MX2007003665A - Ecteinascidin compounds as anti -inflammatory agents - Google Patents

Abstract

We have found anti-inflammatory activity in the ecteinascidin compounds. Such compounds have been widely described, and may have the following general formula (I), wherein:R5is OH, alkoxy or alkanoyloxy;R6is hydrogen, alkyl, alkenyl, alkynyl or aryl;R12is hydrogen, alkyl, alkenyl, alkynyl or aryl;R16is hydrogen, alkyl, alkenyl, alkynyl or aryl;R17is OH, alkoxy or alkanoyloxy;R18is OH, alkoxy or alkanoyloxy;R21is H, OH, CN or another nucleophilic group;and Rais hydrogen and Rbis optionally substituted amino, or Rawith Rbform a carbonyl function=O, or Ra, Rband the carbon to which they are attached form a tetrahydroisoquinoline group.

Description

COMPU THESE OF ECTEI NASC I DI NA AS AGENTS ANTI I N FLAMATORIES The present invention relates to anti-inflammatory agents. More particularly, the present invention relates to the discovery of anti-inflammatory activity in a known class of compounds. Background of the invention Monocytes / macrophages are very important recognized components of innate and adaptive immune units. The circulating monocytes are versatile precursors with the ability to differ in the various forms of tissue macrophages. The macrophages stand guard against external invaders and are able to instantly defend the body against pathogens, as well as send signals for the recruitment of other immunocompetent cells and present antigen to the T lymphocytes. On the other hand, it has also implicated the macrophages at the beginning or progression of many diseases, most of them via the production of pro-inflammatory or pro-angiogenic mediators. Such conditions include, for example, pronounced inflammation present in various chronic diseases (e.g., rheumatoid arthritis, areteriollosclerosis, lupus erythematosus) and tumors. At tumor location, tumor-associated macrophages (TAM) represent a major component of infiltrated stromal cells. TAMs have a complex and ambiguous role within tumors, as suggested in the macrophage balance hypothesis. In fact, although macrophages stimulated with LPS and I FN gamma (also called M 1 macrophages or classical activation macrophages) have the potential to kill tumor cells, many lines of evidence support the idea that macrophages within the microenvironment of the tumor are twisted towards the alternatively activated macrophages, or M2 macrophages. Most often, TAMs are non-cytotoxic and produce many growth factors and nyrogens. TAMs also produce immunosuppressive molecules (for example, I L-1 0, TG Fb) and a variety of inflammatory mediators, including chemokines. Chemokines activate matrix metalloproteases that digest matrix proteins and promote tumor dissemination. Therefore, the accumulation of TAM in the location of the tumor and the continuous expression of the inflammatory molecules may in fact favor the development of the tumor. Brief description of the invention The ecteinascidin compounds include natural and synthetic compounds. They have a fused five-ring system, in addition to a bridge 1, 4. We have discovered anti-inflammatory activity in the ecteinascidin compounds. Said compounds have been widely described, and may have the following general formula (I): wherein: R5 is OH, alkoxy or alkanoyloxy; R6 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R 12 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R16 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R17 is OH, alkoxy or alkanoyloxy; R18 is OH, alkoxy or alkanoyloxy; R21 is H, OH, CN or another nucleophilic group; and Ra is hydrogen and Rb is an optionally substituted amino, or Ra with Rb form a carbonyl function = 0, or Ra, Rb and the carbon to which they are attached form a tetrahydroisoquinoline group. Therefore, the present invention provides a method for treating an inflammation comprising administering an effective amount of an ecteinascidin having the general formula (I). The invention also provides medicaments containing an ecteinascidin having the general formula (I), together with a pharmaceutically acceptable carrier or diluent. The invention further provides the use of an ecteinascidin having the general formula (I) in the preparation of a medicament for use in the treatment of inflammation. DETAILED DESCRIPTION OF THE INVENTION We have discovered that ecteinascidin compounds possess anti-inflammatory activity. Thus, the present invention relates to a medical indication for compounds of the general formula (I) as defined above. In these compounds, the substituents can be selected according to the following guidance: The alkyl and alkoxy groups preferably have from 1 to 12 carbon atoms. A more preferred class of alkyl and alkoxy groups has from 1 to 6 carbon atoms, and more preferably 1, 2, 3 or 4 carbon atoms. The methyl, ethyl and propyl groups, including isopropyl, are particularly preferred alkyl groups in the compounds of the present