ES2358787T3 - COMPOSITIONS FOR ANTITUMORAL TREATMENT CONTAINING ECTEINASCIDINE 743. - Google Patents

Abstract

The use of a synergistic combination of ET-743 and another drug selected from an anthracyclinic drug, a platinum drug, a drug that targets a topoisomerase, a taxane group drug, an antimetabolite drug or an oral antifungal drug in the preparation of a medication for the treatment of a tumor.

Description

Compositions for antitumor treatment containing ecteinascidin 743 The present invention relates to effective antitumor treatments. Ecteinascidin 743, ET-743, is an anticancer agent from a marine source. BACKGROUND OF THE INVENTION The reader is sent to document WO0069441 published on November 23, 2000 for information on the

Compositions and uses of ET-743 to treat cancer considered in accordance with Art.54 (3) EPC SUMMARY OF THE INVENTION In accordance with one aspect of this invention, its authors provide effective combination therapies based on

ecteinascidin 743, using other drugs. The object of the invention is defined in the claims. PREFERRED EMBODIMENTS The other drugs may be part of the same composition or be provided as a composition.

separate for administration at the same time or at a different time. The identity of the other drug is not

Particularly limited and suitable candidates include: a) drugs with antimitotic effects, especially those whose target cytoskeletal elements, including microtubule modulators, such as taxane group drugs (such as taxol, paclitaxel, taxotero, docetaxel), podophyllotoxins or alkaloids of vinca (vincristine, vinblastine);

b) antimetabolite drugs, such as 5-fluorouracil-cytarabine, gemcitabine, purine analogues, such as pentostatin, methotrexate);

c) alkylating agents, such as nitrogen mustards (such as cyclophosphamide or ifosfamide); d) drugs that target DNA, such as anthracyclinic drugs, adriamycin, doxorubicin, farmorubicin or epirubicin;

e) drugs having topoisomerase targets, such as etoposide;

f) hormones and hormone agonists or antagonists, such as estrogens, antiestrogens (tamoxifen and related compounds) and androgens, flutamide, leuprorelin, goserelin, cyprotron or octreotide; g) drugs that target the transduction of signals in tumor cells including derivatives of

antibodies, such as herceptin;

h) alkylating agents, such as platinum drugs (cis-platinum, carboplatin, oxaliplatin, paraplatin) or nitrosoureas; i) drugs that potentially affect tumor metastasis, such as metalloproteinase inhibitors

of matrix; j) genotherapy and antisense agents; k) therapeutic agents consisting of antibodies; l) other bioactive compounds of marine origin, mainly didemnins, such as aplidine; m) steroid analogues, in particular dexamethasone; n) anti-inflammatory drugs, in particular dexamethasone; Y o) anti-emetic drugs, in particular dexamethasone.

As part of this patent specification, the authors include a series of examples referred to below. These examples demonstrate the greater efficacy of ET-743 when used in combination with other drugs and refer to different combinations that use ET-743.

Example 1 refers to effective combinations of ET-743 and doxorubicin for tumor growth inhibitions and used against human and murine sarcomas in athymic mice.

Example 2 shows that ecteinascidin 743 (ET-743) and doxorubicin produce synergistic cytotoxic effects on the soft tissue sarcomas lines HT-1080 and HS-18.

These two examples show more than additive effects of the combination of ET-743 with anthracyclines (in particular doxorubicin) that is more effective than alone against human tumors (in these specific experiments, sarcoma), whose effects appear independently of the administration sequence. These results are a good perspective for the treatment of patients.

Example 3 shows a synergistic cytotoxic effect of ET-743 and cisplatin.

Example 4 provides a sequential evaluation of ET-743 in combinations with chemotherapeutic agents against a panel of human tumor cell lines, in particular combinations of ET-743 with doxorubicin, taxol, SN-38, cisplatin and gemcitabine.

These two examples show more than additive effects of the combination of ET-743 with platinum antitumor compounds (in particular cis-platinum) with the gemcitabine nucleoside analogue and with a topoisomerase II inhibitor (SN38, which is the active agent produced from the pro-drug CPT-11, a drug from the camptothecin group). Again these combinations are more effective than the drug only against human tumors (in these specific experiments against a variety of tumor cells: ovarian, colon, lung, breast and bone sarcoma), the effects of which in some cases depended on the sequence of exposition. Again there are good prospects for the treatment of patients.

Interestingly, the synergistic action was not clearly predictable: Example 4 indicates that in most of the combinations analyzed, no synergy was observed (in fact, in some cases some antagonism was recorded).

Example 5 refers to the evaluation of combinations of ET-743 with doxorubicin or trimetrexate or paclitaxel.

This example shows more than additive effects of the combination of ET-743 with anthracyclines (in particular doxorubicin) which is more effective than alone against human tumors (in these specific experiments, sarcoma), whose effects appear independently of the administration sequence. These results show a good promise for the treatment of patients.

Examples 6 to 8 reinforce and complement the previous examples and especially show the synergy of ET-743 and doxorubicin and also ET-743 with cisplatin.

Example 9 demonstrates a different kind of efficacy of the combinations of this invention, in which high doses of dexamethasone protects against the hepatotoxicity of ecteinascidin-743 (ET-743).

