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US 20030087900A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0087900 A1 Telerman et al. (43) Pub. Date: May 8, 2003

(54) DRUGS WHICH CAN BE USED IN THE Related U.S. Application Data TREATMENT OF CANCER (63) Continuation of application No. 09/885,031, filed on Jun. 20, 2001, now abandoned. (75) Inventors: Adam Telerman, Paris (FR); Robert Amson, Paris (FR); Marius Tuijnder, (30) Foreign Application Priority Data Mechelen (BE) Jun. 1, 2001 (FR)...... 01/07285 Correspondence Address: Publication Classification PENNIE & EDMONDS LLP 7 1667 K Street, N.W. (51) Int. Cl...... A61K 3.1/542; A61K 31/495 Washington, DC 20006 (US) (52) U.S. Cl...... 514/225.2; 514/255.04 (57) ABSTRACT (73) Assignee: Molecular Engines Laboratories The present invention relates to the use of a compound (21)21) AppAppl. No.: 10,304,964/304, Inhibitinginhibiting ththe eXpreSSLOnpressi Off the TPT1 g ene, or off theth products which it controls, for producing a drug which is (22) Filed: Nov. 27, 2002 intended for treating cancer. Patent Application Publication May 8, 2003 Sheet 1 of 10 US 2003/0087900 A1

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Patent Application Publication May 8, 2003 Sheet 4 of 10 US 2003/0087900 A1

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Patent Application Publication May 8, 2003 Sheet 5 of 10 US 2003/0087900 A1

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Patent Application Publication May 8, 2003 Sheet 6 of 10 US 2003/0087900 A1

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Patent Application Publication May 8, 2003 Sheet 7 of 10 US 2003/0087900 A1

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Patent Application Publication May 8, 2003 Sheet 8 of 10 US 2003/0087900 A1

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DRUGS WHICH CAN BE USED IN THE 0013 Those derivatives which may be TREATMENT OF CANCER mentioned are dimethothiazine, hydroxyethylpromethazine, , mecquitazine, , , 0001. The present invention relates to the use of novel , , thiazinamium and trimepra classes of compounds which are designed for producing a drug which is intended for treating cancer. ZC. 0014 Those derivatives which may in particu 0002 Research carried out by the applicant, in particular lar be mentioned are , , , in the context of the phenomenon of tumor reversion, has led , , , flumarizine, homochlor to the demonstration that certain genes are overexpressed cycline, hydroxy Zine, meclozine, niaprazine and . during the tumor phase as compared with the reversion phase. 0015 Those ethanolamine derivatives which may be mentioned, in particular, are the phenhydramine derivatives 0003. The overexpression of one of these genes, i.e. the Such as bromodiphenhydramine and and gene TPT1, standing for “translationally controlled tumor their homologues. protein encoding releasing factor', demonstrated that the expression of this gene was Strongly decreased 0016. The ethylendiamines which may be mentioned are, during tumor reversion. for example, the compounds of the type. 0004 Thus, a tumor cell line designated U937 is capable 0017 Finally, examples of the various compounds which of reverting and thereby resulting in what is termed a US do not have a Sedative component and which may be cell, that is to Say a cell line which no longer exhibits Such mentioned are , , taZifyline and terfena a pronounced malignant phenotype characteristic. dine. 0005. In the U937 cell line, out of 2 000 sequences, for 0018. These compounds only represent the basic mol example, the number of clones for the TPT1 gene was 248 ecules which can be used in accordance with the present whereas it was no more than 2 in the US cell line. invention; it is also possible to use derivatives, in particular