invention. The methoxy, ethoxy and propoxy groups, including isopropoxy, are preferred alkyl groups in the compounds of the present invention. Another more preferred class of alkyl and alkoxy groups has from 4 to about 12 carbon atoms, still more preferably from 5 to about 8 carbon atoms, and most preferably 5, 6, 7 or 8 carbon atoms. As used herein, the term alkyl, unless otherwise modified, refers to cyclic and non-cyclic groups, although the cyclic groups will contain at least three members in the carbon ring. Preferred alkenyl and alkynyl groups in the compounds of the present invention have one or more unsaturated bonds and from 2 to about 12 carbon atoms. An even more preferred class of alkenyl or alkynyl groups has from 2 to about 6 carbon atoms, and ideally 2, 3 or 4 carbon atoms. Another more preferred class of alkenyl or alkynyl groups has from 4 to about 12 carbon atoms, and yet another more preferable 5, 6, 7 or 8 carbon atoms. carbon. The terms alkenyl and alkynyl, as used herein, refer to both cyclic and non-cyclic groups. Suitable aryl groups in the compounds of the present invention include single-ring or multi-ring compounds, including multiple ring compounds containing separate and / or fused aryl groups. Typical aryl groups contain from 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms. Especially preferred aryl groups include substituted or unsubstituted phenyl, naphthyl, biphenyl, phenanthryl and anthracyl. Suitable alkanoyloxy and alkanoyl groups have from 2 to about 20 carbon atoms, more preferably from 2 to about 8 carbon atoms, even more preferably from 2 to 6 carbon atoms and ideally 2 carbon atoms. Another preferred class of alkanoyloxy groups has from 12 to 20 atoms carbon, even more preferably from 14 to 18 carbon atoms and ideally 15, 16, 17 or 18 carbon atoms. The aforementioned groups can be substituted in one or more available positions with one or more suitable groups such as OR \ = 0, SR \ SOR ', S02R', N02, NHR ', N (R') 2, = NR \ NHCOR ' , N (COR ') 2, NHS02R', CN, halogen, C (= 0) R \ C02R ', OC (= 0) R' where each of the groups R 'is independently selected from the group consisting of H, OH, N02, NH2, SH, CN, halogen, = 0, C (= 0) H, C (= 0) CH3 > C02H, substituted or unsubstituted Ci-C12 alkyl, substituted or unsubstituted C2-Ci2 alkenyl, C2-C12 alkynyl and substituted or unsubstituted aryl. Suitable halogen substituents in the compounds of the present invention include F, Cl, Br and I. Preferred compounds of the invention are those of the general formula (I), wherein one or more of the following definitions will apply: R 5 is an alkanoyloxy; R6 is methyl; R12 is methyl; R16 is methyl; R17 is methoxy; R18 is OH; R21 is H, OH or CN; and Ra is hydrogen and R is an amido group, or Ra with R forms = 0, or Ra, Rb and the carbon to which they are attached form a group of the formula (II): Examples of compounds of the present invention include natural ecteinascidins, such as ecteinascidin 743 and other fused and bridged ecteinascidin compounds described, for example, in US Pat. Nos. 5,089,273; US 5,478,932; US 5,654,426; US 5,721,362; US 6,124,293; US 5,149,804; US 09 / 546,877; US 5,985,876 and WO 01/77115. The ecteinascidin 743, also known as ET743 or ecteinascidin 143 is particularly preferred. ET743 is a natural product derived from the marine tunicate Ecteinascidinia turbinata, with a potent antitumor activity. It is a novel and effective drug that is currently in clinical trials and has shown anti-cancer activity in some solid human tumors, including soft tissue sarcomas, breast and ovarian cancer. The compounds of the following formula (III) are particularly preferred: where Ra is hydrogen and Rb is amido of formula -NHRf where Rf alkanoyl, or Ra with Rb form = 0, or Ra, Rb and the carbon to which they are attached form a group of formula Rd is alkanoyl; and R21 is H, OH or CN. The alkanoyl groups may be acetyl or greater, per pio to C2o. Thus, the preferred compounds of this invention include: ET637 derivative B or ET745 and compounds related to different acyl groups. The medicaments provided by this invention are pharmaceutical compositions containing the ecteinascidin compound and a pharmaceutically acceptable carrier. Medication can be in conventional form, and can be develop suitable dosing procedures. As indicated, the compounds of the invention are useful as anti-inflammatory agents. Thus, these compounds can be used in the treatment of diseases that have to do with inflammation, particularly in the treatment of chronic inflammatory and autoimmune diseases (for example, rheumatoid arthritis, Sjogren's disease and Crohn's disease) and for arteriosclerosis. . Brief description of the drawings Figure 1. Panel A: cell viability of blood monocytes, lymphocytes and thymocytes cultured with ecteinascidin 743. Figure 1. Panel B: apoptosis of monocytes treated with ecteinascidin 743. Figure 2. Pretreatment with