In summary, this invention therefore provides compositions for use in treatments, processes for preparing said compositions and related embodiments.

The present invention also extends to the compounds of the invention for use in a method of treatment and to the use of the compounds in the preparation of a composition for cancer treatment.

Therefore, the present invention can be used to treat any mammal, primarily a human being, affected by cancer, which comprises administering to the affected individual a therapeutically effective amount of a compound of the invention or one of its pharmaceutical compositions.

The present invention also relates to pharmaceutical preparations that include a pharmaceutically acceptable carrier, which contains as active ingredient a compound or compounds of the invention, as well as to the processes for their preparation.

Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) with suitable composition and for oral, topical or parenteral administration and may contain the pure compound or in combination with any vehicle or other pharmacologically active compounds. It may be necessary for these compositions to be sterile when administered parenterally.

The administration of the compounds or compositions of the present invention can be carried out by any suitable method, such as by intravenous infusion, preparations for oral, intraperitoneal and intravenous administration. The authors of the present invention prefer that infusion times of up to 24 hours are used, more preferably 2-12 hours, and even more preferably 2-6 hours. Especially desirable are short infusion times that allow treatment to be performed without hospital stay. However, if perfusion is required it can be 12 to 24 hours or even longer. The infusion can be performed at suitable intervals of for example 2 to 4 weeks. Pharmaceutical compositions containing the compounds of the invention can be delivered by encapsulation in liposomes or nanospheres, in prolonged release formulations or by standard means of administration.

The correct dosage of the compounds will vary according to the particular formulation, the mode of application and the particular site, host and tumor to be treated. Other factors such as age, weight, sex, administration time, excretion rate, host status, drug combinations, reaction sensitivities and disease severity should be taken into account. Administration can be carried out continuously or periodically with the maximum tolerated dose.

The combinations of this invention can be used in patients who do not respond to treatment. The reader is referred to WO0069441 for information on the dosage schedules for ET-743 and other information on the use of the combination therapy of this invention.

EXAMPLES OF THE INVENTION

Example 1

Effective combinations of ET-743 and doxorubicin for inhibition of tumor growth against human and murine sarcomas in athymic mice.

ET-743 has a confirmed clinical activity in patients with soft tissue sarcoma and osteosarcoma who do not respond to previous chemotherapy including doxorubicin (Dx) and isosfamide. In view of the potential clinical value in combining ET-743 with Dx, the authors of the present invention have investigated this combination against UV2237 murine fibrosarcoma, its UV2237 / ADR mdr resistant subline and TE671 human rhabdomyosarcoma xenograft. Both ET-743 and Dx alone were effective against murine UV2237 fibrosarcoma, while each was inactive or marginally active against both UV2237 / ADR and TE671. However, the combination of ET-743 and Dx was effective in all 3 models. The synergy was particularly noticeable in human TE671 rhabdomyosarcoma and seemed independent of the sequence or combination of drugs.

After treatments i.v. of each drug made when the TE671 tumor weighed approximately 100 mg, the tumor weight inhibition (hereinafter TWI by the English expression Tumor Weight Inhibition) and the logarithmic values of cell mortality (abbreviated LCK by the English expression Log Cell Kill ) were respectively 46% and 0.132 for ET-743 (0.1 mg / kg) alone, 50% and 0.33 for Dx (10 mg / kg) alone, 77% and 0.924 for ET743 (0.1 mg / kg ) and Dx (10 mg / kg) administered simultaneously, 82% and 1.12 for the combination of ET-743 (0.1 mg / kg) administered 1 hour before Dx (10 mg / kg) and 75% and 0 , 85 for the combination of ET-743 (0.1 mg / kg) administered 1 h after Dx (10 mg / kg).

These data suggest that the combination of ET-743 and Dx may also be effective in tumors that are not sensitive or marginally sensitive to these drugs administered alone, thus providing a strong basis for clinical research using this combination.

Example 2

Ecteinascidin 743 (ET-743) and doxorubicin produce synergistic cytotoxic effects on the soft tissue sarcomas lines HT-1080 and HS-18.

Two sarcoma cell lines, HT 1080, an ET-743 sensitive fibrosarcoma cell line [50% inhibitory concentration (hereinafter abbreviated, IC50) = 10 pm] and HS-18, one less liposarcoma cell line were evaluated sensitive to ET-743 (IC50 = 270 pm) to determine the toxicity to ET-743 in combination with each of doxorubicin, trimetrexate or paclitazel. When ET-743 was used in combination with each of these drugs at a constant molar ratio and analyzed by the method of Chou and Talalay, synergistic effects (incubation for 72 h) were obtained with the ET-743-doxorubicin combination, but not with the combination of ET-743 with trimetrexate or paclitaxel. When cells were exposed to ET-743 for 72 h and each of doxorubicin, trimetrexate or taxol during the last 48 hours of incubation, synergistic effects with doxorubicin against both sarcoma cell lines were also obtained. It is interesting to note that the paclitaxel sequence followed by ET-743 was more effective than the opposite sequence. These results encourage clinical trials of doxorubicin in combination with ET-743 to treat patients with soft tissue sarcoma, since both of these drugs have shown activity against this disease.

