviral and antiprotozoal activity, having a polypyrrole fiamework [Nature 203: 1064 (1964); J. Med. Chem. 32: 774-778 (1989)]. Distamycin A and several analogues thereof, such as the acryloyl -distamycin derivatives, are known in the art as cytotoxic agents for use in antitumor therapy. For a general reference to aciyloyl-distamycin derivatives see, in particular, the international patent applications WO 90/11277, WO 98/04524, WO 98/21202, WO 99/50265, WO 99/50266 and WO 01/40181, all in the name of the applicant itself and herewith incorporated by reference.

Unlike other well known cytotoxic distamycin derivatives, those bearing an -chloro- or α-bromo-acryloyl moiety resulted to be particularly effective in the treatment of tumors associated with high levels of glutathione (GSH) and/or glutathione-S- transferases (GSTs) family, hence known to be poorly responsive to conventional antitumor therapies and/or to cause resistance once therapeutic cytotoxic agents are administered [see, for a reference, Cancer Res. 62:2332-2336 (2002), and the international patent application WO 01/85144 in the name of the Applicant itself, herewith incorporated by reference] .

Likewise, γ-glutamyl- -amino-β-[[[2-[[bis[bis(2-cMoroemyl)amino]phosphoryl]oxy] e l]sulfenyl]propionyl]-(R)-(-)-phenylglycine, also known as TLK-286, is a Glutathione S-transferase (GST)-activated latent alkylating agent which exhibits as well increased toxicity towards cell lines over expressing GST Pl-1 isozyme [Cancer. Res. 58:2568-2575, (1998)]. For a general reference to GSH and GSTs and to the existing correlation between GSH/GSTs over expression and cancer or cancer response-rate to chemotherapy see, as an example, Cancer Res. 54:4313-4320 (1994); Brit. J. Cancer. 72(2):324-326 (1995); DDT 3:113-121 (1998); Cancer Res. 49:5225-5229 (1989); Clinical Reviews in Biochemistry and Molecular Biology 27(4.5):337-386 (1992).

Although GSH and GSTs are ubiquitously present in several human tissues, including pathologic (tumor) tissues, it is known that among the distinct classes of human GSTs, the π-class (GSTπ) is expressed at higher levels in neoplastic tissues (for instance Head & Neck, gastrointestinal, lung and mammary cancers, sarcomas and leukemias) than in normal tissues [Crit. Rev. Biochem. Mol. Biol.27: 337-348, 1992].

In contrast to this, however, recent findings obtained studying the expression of GSTπ in tumor samples by immunoistochemistry, showed that given malignant transformations such as prostate, liver, renal, and breast carcinomas were associated with loss of expression of GSTπ [Cancer 1997, 79:1595-9; Prostate, 39(3):166-174, 1999; Clinical Cancer Research 8(4):1087-92, 2002; Cancer. Res., 58(20): 4515-4518, 1998].

More in particular, aberrant methylation of GSTπ (CpG island hypermethylation) is the most common somatic genome alteration, described for these tumors, that appears to be responsible for the aforementioned lack of GSTπ expression. In this respect, it has been reported that in a high percentage of human prostate cancers, the levels of GSTπ are negligible because of hypermethylation of the promoter region of GSTπ gene.

This was further confirmed by experimental evidence showing that by treating prostate cancer cells with DNA methyltransferase inhibitors, demethylation and activation of GSTπ gene occurred and, consequently, the intracellular level and activity of the GSTπ protein increased.

In particular, by treating prostate cancer cells with an inhibitor of DNA methyltransferase such as 5'-aza-2'-deoxycytidine, the GSTπ promoter DNA hypermethylation was reversed, GSTπ transcription activated and GSTπ expression restored [American Journal of Pathology, November 2001, Vol. 159, No. 5, 1815-26]. Therefore, prostate cancer cells with GSTπ CpG island hypermethylation and loss of

GSTπ expression seemed to have been selected during human prostatic carcinogenesis.

We have now found that tumors such as prostate, liver, renal, breast and gastric carcinomas which are all associated with aberrant methylation of GSTπ (CpG island hypermethylation), hence loss of GSTπ expression, could be advantageously treated by administering to a patient in need thereof an α-bromo- or α-chloro-acryloyl distamycin derivative, or a Glutathione S-transferase (GST)-activated drug, and a pharmacologically active demethylating agent.

The present invention thus 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 α-bromo- or α-chloro-acryloyl distamycin derivative of formula (I):

wherein R is a bromine or chlorine atom, or a pharmaceutically acceptable salt thereof; or a Glutathione S-transferase (GST)-activated drug; and a pharmacologically active demethylating agent. The present invention includes, within its scope, the pharmaceutical compositions comprising any of 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).