M-CSF protects partially to monocytes of the pro-apoptotic effect of ecteinascidin 743. Figure 3. Panel A: kinetics of the cytotoxic effect of ecteinascidin 743 on monocytes. Figure 3. Panel B: inhibition of macrophage differentiation. Figure 4. Panel A: susceptibility to ET743 of monocytes and macrophages from the same donor. Figure 4. Panel B: susceptibility to ET743 of macrophages classically activated by LPS and IFNgamma or by IL-4. Figure 4. Panel C: Susceptibility to ET743 of monocytes of macrophages associated with tumors. Figure 5. In vivo infusion of ecteinascidin 743 in patients with tumors induces transient monocytopenia. Figure 7. Ecteinascidin 743 inhibits the production of CCL2 (Panel A) and IL-6 (Panel B) by monocytes and macrophages. Figure 8. Panel A: ecteinascidin 743 does not affect the production of TNF by monocytes, macrophages and TAM. Figure 8. Panel B: Real-time PCR of CCL2 and TNF records in monocytes stimulated by LPS exposed to ecteinascidin 743. Figure 9. Panel A: cytotoxicity of ecteinascidin 743, Doxorubicin, taxol and Cis-DDP on monocytes. The asterisk indicates the IC50 for each drug on tumor cell lines cultured in vitro. Figure 9. Panel B: production of CCL2 and TNF by monocytes stimulated by LPS treated with the indicated doses of anti tumor agents. Figure 10. Secretion of CCL2 by LPS monocytes previously treated with ecteinascidin 743 and other ecteinascidin compounds. EXAMPLES OF THE INVENTION In this study we demonstrated that, in concentrations within the pharmacological range, ecteinascidin 743 showed selective toxicity for the myeloid lineage and induced monocyte / macrophage apoptosis. In non-toxic concentrations, the ecteinascidin 743 significantly inhibited macrophage differentiation in vitro and reduced the production of select inflammatory cytokines. These findings may be relevant for therapeutic approaches aimed at attacking monocytes / macrophages in various human diseases. Additionally, ET743, derivative A of ET637, derivative B of ET637, ET594, derivative A of ET743 and ET745 were also analyzed. It has also been shown that they reduce the production of select inflammatory cytokines. Materials and methods Cell preparation Purified populations of human blood monocytes were prepared as described above by differential density centrifugation on Ficoll and Percoll gradients (see Allavena, P., Piemonti, L., Longoni, D., Bernasconi, S., Stoppacciaro, A., Ruco, L. and Mantovani, A. IL-10 prevent the differentiation of monocytes to dendritic cells but promote their maturation to macrophages, Eur J Immunol, 28: 359-369, 1998). Monocytes were usually > 85% CD 14+ cells. Purified T lymphocytes (CD3 +> 95%) were obtained in Percoll gradients as described previously (see Chieppa, M., Bianchi, G., Doni, A., Del Prete, A., Sironi, M., Laskarin , G., Monti, P., Piemonti, L., Biondi, A., Mantovani, A., Introna, M. and Allavena, P. Cross-linking of the mannose receptor on monocyte derived dendritic cells activates an anti-inflammatory immunosuppressive program J Immunol, 171: 4552-4560, 2003). Human thymocytes were isolated from the resected thymus of pediatric patients in surgery. The thymocytes were obtained by exciting and isolating in Percoll gradient. The cells were cultured in an amount of 106 cells / mL in a complete RPMI medium (Biochrom, Berlin, Germany) + 10% FCS (Hyclone, Logan, UT). Differentiated macrophages in vitro were obtained by stimulating monocyte monocyte Colony (M-CSF) Peprotech (20 ng / mL), for 5 days. In some experiments, macrophages were treated with LPS (100 ng / mL) Sigma Aidrich, IFN gamma (500 IU / mL) or IL-4 (20 ng / mL) (Schering Plow) for 24 hours. Macrophages associated with tumors (TAM) and tumor cells were isolated from the ascitic acid fluid of patients with diagnosed ovarian adenocarcinoma, admitted to the Clinic of Obstetrics and Gynecology of the University of Milan-Bicocca, S Gerardo Hospital. The cells contained in the ascitic acid fluid were centrifuged and isolated by differential gradients of Ficoll and Percoll and plastic adhesion, as already described (see Allavena, P., Peccatori, F., Maggioni, D., Error, A Sironi, Colombo, N., Lissoni, A., Galazka, A., Meiers, W., Mangioni, C, et al. Interperitoneal recombinant gamma-interferon in patients with recurrent ascitic ovarian carcinoma; modulation of cytotoxicity and cytokine production in tumour-associated effectors and of major histocompatibility antigen expression on tumor cells Cancer Res, 50: 7318-7323, 1990). The purity of the preparations of the TAM and tumor cells was usually > 65 ± 10% as defined by morphology and phenotype analysis. The cells were treated with ecteinascidin 743 at the indicated concentrations and cultured for 1 to 5 days, as specified in the legends of the figure. At the end of the incubation period, the cells were harvested, washed and used for DNA analysis or functional analysis. Determination of cell viability. The