Example 3

Synergistic cytotoxic effect of ET-743 and cisplatin

Ecteinascidin 743 (ET-743) has shown surprising antitumor activity in various preclinical systems and promising clinical activity. ET-743 binds to N2 guanines in the minor groove and affects transcription regulation (Minuzzo et al., PNAS, Vol. 97,6780-84, 2000).

Previous studies have indicated that cells deficient in mating error repair (abbreviated MMR by the English expression MisMatch Repair) are equally sensitive to ET-743 as competent cells. Nucleotide excision repair-deficient cells (abbreviated NER by the English expression Nucleotide Excision Repair) very sensitive to cisplatin are 6-8 times less sensitive to ET-743. Based on the different mechanisms involved in the repair of ET-743 and cisplatin and due to the potential clinical interest of this combination, the authors of the present invention have conducted studies to evaluate the cytotoxic effects of ET-743 and cisplatin on various cell lines Human tumor In this study, a human ovarian cancer Igrov-1 cell line, an ET-743-resistant subline (IG / PSC / ET) and human colon cancer cell lines HCT 116 (MMR-deficient) and HCT11 were used -ch3 (competent in MMR).

Cells were treated for 1 or 24 hours with different concentrations of ET-743 or cisDDP, alone or in combinations, and cytotoxicity was evaluated using a colorimetric analysis after staining with sulphordamine B. A synergistic effect was observed in all cell lines both in the 1 h and 24 h exposure. It should be noted that in HCT116 resistant to cisDDP, ET-743 was obviously able to reverse sensitivity even at concentrations of ET-743 that were only marginally effective. Taken together, the data provides a basis for undertaking clinical studies that combine ET-743 with cisDDP.

Example 4

Combinations of ET-743 with doxorubicin, taxol, Sn-38, cisplatin and gemcitabine

ET-743 was evaluated in combination with doxorubicin, taxol, SN-38, cisplatin and gemcitabine against a panel of human tumor cell lines. These studies were designed to determine the type of drug-drug interaction between ET-743 and standard chemotherapeutic agents and the influence of the exposure sequence on antitumor activity. Multiple combinations of ET-743 with standard cytotoxic agents with a modelless design (Laska, et al. Biometrics 50: 834, 1994) were used to describe the type of drug-drug interaction. These studies suggest that regardless of exposure, the most commonly observed is an additive model of drug-drug interaction.

A synergistic drug-drug interaction was observed when ET-743 was combined against tumor cell lines of non-small cell lung carcinoma (pre-exposure to SN-38), osteosarcoma (pre-exposure to ET-743 followed by cisplatin), breast (pre-exposure to ET-743 followed by gemcitabine) and colon (pre-exposure to ET-743 followed by SN-38 and simultaneous exposure to SN-38). An additive / synergistic model was observed (pre-exposure to ET-743 followed by SN-38 against NSCL; pre-exposure to SN-38 against colon and NSCL; simultaneous exposure to cisplatin against osteosarcoma and with SN-38 against NSCL lines) of drug-drug interaction. An antagonism test was observed when taxol was used simultaneously against two NSCL lines and doxorubicin against a rhabdomyosarcoma cell line.

These studies suggest that ET-743, which is in Phase II clinical trials, could be combined with various cytotoxic agents against a wide range of tumor types.

Material and methods

Cell culture:

In human RPMI-1640 supplemented with 10% fetal bovine serum and 2mM L-glutamine, human breast tumor cell lines (MDA-435, MDA-231, T-470), non-small cell lung carcinoma were grown (NCI-H522, NC1-H226, NCI-H23), colon (HCT-116, HT-29, Colo-320), osteosarcoma (HOS, U-2, OS, SaOS-2), rhabdomyosarcoma (RH1 , RH30, RD). All stock cultures were maintained in 75 cm-2 flasks at 37 ° C in humidified incubators with an atmosphere of 5% CO2-95% air.

IC50 analysis:

A predetermined number of exponentially growing tumor cells were inoculated in 96-well plates for tissue culture and allowed to stabilize for 24 hours. Next, a drug plaque consisting of serially diluted concentrations of ET-743 or standard chemotherapeutic agents was added to the cells. The cells were incubated as a 24-hour exposure for three days followed by the addition of MTT for 4 hours. The resulting formazan crystals were then solubilized with acid / alcohol, determining the absorbance (570 nm-assay / 630 nm-reference) using a microplate reader. The results were expressed as percentage of tumor cell mortality compared to controls.

Combination studies:

For combination studies, the concentration scheme (expressed as a percentage of the IC50 of the individual agent) used to characterize the type of interaction is shown below:

Drug concentration (expressed as a percentage of IC50)

ET-743

Standard agents

100

0

75

25

60

40

fifty

fifty

40

60

25

75

0

100

0

0

Statistical analyzes of combinations studies:

Statistical comparisons are made with each test combination (75: 25-ET-743 / standard agents) and the

5 endpoints (100: 0-ET-743 and 0: 100-standard agents). A statistically significant observation requires that there is a difference between the absorbance value of the combinations (ET-743 and standard agents) and the values of both endpoints (ET-743 and standard agents alone). If the majority of (≥3 of 5) values are statistically above or below the line, then antagonism or synergy is described, respectively. Otherwise the model is more consistent with an additive interaction. The interpretation is very

10 difficult if there is a considerable slope of the line connecting the endpoints. If the slopes of the IC50 curves for the individual agents are identical (unlikely) then the type of interaction can sometimes be determined.