According to a preferred embodiment of the invention, herewith provided are the above pharmaceutical compositions wherein, within the acryloyl distamycin derivative of formula (I), R is a bromine atom.

Pharmaceutically acceptable salts of the compounds of formula (I) are those with pharmaceutically acceptable inorganic or organic acids such as, for instance, hydrochloric, hydrobromic, sul&ric, nitric, acetic, propionic, succinic, malonic, citric, tartaric, methanesulfonic, p-toluenesulfonic acid and the like. Specific examples of acryloyl distamycin derivatives of formula (I), within the compositions object of the invention, for instance in the form of pharmaceutically acceptable salts, preferably with hydrochloric acid, are: N-[5-[[[5-[[[2-[(ammoimmomemyl)amino]ethyl]armno]carbonyl]-l-meth^ 3-yl]amino]carbonyl]- 1 -methyl- lH-pyrrol-3-yl]-4-[[[4-[(2-bromo- 1 -oxo-2- propenyl)amino]- 1 -methyl-lH-pyrrol-2-yl]carbonyl]amino]- 1 -methyl- lH-pyrrole-2- carboxamide hydrochloride (internal code PNU 166196A); and N-[5-[[[5-[[[2-[(aminoiminometbyl)ammo]ethyl]amino]carbonyl]- 1 -methyl- lH-pyrrol- 3-yl]amino]carbonyl]-l-memyl-lH-pyrrol-3-yl]-4-[[[4-[(2-chloro-l-oxo-2- propenyl)amino]- 1 -methyl-lH-pyτrol-2-yl]carbonyl]amino]- 1 -methyl- lH-pyrrole-2- carboxamide hydrochloride.

The above compounds of formula (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.

According to another embodiment of the invention, the Glutathione S-transferase (GST)-activated drug is γ-glutamyl-α-ammo-β-[[[2-[[bis[bis(2-cMoroethyl)amino] phosphoιyl]oxy]ethyl]sulfonyl]propionyl]-(R)-(-)-phenylglycine (TLK-286). Demethylating agents, according to the present invention, are preferably selected from the group consisting of 5'-aza-cytidine, 5'-aza-2'-deoxycytidine, l-(beta-D- riboftLranosyl)-l,2-ά^ydropyrimidin-2-one, L-methionine, inhibitors of bistone deacetylase (HDAC) such as, for instance, valproic acid or trichostatin A, apicidine, hydralazine, procainamide, their admixtures and derivatives thereof. The present invention further provides a product, otherwise referred to as kit of parts, comprising an acryloyl distamycin derivative of formula (I) as defined above or a Glutathione S-transferase (GST)-activated drug, and a pharmacologically active demethylating agent, as a combined preparation for simultaneous, separate or sequential use in antitumor therapy. As formerly indicated, both the pharmaceutical composition or the above kit according to the invention are useful, in therapy, in the treatment of tumors associated with aberrant GSTπ gene such as, prostate, liver, renal, gastric and breast carcinomas . A further aspect of the present invention is to provide a method of treating a mammal, including humans, suffering from a neoplastic disease state presenting aberrant methylation of GSTπ gene, which method comprises administering to said mammal the above acryloyl distamycin derivative of formula (I) or a Glutathione S-transferase (GST)-activated drug, and a pharmacologically active demethylating agent, 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) or a Glutathione S-transferase (GST)-activated drug, and a pharmacologically active demethylating 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.

According to the method of the present invention, the acryloyl distamycin derivative or the Glutathione S-transferase (GST)-activated drug may be administered simultaneously with the demethylating agent or, alternatively, both compounds may be 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) or of the Glutathione S-transferase (GST)-activated drug being used, the particular formulation of the demethylating agent being used, the particular tumor model being treated as well as the particular host being treated. According to a preferred embodiment, however, the course of therapy may comprise a pretreatment with the demethylating agent, for instance a week before the administration of the distamycin derivative of formula (!) or of the Glutathione S- transferase (GST)-activated drug, followed by the administration of one of these two latter agents. The dosage of any active ingredient will depend from several factors, also including the selected schedule of administration which may comprise repeated doses, for instance once a day, once a week, twice a week, and the like, as the case may be. As a non limiting example, suitable dosages of the acryloyl-distamycin derivatives of formula (I) may range from about 0.05 mg/m² 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 any indication concerning suitable pharmaceutical forms for administering the acryloyl-distamycin derivatives in re, hence including any pharmaceutically acceptable excipient, see the international patent application WO 02/28389, in the name of the Applicant itself and herewith incorporated by reference.