Substitution derivatives, of the abovementioned compounds, 0006. This has led the applicant to focus on the impor as well as physiologically acceptable Salts. tance of the histamine activation pathway in the phenom 0019. According to the present invention, treatment of enon of tumor reversion and to demonstrate the activity of the cancer is essentially understood as being the ability of a products which interfere with this pathway as a way of compound to Selectively destroy the tumor cells without treating cancer. appreciably affecting healthy cells, it being understood that 0007 More specifically, the present invention relates to this Selectivity can vary depending on the compounds and the use of compounds which totally or partially inhibit the depending on the condition of the patient and the type of expression of the TPT1 gene, or of the products which it cancer being treated. controls, for the purpose of producing a drug which is 0020. As will be observed in the examples which follow, intended for treating cancer. the products according to the present invention exhibit 0008. The present invention naturally relates not only to remarkable Selectivity and, in particular, a very great ability the possibility of inhibiting the expression of the TPT1 gene to destroy tumor cells, in particular, the results have been in the cell but also to that of totally or partially inhibiting the found to be particularly impressive in relation to cells of the expression of the products whose metabolic chain it controls leukemic type and in relation to breast cancer, ductal carci directly or indirectly, up to and including the release of noma and carcinoma of the mammary glands. histamine, in particular. 0021. The compounds according to the present invention 0009 Among the compounds which can be used within can be used in the form of pharmaceutical compositions the context of the present invention, those which may more which may be employed by any route of administration; Specifically be mentioned are the , that is to however, in a general manner, preference will be given to Say the antagonists of the histamine H1 receptors, in par using injectable routes, in particular, for treating tumors. It ticular. is, of course, possible to use other galenic forms, in particu lar the oral route. The daily doses have to take account of the 0010. It should be clearly understood that the antine compound, the condition of the patient and the nature and oplastic function of these compounds may be linked to Stage of the cancer being treated. Subsidiary elements of the cell, taking into account the fact that, for example, one of the prototypes, i.e. 0022. The appended results demonstrate that there is a polaramine, is inactive within the context of the present dose above which the product loses most of its activity. invention. 0023. It is also possible to envisage using the compounds 0.011 Among these compounds which can be used within according to the present invention in combination with other the present invention, those which may very particularly be antineoplastic agents, whether these be antimetabolites, mentioned are the phenothiazine derivatives and the deriva alkylating agents, Spindle poisons, intercalating agents or tives of the piperazine type. other types of hormonal cytolytic agents or antineoplastic agents, as well as certain proteins Such as interferons in 0012. It is also possible to use other derivatives of the accordance with additional chemotherapy processes, with ethanolamine or ethyldiamine type, or else the new genera the Said compounds being used together or Separately in tion of antagonists of the histamine H1 receptors which do accordance with a protocol which is to be determined for not have a Sedative component. each combination. US 2003/0O87900 A1 May 8, 2003