Sequential combination of ET-743 with chemotherapeutic agents

Type of tumor / Cell line

Exposure conditions / Agents Drug-drug interactions observed

Osteosarcoma

US

24-hour exposure to ET-743 followed by 24-hour exposure to cisplatin 24-hour exposure to cisplatin followed by 24-hour exposure to ET-743 Synergistic Additive

U2-OS

24-hour exposure to ET-743 followed by 24-hour exposure to cisplatin 24-hour exposure to cisplatin followed by 24-hour exposure to ET-743 Additive Additive

Sa06

24-hour exposure to ET-743 followed by 24-hour exposure to cisplatin 24-hour exposure to cisplatin followed by 24-hour exposure to ET-743 Additive Additive

Non-small cell lung

24-hour exposure to ET-743 followed by 24-hour taxol exposure

Additive

NCB-H226

24 hours exposure to taxol followed by 24 hours exposure to ET-734 Simultaneous exposure 24 hours to ET-743 / taxol 24 hours exposure to ET-743 followed by 24 hours exposure to SN38 24 hours exposure to SN-38 followed by exposure 24 Additive Antagonic Additive / Synergic

NCB-N522

24 hours exposure to ET-743 followed by 24 hours exposure to taxol 24 hours exposure to taxol followed by 24 hours exposure to ET-734 Simultaneous exposure 24 hours to ET-743 / taxol 24 hours exposure to ET-743 followed by 24 hours exposure to SN38 Additive Additive Antagonic Additives / Synergic

NCB-N23

24 hours exposure to ET-743 followed by 24 hours exposure to taxol 24 hours exposure to taxol followed by 24 hours exposure to ET-734 Simultaneous exposure 24 hours to ET-743 / taxol 24 hours exposure to ET-743 followed by 24 hours exposure to SN38 Additive / Antagonic Additive Antagonistic Additive

Breast

MDA-435

24-hour exposure to ET-743 followed by 24-hour exposure to gemcitabine 24-hour exposure to gemcitabine followed by 24-hour exposure to ET-743 Additive Additive

MDA-231

24 hours exposure to ET-743 followed by 24 hours exposure to gemcitabine 24 hours exposure to gemcitabine followed by 24 hours exposure to ET-743 Simultaneous exposure 24 hours to ET-473 / gemcitabine Additive Additive Additive

S47-8

24-hour exposure to ET-743 followed by 24-hour exposure to gemcitabine 24-hour exposure to gemcitabine followed by 24-hour exposure to ET-743 Additive Additive

Colon

HCT-116

24-hour exposure to ET-743 followed by 24-hour exposure to SN-38 24-hour exposure to ET-743 followed by 24-hour exposure to SN-38 Synergistic Additive

NT-29

24-hour exposure to ET-743 followed by 24-hour exposure to SN-38 24-hour exposure to ET-743 followed by 24-hour exposure to SN-38 Additive Additive

Colo-320

24-hour exposure to ET-743 followed by 24-hour exposure to SN-38 24-hour exposure to ET-743 followed by 24-hour exposure to SN-38 Additive Additive / Synergic

Rhabdomyosarcoma

RN1

24 hours exposure to ET-743 followed by 24 hours exposure to doxorubicin 24 hours exposure to doxorubicin followed by 24 hours exposure to ET-743 Simultaneous exposure 24 hours to ET-743 / doxorubicin Additive Additive

RD

24 hours exposure to ET-743 followed by 24 hours exposure to doxorubicin 24 hours exposure to doxorubicin followed by 24 hours exposure to ET-743 Additive Additive

RN30

24 hours exposure to ET-743 followed by 24 hours exposure to doxorubicin 24 hours exposure to doxorubicin followed by 24 hours exposure to ET-743 Additive Additive

5

10

fifteen

twenty

25

30

35

Conclusions-Summary

These studies suggest that regardless of the sequence of exposure between ET-743 and standard chemotherapeutic agents, an additive model of drug-drug interaction is typically observed.

Synergy tests were observed when the NC1-H522 and NC1-H23 NSCL lines were previously exposed to SN-38, prior exposure to ET-743 with cisplatin against osteosarcoma HOS, the T-470 breast cell line with gemcitabine, SN- 38 against colon HCT-116 and simultaneous exposure with SN-38 against the Colo-320 colon tumor cell line.

Antagonism tests were observed when taxol was used simultaneously against the NC1-H226 and NC1-H23 NSCL cell lines and doxorubicin against the RHI rhabdomyosarcoma tumor cell line.