For the administration of the Glutathione S-transferase (GST)-activated drug such as TLK-286, dosages may range from about 60 mg/m² to about 1280 mg/m² of body surface area. For the administration of the demethylating agent, according to the method of the invention, the course of therapy generally employed may be as follows.

For the administration of 5'-aza-2'-deoxycytidine, doses varying from about 5 mg day to about 2.000 mg/day and, more preferably, from about 50 to about 400 mg/day. For the administration of valproic acid, doses varying from about 5 mg/day to about 1.000 mg/day and, more preferably, from about 10 to about 250 mg/day. For the administration of procainamide, doses varying from about 100 mg/day to about 10.000 mg/day and, more preferably, from about 500 to about 4.000 mg/day. According to an additional embodiment of the invention, the acryloyl distamycin derivative or the Glutathione S-transferase (GST)-activated drug may be also administered with additional antitumor agents such as, for instance, topoisomerase I or II inhibitors, e.g. CPT-11, topotecan, 9-amino-camptothecin, 9-nitiO-camptothecin, 10,11-methylenedioxy-camptothecin, doxorubicin, daunorubicin, epirubicin, nemorubicin, idarubicin, etoposide, teniposide, mitoxanthrone, losoxantrone, amsacrine, actinomycin D; alkylating agents, e.g. melphalan, chlorambucil, mechlorethamine, cyclophosphamide, ifosfamide, busulfan, carmustine, lormustine, semustine, fotemustine, decarbazine, temozolide, thitepa, mitomycin C, cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin; antimicrotubule agents, e.g. paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbine, estramustine; antimetabolites, e.g. metotrexate, trimetrexate, tomudex, 5-FU, floxuridine, ftorafur, capecitabine, cytarabine, azacitidine, gemcitabine; protein kinase inhibitors, e.g. SΗ571 (Gleevec), ZD-1839 (Iressa), OSI-774 (Tarceva), SU 5416 (Semaxanib), SU 6668, SU 11248; retinoid derivatives, e.g. cis-retinoic acids, trans-retinoic acids; cyclooxygenase inhibitors such as COX-2 inhibitors, e.g. celecoxib, rofecoxib, parecoxib, valdecoxib; hormonal agents, e.g. exemestane, formestane, atamestane, letrozole, fadrozole, anastrozole and radiotherapy. The antitumor therapy of the present invention is particularly suitable for treating prostate, liver, breast and renal cancers in mammals, including humans.

In a further aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of an acryloyl distamycin derivative of formula (I) as defined above or of a Glutathione S-transferase (GST)-activated drug, and an effective amount of a pharmacologically active demethylating 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. Pharmacology

As the effect of the acryloyl distamycin derivative of formula (I) or of the Glutathione S-transferase (GST)-activated drug and of the demethylating agent is significantly increased without a parallel increase of toxicity, the combined therapy of the present invention enhances the antitumoral effects of the single antitumor agents and, hence, provides the most effective and least toxic treatment for the above tumors. As formerly indicated, among the GST isoenzymes family, GSTπ appears to be the strongest activator of distamycin derivatives bearing an α-bromo- or α-chloro-acryloyl moiety, for instance PNU166196A (brostallicin). The cytotoxic activity of brostallicin has been tested against the non GSTπ-expressing human prostate cancer cell line (LNCaP) where the GSTπ promoter is completely methylated.

Brostallicin resulted to be five times less cytotoxic on LNCaP cells compared with the GSTπ-expressing Dul45 human prostate cancer cells with methylated promoter (IC₅₀ 200 and 38 ng/ l, respectively). To verify whether the treatment with agents releasing the expression of GSTπ. such as, for instance, 5'-aza-2'-deoxycytidine, procainamide or HDAC inhibitors, could activate the expression of GSTπ in LNCaP cells and, consequently, increase the antitumor activity of brostallicin, the brostallicin cytotoxicity has been tested in vitro against LNCaP cells being pretreated with 5'-aza-2'-deoxycytidine or procainamide or l-(beta- D-ribofuranosyl)- 1 ,2-dihydropyrimidin-2-one.

Obtained results indicated that a pre-treatment with the demethylating agents, according to a known scheme previously reported to induce hypomethylation of GSTπ promoter, resulted in an increased efficacy of brostallicin compared to untreated LNCaP cells, hence indicating that the association of brostallicin with hypomethylating agents is synergistic in prostate cancer cells.

To better illustrate the present invention, without posing any limitation to it, the following examples are now given.