cell viability was analyzed by means of DNA content in flow cytometry. The cells exposed to the treatments were fixed with 70% ethanol, washed in PBS and stained with a propidium iodide (Pl) solution containing 10 μg / mL of Pl in PBS and 25 μ? of RNAse 10,000 units, during the night, in the dark. The incorporation of Pl was evaluated in at least 20,000 cells / samples using a Facs Calibur instrument (Becton Dickinson, Sunnyvale, CA, United States), with a bandpass filter at 620 nm. Apoptosis was detected by staining with AnnexinV and Pl. A FACS analysis was carried out using a bandpass filter of 530 and 620 nm for green (AnnexinV) and red (Pl) fluorescence respectively, in combination with a dichroic mirror at 570 nM. Phenotype analysis The expression of cell membrane markers was carried out by immunofluorescence and analyzed by flow cytometry. The cells were incubated with anti-CD14, anti-CD16, anti- CD68, anti-CD206 (handy receptor) and then with FITC-lg anti-mouse goat as described. At least 10,000 cells were analyzed. Production of cytokines Supernatants from untreated cells or cells treated with ecteinascidin 743 or other antineoplastic agents were harvested after 24 hours of culture and frozen. Monocytes, macrophages and TAM were stimulated with 100 ng / mL of LPS to induce the maximum production of cytokines. The determination of cytokines CCL2, TNF and IL-6 was measured by a specific ELISA, following the manufacturer's instructions. Patients with tumors Patients with sarcoma or ovarian cancer who are in Phase II trial with ecteinascidin 743 were admitted to the European Institute of Oncology, in Milan, Italy. Patients received ecteinascidin 743 (1300 mg / mL) in a 3-h infusion. Blood samples (40 mL) were collected immediately before treatment and at the end of the infusion (+ 3 h). The blood samples were processed immediately and the monocytes purified by Percoll (usually 160 cells) were cultured with M-CSF (20 ng / mL) for 5 days. The differentiated cells were harvested, counted and analyzed in phenotype expression, as described above. The results are presented in absolute amounts of marker positive cells / 10,000 cells. The significant inhibition of macrophage differentiation was considered as a 50% reduction of positive cells to the marker, in relation to the cells collected before the therapy, from the same patient. Example 1 Ecteinascidin 743 shows a selective cytotoxic effect on mononuclear phagocytes We first studied the effect of ecteinascidin 743 treatment on viability in subgroups of human leukocytes in vitro. Purified preparations of monocytes, lymphocytes and blood thymocytes were cultured with different concentrations of ecteinascidin 743 for 48 hours. Cell viability was established by DNA analysis and staining with propidium iodide (Pl) in flow cytometry. Purified preparations of blood monocytes were highly susceptible to the cytotoxic effect of the drug. There was a dose-dependent mortality with a lethal dose of 50% (IC50 of 2.5-5 nM after 48 hours of culture (Figure 1A) .The purified T lymphocytes were much less susceptible, and all remained alive at 5 nM. The IC 50 for lymphocytes was 20 nM Even more resistant were thymocytes freshly isolated (IC50> 40 nM, Figure 1A) Virtually all monocytes that died and were exposed to ecteinascidin 743 stained positively with Annexin V , indicating that the drug induces apoptosis (Figure 1B) The mortality of monocytes was further confirmed by DNA analysis by means of flow cytometry (Figure 2) In the presence of M-CSF a growth factor was observed and d iference for monocytes, partial protection against the toxic effect of ecteinascid ina 743. M-CSF changed the death of monocytes from 55% to 30% with 5 nM of ecteinascidin 743, after 48 hours of incubation, and 65 % to 35% at 10 0 n M after 24 hours of treatment (Fig. 2). M-CSF was effective only if it was added simultaneously or before ecteinascidin 743, but was no longer effective when administered 4 hours after the drug. A kinetic analysis of the cytotoxic effect of ectei nascid ina 743 was carried out in the presence of M-CSF. The cells were treated with the M-CSF (20 ng / mL) and the different concentrations of ecteinascidin 743. The samples were collected at the indicated times and tested in a DNA analysis. At higher concentrations, significant toxicity was observed after 24 hours of incubation, and this increased with time (Figure 3A). The lower concentrations (2.5 nM) induced 40-50% mortality after 5 days. Next, we studied the effect of ecteinascidin 743 on differentiated macrophages, obtained from monocytes quantified in vitro for 5 days with M-CSF. The addition of ecteinascidin 743 in the last 48 hours resulted in a significant mortality, but lower compared to that of the newly isolated monocytes. Figure 4A shows a representative experiment comparing the susceptibility of monocytes and macrophages of the same donor. The monocytes were