Example 5

Interaction between ET-743 and other antineoplastic agents

Although ET-743 is currently undergoing clinical trials in human cancers, the mechanisms of ET-743 antitumor activity have not been fully clarified. The objective of this study was to assess the nature of the interaction between ET-743 and other antineoplastic agents (doxorubicin; DXR, trimetrexate; TMTX and paclitaxel; taxol) using the combination index method (hereinafter referred to as IC) of Chou and Talalay To better understand how ET-743 could be used clinically, the present study used SRB (sulphordamine B) assays to examine the cytotoxicity that results from combining ET-743 with three other antineoplastic agents in the different administration programs in the two cell lines of Soft tissue sarcomas, HT-1080 and HS-18, in vitro. DXR was the only agent that resulted in a sequence-independent synergy when combined with ET-743. Simultaneous exposure of ET-743 with DXR resulted in synergistic interactions in both cell lines.

The IC values (mean values) with said program were 0.86, 0.83, 0.84 and 0.85 at a cell mortality of 50, 75, 90 and 95%, respectively, in HT-1080 and 0 cells , 89, 0.74, 0.64 and 0.60 at a cell mortality of 50, 75, 90 and 95%, respectively, in HS-18 cells. The ET-743 sequence for 24 h before DXR was the most effective regimen against both cell lines; resulted in a consequently low CI of up to a 90% cell mortality level for both cell lines. Exposure to taxol before ET-743 was also an effective regimen. These results suggest that the combination of ET-743 and DXR should be further explored in clinical trials in the treatment of soft tissue sarcoma.

Materials and methods

Chemical products

ET-743 was provided by Pharma-Mar S.A (Tres Cantos, Madrid, Spain) and was prepared as a 2 mM stock solution in dimethylsulfoxide. Paclitaxel and DXR were purchased from Sigma Chemical Co. (St. Louis, MO). TMTX was supplied by Warner-Lambert (Parke-Davis, Ann Arbor, Mich).

Cell culture

The soft tissue sarcoma cell lines, HT-1080 and HS-18 were maintained as monolayer cultures in RP <I-1640 containing 10% fetal bovine serum.

Cytotoxicity test with SRB -8 Drug cytotoxicity was determined by the cytotoxicity test with SRB performed on 96-well microtiter plates as described. The cells were plated in duplicate wells (5000 cells / well) and exposed to drugs at different concentrations. The cells were fixed with a 50% TCA solution for 1 h and 0.4% SRB (Sigma) was added to each well. After a 30 min incubation, the plate was washed with 1% acetic acid and a reading at 570 nm was performed on a Biowhitaker 2001 microplate reader. As positive and negative controls, wells were respectively used with cells that did not contain drugs and wells that contained the medium plus drugs but without cells.

Simultaneous exposure to ET-743 and DXR, TMTX or Paclitaxel

Cells were seeded in 96-well plates, as described above. The cells were treated with seven different concentrations of the drugs alone or combinations at a 1: 100 molar ratio (ET-743: the other drugs). After a 72 h exposure, growth inhibition was measured using the SRB assay.

Sequential exposure to ET-743 and DXR, TMTX or Paclitaxel

Using the same experimental system described above, the cells were exposed to three different concentrations of the drugs represented by IC25, IC50, CI75 of ET-743, DXR, TMTX and paclitaxel, respectively. After 24 hours of prior treatment with ET-743 or a combination drug, the second drug was added to the respective wells for 48 h. Growth inhibition was determined using the SRB assay.

Cell cycle analysis

Cells with exponential growth were treated with or without drugs for several hours. The cells were then collected and fixed with 70% methanol cooled with ice and the DNA stained with propidium iodide as described above. Ten thousand cells stained with a fluorescence activated cell sorter (FACS) Becton Dickinson were analyzed.

Determination of synergy and antagonism and construction of isobolograms

The IC was calculated by the Chou-Talalay equation, which takes into account both the power (Dm or IC50) and the shape of the dose-effect curve (the m value). The general equation of the classical isobologram (CI = 1) is:

IC = (D) 1 / (Dx) 1 + (D) 2 / (Dx) 2 (A)

where (Dx) 1 and (Dx) 2 in the denominators are the doses (or concentrations), being D1 (ET-743) and D2 (another drug) alone the doses that provide an inhibition of X%, while (D) 1 and (D) 2 in the numerators are the doses of ET-743 and another drug in combination that also inhibit X% (i.e. the same efficacy). CI <1, CI = 1, CI> 1 indicated synergy, additive effect and antagonism, respectively.

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The (Dx1 or (Dxepecrfmeeap

or

iand

lfteeec

oicuac

rdelaendomeadeChuy

:

lta

Chue

or

Dx = Dm [fa / (1-fa)] 1 / m (B)

where Dm is the dose of the median effect that is obtained from the antilogarithm of the abscissa at the origin of the graph of the median effect, X-log (D) versus Y -log [fa / (1-fa)] or Dm = 10- (ordered in origin) / m, and m is the slope of the graph of the median effective dose. The Chou and Chou software allows automatic calculation of the values of m, Dm, Dx and CI. From (Dm) 1, (Dx) 2 and D1 + D2, the isobolograms are easily constructed based automatically on equation A.

For mutually non-exclusive conservative isobolograms of two agents, a third term is added to the equation

(D1) (D2) / (DX) 1 (DX) 2 (C)

For simplicity, this third term is generally omitted and therefore the mutually exclusive assumption or classical isobologram is indicated. In results 2 and 3, the IC values obtained from the classical calculation (mutually exclusive) are given.