Example 1 The results obtained testing the cytotoxicity of brostallicin against the two human prostate cancer cells: Dul45 expressing GSTπ with demethylated promoter, and LNCaP (derived from a patient with diagnosis of metastatic prostate carcinoma) that do not express the GSTπ because of hypermethylation at CpG islands in the 5' region of the gene, are reported in Table 1. Cells were incubated with the compound for 1 hour, the growth inhibition was evaluated after 72 hours recovery in drug-free medium. Growth inhibition was determined according to conventional techniques, through SRB colorimetric assay.

Table 1 : Cytotoxicity of brostallicin against human prostate cancer cells

As a result, the over expression of GSTπ is correlated with a higher cytotoxic effect of brostallicin (38 ng/ml and > 200 ng/ml on DU145 and on LNCaP cells, respectively). Example 2

LNCaP cells have been then used for testing the cytotoxicity of brostallicin before and after pretreatment with the demethylating agent 5'-aza-2'-deoxycytidine. Cells were pretreated with 0.05 μM of 5'-aza-2'-deoxycytidine for 6 days, so as to allow efficient demethylation of the CpG islands.

5'-Aza-2'-deoxycytidine inhibited DNA methylation by reducing the biochemical activity of DNA methyltransferase via the formation of a covalent complex with this enzyme; this is believed to deplete methyltransferase activity, hence resulting in DNA demethylation. To determine whether this activation was associated with a change in the methylation pattern of the GSTπ promoter, DNA was isolated from drug-treated LNCaP cells and tested according to conventional methods by MS-PCR.

5'-Aza-2'deoxycytidine treatment resulted in a partial demethylation of the GSTπ promoter. Table 2 reports data obtained testing the cytotoxic effect of brostallicin on LNCaP cells pretreated with 5'-aza-2'deoxycytidine. Cells were incubated with the compound for lh then the growth inhibition was evaluated after 72 hours recovery in a drug-free medium. Growth inhibition was determined according to conventional techniques by SRB colorimetric assay.

Table 2: cytotoxicity of brostallicin against human prostate LNCaP cancer cells pretreated with 5'-aza-2'-deoxycytidine.

As a result, the pretreatment with 5^,-aza-2'-deoxycytidine increased at least ten times the cytotoxicity of brostallicin, hence indicating a synergistic effect of this combination against tumors presenting methylation of the GSTπ gene.

Example 3 Similar results have been obtained with the human breast cancer cell line (MCF-7). This tumor model does not express the GSTπ because of hypermethylation at CpG islands in the 5' region of the gene. Hypomethylating treatment with 5'-aza-2'-deoxycytidine (5- AZA) was performed for one week before adding brostallicin to cell cultures. MTT colorimetric assay has been used for the evaluation of cell growth inhibition of brostallicin in untreated or pretreated cells. GSTπ activity increased by two-fold in cell cultures treated with 5'-aza-2'-deoxycytidine in respect to untreated ones. The cytotoxic effect of brostallicin was higher in tumor cells pretreated with 5-AZA [Figure 1: Cytotoxic effect of brostallicin on MCF-7 cells pretreated with 5'-aza-2'- deoxycytidine (5-AZA)] .

Example 4 LNCaP cells have been then used for testing the cytotoxicity of brostallicin before and after pretreatment with the demethylating agent l-(beta-D-ribofuranosyl)-l,2- dihydropyrimidin-2-one (ZEB). Figure 2 shows the dose-dependent effect of ZEB pretreatment on the cytotoxic effect of brostallicin against LNCaP cells. Cells were incubated with different concentrations of ZEB (50, 75, 100, 125μM) for 96 hours and then with brostallicin. Cell growth inhibition was evaluated after 96 hours co-treatment according to conventional techniques (SRB colorimetric assay). Figure 2: Cytotoxic effect of brostallicin on LNCaP cells pretreated with l-(beta-D- ribofuranosyl)- 1 ,2-dihydropyrimidin-2-one (ZEB).

As a result, the pretreatment with l- eta-D-π^ofuranosyl)-l,2-ά ydropyrimidin-2-one increased in a dose-dependent manner the cytotoxicity of brostallicin, hence indicating a synergistic effect of this combination against tumors presenting hypermethylation of the GSTπ gene.