Differentiated to macrophages by culture with M-CSF (20 ng / mL). On day 3, ecteinascidin 743 was added to the cultures and incubated for 48 hours. The results show the comparison of monocytes and macrophages obtained from the same donor. Viability was demonstrated by Pl staining and analyzed by flow cytometry. Similar results were obtained in 4 other experiments. In a series of 4 different experiments, the IC 50 for the differentiated in vitro was 10 nM. The susceptibility to ecteinascidin 743 of classically activated macrophages was then tested by LPS and IFN gamma (or M1 macrophages) and those activated alternately by IL-4 (or M2 macrophages). Differentiated macrophages in vitro were stimulated by LPS (100 ng / mL) + IFNgamma (500 IU: mL), IL-4 (20 ng / mL), in the presence or absence of ecteinascidin 743 for 48 hours. Viability was demonstrated by Pl staining and analyzed by flow cytometry. Both macrophages stimulated by LPS and those stimulated by IL-4 were susceptible to treatment, as were unstimulated macrophages (Figure 4B). Tumor-associated macrophages (TAMs) isolated from the hydroperitoneuses of patients with untreated ovarian adenocarcinoma were also tested. The preparations enriched with TAM isolated from three different patients with ovarian cancer were treated in vitro with ecteinascidin 743 for 48 hours. Viability was demonstrated by staining with Pl and analyzed by flow cytometry. The TAMs were significantly killed in vitro by ecteinascidin 743 with a mortality of 40-70% at 10 nM. The results of three different patients are shown in Figure 4C. Taken together, these experiments demonstrate that human mononuclear phagecytes are highly susceptible to the cytotoxic effect of ecteinascidin 743 at concentrations within the therapeutic range. It should be noted that even in the presence of M-CSF, monocytes never experienced cell cycle progression, which was verified by DNA analysis with flow cytometry. The toxic effect of ecteinascidin 743 on monocytes is, therefore, independent of the cell cycle and provides the unique opportunity to study the biological effects of this drug on non-replicating cells. Example 2 The non-cytotoxic concentrations of ecteinascidin 743 inhibit the in vivo and in vitro differentiation of macrophages. In order to study the effect of ecteinascidin 743 on the differentiation of macrophages, non-cytotoxic doses of the drug were used. Monocytes were cultured with M-CSF (20 ng / mL) and with sub-cytotoxic concentrations of ecteinascidin 743 for 5 days. Phenotype analyzes were performed by means of indirect immunofluorescence, as well as by flow cytometry, restricting large cells. Usually, an average of 65 ± 15% (mean ± SD of> 10 experiments) of input monocytes is they differentiate into large cells that express typical macrophage markers, including CD 1 6, C D68 and CD206 (handy receptor). After 5 days of culture, the viability of monocytes, evaluated by means of staining with propyl iodide in flow cytometry, was 92% and 70% of untreated cells at 0.5 and 1 n M of ecteinascidin 743, respectively. The process of differentiation of macrophages was partially inhibited to the network of the new expression of C D68, CD 1 6 and CD206 to 1 n M of ecteinascidin 743 (Fig. 3B). To validate the aforementioned in vitro findings we tested whether the in vivo administration of ecteinascidin 743 in patients with tumors could have measurable effects on the viability of monocytes and the ability for macrophage differentiation in vitro. A phase I I trial with ecteinascidin 743 is currently underway in patients with advanced ovarian adenocarcinoma who had not failed in two different cycles of cis-platinum chemotherapy and taxol-based chemotherapy. Patients with tumors selected for this study were treated with 1 300 ug / mL / m2 of ecteinascidin 743. Patient blood samples were drawn just before administration of the drug, and at the end of a 3-hour infusion. The purified monocytes were isolated immediately and cultured with M-CSF (20 ng / mL) for 5 days to induce the differentiation of macrophages and then analyzed in phenotype expression. Of 1 2 evaluable patients, the monocytes of 6 subjects showed reduced macrophage differentiation after the treatment of ecteinascidin 743. Table 1 shows the phenotype analysis of the in vitro differentiated macrophages from patients whose post-therapy cells showed at least 50% inhibition of expression of CD206, CD16 and CD68, compared to cells collected before therapy. The data shown are the whole numbers of positive cells in a marker for a total of 10,000 input cells. The monocytes collected in the other six patients did not show a significant reduction in the ability to differentiate. Table 1. Effect of in vitro treatment with ecteinascidin 743 on the in vitro differentiation of macrophages in patients with tumors *% inhibition of differentiation of macrophages with reference to the cells before the infusion.