Result 1 This table showed that both HT-1080 and S18 cell lines were more sensitive to ET-743 than other antineoplastic agents.

Cytotoxicity of four drugs on HT-1080 and S 18

IC50 of human soft tissue sarcoma cells

HT-1080

HS-18

ET-743

(nM) 0.01 0.27

DXR

(nM) 25 225

TMTX

(nM) 6 70000

Paclitaxel

(nM) 1: 3 10

Effect of each agent on the distribution of the cell cycle against HS-18 cells 24 h and 72 h after treatment with doses of approximately IC50

Drugs

Dose Hours % of G1 % of Phase S % of G2M

Control

76.3 11.2 12.5

ET-743

270 pM 24 72 32.4 86 7 47.6 8 4 20.0 4 9

DXR

225 nM 24 72 10.1 1 3 64.9 63 8 25.0 34 9

TMTX

70 µM 24 72 44.2 35 5 53.8 57 6 1.9 7 0

Paclitaxel

10 nM 24 32.8 52.5 15.5

72

23.5 58.7 26.2

Effect of each agent on the distribution of the cell cycle against HT-1080 cells 24 h and 72 h after treatment with doses of i d CI

Drugs

Dose Hours % of G1 % of Phase S % of G2M

Control

47.5 35.8 16.7

ET-743

10 pM 24 72 42.6 83 1 36.1 10 2 21.3 6 7

DXR

25 nM 24 72 36.1 46 2 17.5 5 3 46.4 48 5

TMTX

6 nM 24 72 31.9 32.0 56.8 53.7 11.3 14.4

Paclitaxel

1.3 nM 24 72 45.4 86 0 37.3 9 0 17.3 5 0

Result 2 shows the IC for HT-1080 and HS-18 cells, respectively, that were simultaneously exposed to ET-743 and one of the antineoplastic drugs, such as DXR, TMTX or paclitaxel, to a combination 10 with a molar ratio from 1 to 100. When the cells were treated with ET-743 and DXR, the lC values were lower than

1, which indicates a synergistic effect on both cell lines. The lC (mean values) with this calendar were 0.86, 0.83, 0.84 and 0.85 at a cell mortality of 50, 75, 90 and 95%, respectively, in HT-1080 and 0.89 cells , 0.74, 0.64 and 0.60 at a cell mortality of 50, 75, 90 and 95%, respectively, in HS-18 cells. This result showed that the simultaneous treatment of ET-743 and DXR produced a synergistic cytotoxic effect. In contrast, when cells were treated with ET-743 and TMTX or paclitaxel, an antagonistic cytotoxic effect was observed.

A graphic representation of the ICs was obtained from both cell lines that were initially exposed to ET-743 for 24 h, followed by exposure to DXR for 48 h. In both cell lines, treatment with ET743 followed by DXR showed a synergistic cytotoxic effect, with the value of 1C HT-1080, at a cell mortality of 80%, 0.64 ± 0.12 and that of HS-18, at a cell mortality level of 88%, 0.24 ± 0.06. In contrast, treatment with DXR followed by ET-743 (Result 3a, lower figure) demonstrated at first sight the good value of Ic. However, the value of the HT-1080 lC at a cell mortality level of 80% was 1.00 ± 0.03, which indicates that the effect of the two agents was additive, in addition, in both cells the lC at a higher mortality fraction was worse than at a medium mortality fraction.

When the cells were exposed to ET-743 followed by exposure to TMTX, the IC values of HT-1080 were shown to be close to one or above one, indicating that the effect of the two agents was antagonistic or additive. In contrast, those of HS-18 were all less than 0.6, which demonstrates that these two drugs have a synergistic effect. When cells were treated with TMTX followed by ET-743, an additive effect was observed in both HT-1080 and HS-18 cell lines.

Treatment with paclitaxel followed by ET-743 produced a synergistic cytotoxic effect. When the cells were exposed to paclitaxel followed by exposure to ET-743, the IC value of HT-1080 at a cell mortality level of 89% was 0.92 ± 0.06 and that of HS-18 at a level of 78% cell mortality was 0.38 ± 0.13.

Summary

ET-743 was very active against human soft tissue sarcoma cells, especially against the HT-1080 malignant fibrosarcoma cell line.

DXR resulted in an independent sequence synergy when combined with ET-743, however, the ET-743 sequence followed by DXR was the most effective against both cell lines.

Exposure to paclitaxel followed by exposure to ET-743 was also an effective regimen against human soft tissue sarcoma cells, while simultaneous exposure was antagonistic.

Example 6

In vivo combinations of chemotherapeutic agents with ecteinascidin 743 (ET-743) against solid tumors.

Various unique mechanisms of action for ET-743 have been described including minor DNA slot binding, N2 alkylation of guanine, transcripctional inhibition of the MDR1 gene (Jin et al., PNAS 97, 6775, 2000; Minuzzo et al ., PNAS 97, 6780, 2000) and the counterbalance of the activation of the SXR nuclear receptor (Synold et al., Nature Med 7, 584, 2001). As a single agent, ET-743 inhibits tumor growth in vivo by achieving complete remissions (RC) against various human tumor strains (Hendriks et al., Ann. Oncol. 10, 1233, 1999) including melanoma (MEXF 989), NSCL carcinoma (LXFL 529), ovarian (HOC 22) and breast (mix-1). The efficacy of ET-743 in combination with drugs that act by alternative mechanisms may provide opportunities to reduce the toxicities of each drug or enhance the efficacy of a drug in resistant or recurrent cancers.