Claims

1) A composition comprising a pharmaceutically acceptable carrier or excipient; an α-bromo- or α-chloro-acryloyl distamycin derivative of formula (I):

wherein R is a bromine or chlorine atom, or a pharmaceutically acceptable salt thereof; or a Glutathione S-transferase (GST)-activated drug; and a pharmacologically active demethylating agent. 2) The composition of claim 1 wherein, within the compounds of formula (I), R is a bromine atom. 3) The composition of claim 1 wherein the compound of formula (I), optionally in the form of a pharmaceutically acceptable salt, is selected from the group consisting of: N-[5-[[[5-[[[2-[(arninoim omethyl)ammo]ethyl]am o]carbonyl]-l-memyl-lH-py^ 3-yl]amino]carbonyl]-l-methyl-lH-pyrrol-3-yl]-4-[[[4-[(2-bromo-l-oxo-2- propenyl)amino]- 1 -methyl- 1 H-pyrrol-2-yl]carbonyl]amino] - 1 -methyl- lH-pyrrole-2- carboxamide; and

N-[5-[[[5-[[[2-[(aminoimmomemyl)ammo]ethyl]amino]carbonyl]- 1 -methyl- lH-pyrrol-

3-yl]ammo]carbonyl]-l-methyl-lH-pyrrol-3-yl]-4-[[[4-[(2-chloro-l-oxo-2- propenyl)ammo]-l-methyl-lH-pyττol-2-yl]carbonyl] carboxamide. 4) The composition according to claim 3 wherein the compound of formula (I) is N-[5- [[[5-[[[2-[(ammoiminomemyl)ammo]ethyl]amino]carbonyl]-l-methyl-lH-pyrrol-3- yl]amino]carbonyl]- 1 -methyl- lH-pyrrol-3-yl]-4-[[[4-[(2-bromo- l-oxo-2- propenyl)amino]-l-methyl-lH-pyrrol-2-yl]carbonyl]ammo]-l-methyl-lH-pyrrole-2- carboxamide hydrochloride. 5) The composition according to claim 1 wherein the Glutathione S-transferase (GST)- activated drug is γ-glutamyl-α-arnmo-β-[[[2-[| is|bis(2-cl3loroe l)amino] phosphoryl]oxy]e l]sulfonyl]propionyl]-(R)-(-)-phenylglycine ILK-286). 6) The composition according to claim 1 wherein the demethylating agent is selected from the group consisting of 5'-aza-cytidine, 5'-aza-2 -deoxycytidine, l-(beta-D- ribofuranosyl)-l,2-dihydropyrimidin-2-one, L-methionine, valproic acid, trichostatin A, apicidine, hydralazine, procainamide, or admixtures thereof.

7) A composition comprising a pharmaceutically acceptable carrier or excipient;

N-[5-[[[5-[[[2-[(arninoimmome yl)ammo]etbyl]ammo]carbonyl]-l-me1byl-l^ pyrrol-3-yl]arnino]carbonyl]-l-methyl-lH-pyrrol-3-yl]-4-[[[4-[(2-bromo-l-oxo-

2-propenyl)amino] - 1 -methyl- 1 H-pyrrol-2-yl]carbonyl]amino] - 1 -methyl- 1H- pyrrole-2-carboxamide hydrochloride of formula (I):

and a pharmacologically active demethylating agent selected from 5'-aza-cytidine, 5'- aza-2'-deoxycytidine or l- eta-D-ribofuranosyl)-l,2-dmydropyrimidin-2-one.

8) A composition as defined in claim 1 for use as a medicament.

9) Use of a composition as defined in claim 1 in the manufacture of a medicament for treating tumors associated with aberrant GSTπ gene expression.

10) The use according to claim 9 wherein the tumor is selected from prostate, liver, renal, gastric and breast carcinomas.

11) A product or kit of parts comprising an acryloyl distamycin derivative of formula (I), as defined in claim 1, or a Glutathione S-transferase (GST)-activated drug, and a pharmacologically active demethylating agent, as a combined preparation for simultaneous, separate or sequential use in antitumor therapy.

12) A method of treating a mammal, including humans, suffering from a neoplastic disease state presenting aberrant methylation of GSTπ gene, which method comprises administering to said mammal an acryloyl distamycin derivative of formula (I), as defined in claim 1, or of a Glutathione S-transferase (GST)-activated drug, and a pharmacologically active demethylating agent, in amounts effective to produce a synergistic antineoplastic effect.

13) The method of claim 12 wherein the neoplastic disease state is selected from prostate, liver, renal, gastric and breast carcinomas. 14) The method of claim 12 comprising subjecting the mammal in need thereof with the demethylating agent first and, subsequently, with the acryloyl distamycin derivative of formula (I), as defined in claim 1, or with the Glutathione S-transferase (GST)-activated drug.

15) The method of claim 14 wherein the demethylating agent is administered a week before the administration of the acryloyl distamycin derivative of formula (I), as defined in claim 1, or the Glutathione S-transferase (GST)-activated drug.