We also investigated whether in vivo treatment with ecteinascidin 743 causes measurable monocytopenia in patients with cancer. The monocyte values were obtained from the blood formula during a routine clinical analysis. Of 9 patients whose morphological analyzes of monocytes were recorded and available, 7 patients showed a reduction (25% inhibition compared to pre-infusion values, in at least one cycle) in the number of monocytes, evaluated as both% of monocytes on total leukocytes, and as a whole number of monocytes / ul of blood. The results of three representative patients are shown in Figure 5. Despite a level of contrast or a transient increase in the total number of leukocytes, in the first few days following the infusion of the drug, monocytes never increased, and in fact they were reduced frequently. Example 3 Ecteinascidin 743 inhibits the production of inflammatory cytokines / chemokines Monocytes / macrophages are potent producers of soluble factors that orchestrate the inflammatory / immune response. Therefore, the effect of the treatment of ecteinascidin 743 on the secretory function of these cells was tested. The chemokine CCL2 is a major chemoattractant for mononuclear phagocytes and is produced by immune cells as well as by many tumor cells. CCL2 derived from tumors attracts circulating monocytes at the location of the tumor and the TAM content of a tumor correlates with CCL2 levels, as has been demonstrated in many tumors. Monocytes and differentiated macrophages in vitro were stimulated with LPS (100 ng / mL). One hour after stimulation with LPS, they were treated with ecteinascidin 743. After 16 hours of incubation, the cell supernatants were harvested and tested in ELISA. Under these treatment conditions, cell viability was usually > 85% for concentrations up to 5 nM. The treatment with ecteinascidin 743 reduced, depending on the dose, the production of CCL2 by monocytes stimulated by LPS and derived macrophages in vitro (Figure 6A). The mean inhibition at 5 nM, for monocytes, was 65% (range of 60-80%, n = 5) and was 50% (range of 25-75%, n = 5) for differentiated macrophages in vitro. The results are the mean +/- standard error of 3-5 experiments. Next, TAMs associated with ovarian carcinomas were tested. Freshly isolated ovarian tumor cells were incubated with ecteinascidin 743 for 16 hours. The TAMs were stimulated by LPS (100 ng / mL). The cell supernatants were harvested and tested in ELISA. The mean results are +/- SE of 4 experiments for TAM and 1 experiment for tumor cells. The production of CCL2 stimulated by LPS was reduced by 50% (range of 40 to 60%, N = 4) (Figure 7A), while its constitutive production was reduced by 43% (range from 30 to 50%, n = 4). Two other cytokines, IL-6 and TNF, produced by macrophages and tumor cells, which have inflammatory properties and also act as growth factors for some tumors, were also tested. The production of IL-6 was always reduced after treatment with ecteinascidin 743, with a total inhibition at 5 nM of 54% (range of 51 to 57%, n = 2) and 69% (range of 66 to 72%, n = 2), in monocytes and macrophages, respectively (Figure 6B). The release of IL-6 in TAM was somewhat more resistant to treatment: at 5 nM the mean inhibition was 35% (range 25-53%, n = 4); to 10 nM was 47% (range of 33 to 63%, n = 4), (Figure 7B). It is also of interest that ecteinascidin 743 also reduced the constitutive production of CCL2 and IL-6 by newly isolated tumor cells. A representative experiment is shown in Figure 7. In contrast, and surprisingly, when monocytes, macrophages and TAM differentiated in vitro stimulated with LPS (100 ng / mL), treated with ecteinascidin 743 preceded by 1 hour of LPS stimulation, and 16 hours after the incubation, the cell supernatants were harvested and tested in ELISA. It was observed that the production of TNF by monocytes / macrophages, as well as that generated by TAM was never inhibited, even up to 10 nM for TAM (Figure 8A), suggesting that ecteinascidin 743 interferes only with select genes. These Results also indicate that, under these conditions, the cells were not damaged by the treatment. To verify if the inhibitory effect of ecteinascidin 743 on cytokine production was at the level of transcription, we analyzed the mRNA of CCL2 and TNF of macrophages stimulated by LPS by means of transcripts of real-time PCR of CCL2 and TNF in monocytes. stimulated by LPS exposed to ecteinascidin 743. As shown in Figure 8B, after treatment with ecteinascidin 734, a reduction consisting of transcripts of CCL2 was observed, whereas TNF and mRNA were not affected, in line with the results obtained by ELISA. Taken together, these results indicate that ecteinascidin 743 at drug concentrations reduces the production of two important inflammatory cytokines in molecular phagocytes and tumor cells. Example 4 Other ecteinascidin compounds also inhibit the production of inflammatory cytokines / chemokines. In addition, five other ecteinascidin compounds (Table 2) were tested in their ability to inhibit the production of CCL2 by human monocytes in vitro. Of the five compounds tested, only derivative A of ET637 showed a marked and consistent ability to modulantly reduce the production of inflammatory cytokine by monocytes, at concentrations of 2.4 and 5 nM. These concentrations did not affect the viability of monocytes after 48 hours of exposure. The extent of inhibition of derivative A of ET637 was even more pronounced compared to ET743. In Table 2 it is shown that the production of CCL2, induced by exposure of monocytes to supernatants of tumor cells is inhibited by up to 80% and 97% at 2.5 and 5 nM, respectively, in two different donors. In the same experiment, ET743 inhibited between 30% and 70%. The other compounds also showed inhibitory activity, but at lower levels than those of the two compounds mentioned hereinabove. Table 2. Inhibitory effect of ET743 and other ecteinascidin compounds on the production of the inflammatory chemotactic cytokine CCL2 Similar results were obtained when the monocytes were stimulated with LPS (100 ng / mL) and treated with ET743 and the other ecteinascidin