Various agents including doxorubicin (DOY, 8 mg / kg), cisplatin (DDP; 12 mg / kg) and vinblastine (VINB; 6 mg / kg) before / after ET-743 (0.2 mg /) were administered for this evaluation. kg) with a 1-hour prior treatment, qdx5, in one or more of the following tumors: chondrosarcoma (CSHA), osteosarcoma (OSA-FH), fibrosarcoma (SW684), ovarian ((MRIH-1834), NSCL ( LX-1) and renal (MRI-H-121) with activity defined as <50% T / C. In the hollow fiber model (FH), the DOX sequence, 1 h of prior treatment with ET-743, was consequently more effective than ET-743 alone in chondrosarcoma (6% vs. 10%), fibrosarcoma (33% vs. 48%) and osteosarcoma (20% vs. 34%). Osteosarcoma xenografts produced similar results of 17% versus 43% FH studies with DPP showed that pre-DDP ET-743 was more effective than ET-743 alone in ovarian tumors (28% vs. 100%) and chondrosarcoma (15% vs. 19%) and equivalent activities in osteosarcoma (36% T / C) .Xe data No graft confirm that the sequence of ET-743 pre-DDP is more effective than ET-743 alone (35% vs. 66%). The only exception was in NSCL where ET-743 alone was not active (62% T / C) but DPP followed by ET-743 produced CR (<1% T / C). In renal xenografts, ET-743 alone was very active (22% T / C) but ET-743 followed by VINB also produced CR (<1% T / C). Separate studies are under way with other standard agents in xenografts of breast, renal, melanoma and gastric tumors.

Example 7

Preclinical activity and biodistribution of combinations of ecteinascidin 743 (ET-743) and doxorubicin (DOX) in human rhabdomyosarcoma.

ET-743 is the first of a new class of antitumor agents that exhibit antitumor activity. ET-743 has shown activity in patients with sarcoma who do not respond to treatment with DOX and ifosfamide. In view of its potential as an effective drug, the authors of the present invention investigated:

(one)

the preclinical antitumor activity of the ET-743 / DOX combination against human rhabdomyosarcoma TE 671 and

(2)

possible interactions between drugs and their biodistribution in athymic mice and tumor xenografts.

In vitro: the effect of each drug or combination after 1 h exposure was evaluated by clonogenic assay. ET743 or DOX alone showed antitumor activity against TE 671 cells. The combination according to the isobologram analysis and the combination index, was at least additive in various tumor cell lines including TE

671.

In vivo: IV treatments alone (ET-743, 0.1mg / kg; DOX, 10mg / kg) were administered to athymic mice when the xenograft tumors weighed approximately 100 mg. The tumor weight / logarithmic cell mortality inhibition values were 46% / 0.132 for ET alone, 50% / 0.33 for DOX alone, 77% / 0.924 for ET-743 and DOX administered simultaneously, 82% / 1.12 for the combination of ET-743 administered 1 hour before DOX and 75% / 0.85 when ET-743 was administered 1 hour after DOX. A synergistic effect was also observed against UV2237 murine fibrosarcoma and against its multi-drug resistant UV2237 / ADR subline.

These data show a synergistic effect of ET-743 / DOX and appear to be independent of the sequence or combination of drugs in the cases studied so far. Neither the concentrations in the plasma nor in the DOX tumors are significantly different when DOX was administered alone or in combination with ET-743. The pharmacokinetic (PK) evaluation of ET-743 administered alone or in combination with DOX is ongoing. The combination of ET743 and DOX seems additive in vitro but synergistic in vivo in rhabdomyosarcoma TE 671. The pharmacokinetic profile of DOX is not influenced by simultaneous treatment with ET-743. These data provide a basis for using this combination in early clinical trials.

Example 8

ET-743 and cisplatin (DDP) show synergy in vitro and in vivo against human ovarian sarcoma and carcinoma cell lines.

The authors of the present invention show that ET-743 improves DDP activity both in vitro and in vivo. In various cancer cell lines including human intestinal carcinoma (HCT116), ovarian carcinoma (Igrov-1, A2780), its resistant sublines (Igrov-1 / PSC-ET and 1A9, respectively) and rhabdomyosarcoma (TE671), lower concentrations of ET -743 used as a single agent could enhance DDP activity at least twice. Concentrations corresponding to IC30 / IC50 of ET-743 resulted in additive or synergistic effects. These results have led to in vivo studies using xenograft models to study effective drug combinations with ET-743.

In TE 671 subcutaneously transplanted, partially sensitive to ET-743 and DDP, the combination of the two drugs produced an antitumor effect much greater than that achieved with each drug used at their respective MTD levels. Ovarian tumor 1A9 that is normally resistant to both ET-743 and DDP as agents alone, in combination produced a tumor growth inhibition greater than 50%. Human orthotopically transplanted HOC8 ovarian carcinoma, which produces tumor nodules in the peritoneal cavity with ascites, which is resistant to ET-743 and partially sensitive to DDP, in combination resulted in a dramatic increase in survival even at the dose of ET -743 of 0.05 mg / kg (¼ MTD) and did not cause any significant toxicity. A dose of ET-743 of 0.15 mg / kg markedly increased survival, but also increased toxicity as indicated by weight loss, which was significantly greater than that observed after treatment with each drug.

These findings offer a strong basis for designing clinical trials using the combination of ET-743 and DDP in sarcomas and ovarian cancers. In vitro and in vivo studies are underway to illustrate the mechanisms that highlight the synergy between ET-743 and DDP in these types of cancer.

Example 9

Dexamethasone (Dex) in high doses protects against the hepatoxicity of ecteinascidin-743 (ET-743) in the rat

ET-743, an agent from a marine tunicate, is currently in phase II clinical trials. It has shown activity against sarcomas and preliminary data suggest activity against breast and ovarian carcinoma. However, in the majority of treated patients there is a hepatotoxicity characterized by reversible transaminitis and in a minority cholestasis. In the most sensitive animal species, the rat, the toxicity of ET-743 is characterized by hepatic necrosis and inflammation of the bile ducts. Due to the anti-inflammatory activity of Dex, the authors of the present invention investigated its effect on liver lesions induced by ET-743 in the rat. Female Wistar rats received a single i.v dose of ET-743 (40 µg / kg). Some rats were previously treated with a single dose of Dex of 1, 5, 10 or 20 mg / kg, 24 hours before treatment with ET-743. Hepatic pathology and plasma concentrations of alkaline phosphatase (ALP), aspartate aminotransferase (GOT) and total bilirubin (TB) were evaluated up to 3 days after administration of ET-743. Conventional histological sections of the livers were examined by optical microscope.

2 days after treatment with ET-743, the livers of rats that received ET-743 alone showed inflammation of the bile ducts, surprising degenerative changes in biliary epithelial cells and areas of necrosis

5 liver. The plasma levels of ALP and GOT rose significantly after 2 days. Cholestasis was reflected by a dramatic increase in plasma TB concentrations, which began 2 days after the administration of ET-743. The histopathological changes induced by ET-743 and the elevation of ALP, GOT and TB in plasma did not occur at all in rats previously treated with 10 or 20 mg / kg of Dex.

Although Dex at 1 mg / kg showed little protection, at 5 mg / kg it was moderately protective. The plasma levels of

10 ET-743 in rats that received daily Dex (50 mg / kg) for 3 days before ET-743 did not decrease compared to those in rats to which ET-743 had been administered alone. In addition, the activity of ET-743 against B16 melanoma implanted in mice was not impeded by dexamethasone. These findings suggest that the addition of dexamethasone in high doses to the ET-743 regimen may improve its hepatotoxicity in cancer patients.

fifteen

Claims (21)

one.

The use of a synergistic combination of ET-743 and another drug selected from an anthracyclinic drug, a platinum drug, a drug that targets a topoisomerase, a taxane group drug, an antimetabolite drug or an oral antifungal drug in the preparation of a medication for the treatment of a tumor.

2.

The use according to claim 1, wherein the combination therapy employs ET-743 and doxorubicin.

3.

The use according to claim 1, wherein the combination therapy employs ET-743 and cisplatin.

Four.

The use according to claim 1, wherein the combination therapy employs ET-743 and paclitaxel.

5.

The use according to claim 1, wherein the combination therapy employs ET-743 and gemcitabine.

6.

The use according to claim 1, wherein the combination therapy employs ET-743 and SN-38.

7.

The use according to claim 1, wherein the combination therapy employs ET-743 and trimetrexate.

8.

The use according to claim 1, wherein the combination therapy employs ET-743 and vinblastine.

9.

The use according to any of claims 2, 3, 4 or 7, for the treatment of sarcoma.

10.

The use according to claim 3 or 6 for the treatment of colon cancer.

eleven.

The use according to claim 3 for the treatment of intestinal carcinoma.

12.

The use according to claim 3 or 6 for the treatment of lung cancer.

13.

The use according to claim 5 for the treatment of breast cancer.

14.

The use according to claim 8 for the treatment of renal cancer.

fifteen.

Use according to any of the preceding claims, wherein ET-743 and the other drug are provided as a single medicament.

16.

Use according to any one of claims 1 to 14, wherein ET-743 and the other drug are provided as separate medications.

17.

The use according to claim 16, wherein the separate medicament containing ET-743 is for administration at the same time as the medicament containing the other drug.

18.

The use according to claim 16, wherein the separate medicament containing ET-743 is for administration at a different time from the medicament containing the other drug.

19.

The use according to any of the preceding claims, wherein the other drug or combination is supplied by liposome or encapsulation in nanospheres.

twenty.

The use according to any of the preceding claims, wherein the combination therapy also employs dexamethasone.

twenty-one.

A synergistic combination of ET-743 and another drug selected from an anthracyclinic drug, a platinum drug, a drug that targets a topoisomerase, a drug from the taxane group, an antimetabolite drug or an oral antifungal drug for use in the treatment of a tumor as described in any of the preceding claims.