compounds, although the overall inhibition was less marked in comparison with the previous experiment where the floating tumor was used as a stimulus inductor of CCL2. In Figure 10 it is confirmed that derivative A of ET637 gives a significant inhibition of CCL2 production. Example 5 Comparison of ecteinascidin 743 with antineoplastic agents currently used in ovarian cancer Since ecteinascidin 743 is being actively studied for the treatment of ovarian adenocarcinoma, it was of interest to compare its anti-inflammatory effects with those of other compounds conventionally used in the treatment of ovarian adenocarcinoma. this disease, more specifically, Doxorubicin, Cisplatin, and Taxol. The monocytes were incubated for 48 hours at the indicated concentrations of ecteinascidin 743, Doxorubicin, Taxol and Cisplatin. Viability was established by Pl staining and analyzed by flow cytometry. Figure 9A shows that, in active concentrations in tumor cells (> 0.5 μ?), Doxorubicin was highly cytotoxic for monocytes 48 hours after treatment, while Cisplatin and Taxol did not. Significant toxicity with Cisplatin was observed only at very high concentrations (40 μ?) While Taxol was ineffective at 300 nM. The production of CCL2 and TNF by monocytes stimulated by LPS treated with the indicated doses of anti tumor agents was also tested. The cell supernatants were harvested after 24 hours of incubation and were tested with ELISA. As shown in Figure 9B, Taxol and Doxorubicin were ineffective, but DDP (Cisplatin) (10 μ?) Reduced the production of CCL2. None of these compounds interfered with the production of TNF. These results indicate that the cytotoxicity of monocytes and the inhibition of CCL2 are not generalized properties of anti-tumor agents conventionally used in the treatment of ovarian cancer. Discussion In this study we have evaluated the cytotoxic effect of ecteinascidin 743 on mononuclear phagocytes. Circulating blood monocytes were highly susceptible to drug, and underwent apoptosis at concentrations of 5 nM / 48 hours. Differentiated macrophages in vitro and tumor-associated macrophages (TAM) were also susceptible to 5-1 0 nM. These values are within the range of therapeutically effective concentrations. At low concentrations of ecteinascidin 743 the differentiation of monocytes into macrophages was inhibited. We have confirmed these results by studying monocytes from patients suffering from tumors and who are receiving ecteinascid therapy 743. In 6 out of 1 2 patients tested, monocytes collected three hours after the infusion (1300 mg / m2) showed > 50% inhibition of macrophage differentiation in vitro compared to monocytes collected before therapy. Furthermore, significant monocytopenia has been observed in the first few days following the infusion of the drug in most patients. These results indicate that a brief in vivo exposure to ecteinascid 743 is sufficient to provide a cytotoxic effect on monocytes. An important finding of this work is the inh ibido activity of ecteinascidin 743 on the production of inflammatory cytokines. Among various inflammatory cytokines produced by monocytes / macrophages the I L-6 has been tested, the TN F and the chemokine CC L2. CCL2 is a chemokine that attracts monocytes and other subsets of leukocytes, and is produced by monocytes / macrophages and various tumor cells. It has been described that ovarian adenocarcinoma cells produce large amounts of CCL2 and that their levels correlate with the macrophage content of the tumors. CCL2 is then one of the most important factors in regulating the recruitment of monocytes / macrophages in the location of the tumor. Ecteinascidin 743 strongly inhibited the release of CCL2 by activated monocytes, macrophages and TAM by LPS. The ecteinascidin 743 also strongly inhibited the constitutive production of CCL2 produced by freshly isolated ovarian tumor cells. Therefore, lower levels of CCL2 produced by TAM and tumor cells are likely to reduce the number of macrophages recruited at the tumor location. In the in vitro experiments described above, ecteinascidin 743 was present throughout the 16-hour culture period. We also reviewed whether a shorter in vitro exposure to ecteinascidin 743 was sufficient to affect cytokine production. The monocytes exposed to ecteinascidin 743 were washed after 1 hour of culture and placed in a fresh medium. Under these conditions, the inhibition of CCL2 production was still significant, although a little lower compared to the cells receiving 16 hours of treatment (57% and 69% inhibition, respectively). IL-6 is a pro-inflammatory cytokine with important effects on the immune / hematopoietic system, and is an adjunctive factor for the production of CCL2. Additionally, many studies have pointed out that IL-6 can act as a factor in growth for some tumor cells, including ovarian cancer. With respect to CCL2, IL-6 induced by LPS was drastically reduced in monocytes / macrophages by ecteinascidin 743. The constitutive production of IL-6 from newly isolated tumor ascitic cells was also reduced. A novel and recently described effect of IL-6 is its ability to rescue T lymphocytes from the regulatory suppression of T cells mediated by (Treg). Treg is a small, but very important subgroup of T-lymphatics that controls T-cell auto-reactivity and maintains homeostasis. The role of Treg in autoimmune diseases is well recognized. The self-reactive T lymphocytes suppressed by Treg can be rescued by IL-6, thus perpetuating the autoimmune reaction. Therefore, the reduction of IL-6 regulated by ecteinascidin 743 could be a favorable therapeutic effect. Ecteinascidin 743 has never been considered for the treatment of chronic inflammatory disorders. The results of this study indicate that, due to its cytotoxic effect on the precursors of antigen-presenting cells (ie, monocytes) and its ability to reduce IL-6, ecteinascidin 743 is an interesting candidate for anti-inflammatory therapy . Unlike CCL2 and IL-6, ecteinascidin 743 had no significant effect on the production of TNF, another important inflammatory mediator, produced by monocytes / macrophages stimulated by LPS.

It has been shown that other ecteinascidin compounds, as well as ET743 are able to inhibit the production of CCL2 by human monocytes. Of the compounds tested, derivative A of ET637 has shown a marked and consistent ability to modulably reduce the production of CCL2. The extent of inhibition of derivative A of ET637 was even more pronounced compared to ET743. The other compounds also showed inhibitory activity, but at lower levels. In conclusion, the discovery that ecteinascidin 743 and other ecteinascidin compounds affect the viability and functions of monocytes / macrophages contains novel effects of these compounds, as well as a new therapeutic indication.

Claims (9)

1. A method for treating inflammation, comprising administering an effective amount of an ecteinascidin compound of the general formula (I): wherein: R5 is OH, alkoxy or alkanoyloxy; R6 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R 2 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R16 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R 7 is OH, alkoxy or alkanoyloxy; R18 is OH, alkoxy or alkanoyloxy; R21 is H, OH, CN or another nucleophilic group; and Ra is hydrogen and R is optionally substituted amino, or Ra with R forms a carbonyl function = 0, or Ra, Rb and the carbon to which they are attached form a group tetrahydroisoquinoline.

2. The method according to claim 1, further characterized in that the inflammation is caused by a disease selected from the group consisting of chronic inflammatory diseases, autoimmune diseases and atherosclerosis.

3. The method according to claim 1, further characterized in that in the ecteinascidin compound of the formula (I), the group Rs is an alkanoyloxy.

4. The method according to claim 1, further characterized in that in the ecteinascidin compound of the formula (I), the R6 group is methyl.

5. The method according to claim 1, further characterized in that in the ecteinascidin compound of the formula (I), the group R12 is methyl.

6. The method according to claim 1, further characterized in that in the ecteinascidin compound of the formula (I), the R16 group is methyl. The method according to claim 1, further characterized in that in the ecteinascidin compound of the formula (I), the R 7 group is methoxy. The method according to claim 1, further characterized in that in the ecteinascidin compound of the formula (I), the group R 8 is OH. 9. The method according to claim 1, further characterized in that in the ecteinascidin compound of the formula (I), the group R21 is H, OH or CN; and Ra is hydrogen and Rb is an amido group, or Ra with Rb form = 0, or Ra, R and the carbon to which they are attached form a group of the formula (II): wherein Ra is hydrogen and Rb is amido of formula -NHRf wherein Rf alkanoyl, or Ra with Rb form = 0, or Ra, Rb and the carbon to which they are attached form a group of formula (II): Rd is alkanoyl; and R2 is H, OH or CN. The method of claim 10, characterized in that ecteinascidin compound is selected from the group consisting of ET594 ET743 derivative A ET637 derivative B ET745 12. The use of an ecteinascidin compound of the general formula (I): wherein R5 is OH, alkoxy or alkanoyloxy; R6 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R 12 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R16 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R 7 is OH, alkoxy or alkanoyloxy; R 8 is OH, alkoxy or alkanoyloxy; R is H, OH, CN or another nucleophilic group; and Ra is hydrogen and Rb is an optionally substituted amino, or Ra with Rb forms a carbonyl function = 0, or Ra, R and the carbon to which they are attached form a tetrahydroisoquinoline group in the preparation of a medicament for use in a method of according to any of the preceding claims. 13. A medicament for the treatment of inflammation containing an ecteinascidin compound of the general formula R6 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R 12 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R16 is hydrogen, alkyl, alkenyl, alkynyl or aryl; R17 is OH, alkoxy or alkanoyloxy; R18 is OH, alkoxy or alkanoyloxy; R21 is H, OH, CN or another nucleophilic group; Y Ra is hydrogen and Rb is an optionally substituted amino, or Ra with Rb form a carbonyl function = 0, or Ra, Rb and the carbon to which they are attached form a tetrahydroisoquinoline group, and a pharmaceutically acceptable carrier.