0024. The figures represent the results obtained, after 48 0039. These products are: hours or 144 hours, by treating either malignant or normal 0040 A) dihydrochloride (Atarax(R) cells with the compounds A, B, C or D. They depict the UCB). Solution: 100 mg/2 ml percentage of Surviving cells as a function of a treatment without compound or without product (control) or with a 0041 B) maleate (Dimegan(R) compound in accordance with a dilution as previously FEDERA S.A.). Solution: 10 mg/1 ml indicated. 0.042 C) Promethazine (Phenergan(R) Medeva Pharma S.A.). Solution: promethazine hydrochlo 0025 FIG. 1 illustrates the treatment of myeloid leuke ride: 2820 g/100 ml mia cells, which are derived from the cell line K562, with 0043 D) maleate compound A, B, C or D (1A: at the end of 48 hours, 1B: at (Polaramine(R) Schering-Plough). Solution: 5 mg/1 the end of 144 hours). ml. 0026 FIG. 2 illustrates the treatment of U937 premono 0044) These products were added, at different concentra cytic leukemia cells with compound A, B, C or D (2A; at the tions, to cultures of various malignant cell lines and also to end of 48 hours, 2B: at the end of 144 hours). normal cells. 0.027 FIG. 3 illustrates the treatment of acute leukemia T cells, derived from the Jurkat cell line, with compound A, B, C or D (3A at the end of 48 hours, 3B: at the end of 144 K562 myeloid leukemia KS revertant of K562 possessing reduced hours). tumorigenicity 0028 FIG. 4 illustrates the treatment of breast ductal U937 premonocytic leukemia US4 revertant of U937 possessing reduced carcinoma cells, derived from the T47-D cell line, with tumorigenicity compound A, B, C or D (4A: at the end of 48 hours, 4B: at Jurkat T lymphocyte, acute leukemia of T cells the end of 144 hours). T47-D breast cancer, ductal carcinoma MCF7 breast cancer, ductal carcinoma 0029 FIG. 5 illustrates the treatment of breast ductal BT2O breast cancer, carcinoma of the mammary carcinoma cells, derived from the MCF7 cell line, with glands LoVo colorectal adenocarcinoma compound A, B, C or D (5A: at the end of 48 hours, 5B; at 184B5 breast, immortalized, non-tumorigenic the end of 144 hours). cells of the luminal epithelium MCF1OA breast, immortalized, non-tumorigenic 0030 FIG. 6 illustrates the treatment of mammary gland luminal epithelium cells carcinoma cells, derived from the BT20 cell line, with Donors 1, 2, 3 T and B cells which have been freshly compound A, B, C or D (6A: at the end of 48 hours, 6B: at isolated from 3 healthy donors. the end of 144 hours). 0031 FIG. 7 illustrates the treatment of immortalized, 0045 Leukemic Cell Lines non-tumorigenic breast epithelium cells, derived from the 0046 All the leukemic cell lines were grown and used in 184B5 cell line, with compound A, B, C or D (7A: at the end logarithmic phase. After 1 day of treatment, the cells were of 48 hours, 7B: at the end of 144 hours). isolated, counted and diluted in a regular growth medium So 0032 FIG. 8A illustrates the treatment of lymphocytes as to obtain cell densities of 75.10 cells/ml and 9.375 from donor 1 with compound A or C, at the end of 48 hours. cells/ml for reading on plates at 48 hours and 144 hours after 0033 FIG. 8B illustrates the treatment of lymphocytes the treatment, respectively. from donor 2 with compound A or C, at the end of 48 hours. 0047. In the case of the leukemic cell lines, each dilution product was added to the wells (12 wells per plate, TPP), and 0034 FIG. 9 illustrates the treatment of lymphocytes 1 ml of cells from the parent solution were added per well from donor 3 with compound A or C, at the end of 48 hours. after a line had been completed. 0035 FIG. 10A illustrates the treatment of colorectal 0048 All the dilutions of each product were tested 4 adenocarcinoma cells, derived from the LoVo cell line, with times and counted manually and tested with the Alamar compound A or C, at the end of 48 hours. reduced assay. The products are diluted in culture medium. 0036 FIG. 10B illustrates the treatment of immortalized, non-tumorigenic breast luminal epithelium cells, derived from the 184B5 cell line, with compound A or C, at the end Control no product of 48 hours. 1:100 dilution 10 ul of product 1:1000 dilution 1 ul of product 0037. The examples given below will demonstrate other 1:2 000 dilution 50 ul of a 1:100 dilution features and advantages of the present invention. 1:5 000 dilution 20 ul of a 1:100 dilution 1:10 000 dilution 10 ul of a 1:100 dilution EXAMPLES 0.038. This study made use of a certain number of human 0049 Lymphocytes From Healthy Donors tumors of different origins and of lymphocytes from healthy 0050. The blood is collected on citrate and diluted 1:1 donors, with these tumors/lymphocytes being treated with with 0.15M NaCl. 6 ml of this blood dilution are loaded onto varying concentrations of compounds A, B, C and D So as 3 ml of lymphoprep (Nycomed) and centrifuged at ambient to determine the cytotoxicity of the latter. temperature for 30 minutes at 800 g. The white cells are US 2003/0O87900 A1 May 8, 2003

isolated and washed with RPMI1640+10% FBS. They are 0056 By contrast, compound B and compound D are diluted to 450 000 cells/ml in a RPMI1640+10 FBS found to possess little activity or to be inactive. medium. The same procedure as in the case of the leukemic 0057 Similarly, assays carried out with dilutions greater cells is then followed. than 1:1000, in particular 1:10 000, show that the compound 0051) Adherent Cells of the Breast and the Colon becomes inactive. 0.052 All the cells are grown on their own propagation 0058. The assays which were carried out using the lym medium and Seeded 24 hours before the products are added. phocytes from healthy donors show that the level of Survival The cells are trypsinized, counted and seeded at 50 000 and is very substantial at concentrations of 1:1000; a differential 10 000 cells/well in order to read the plates at 48 hours and effect between the lymphocytes from healthy donors and the 144 hours after the treatment. cancerous cells does therefore exist. 0053) On the day of the treatment, the medium is replaced 0059. In order to demonstrate that this phenomenon is not (1 ml/well) with the following dilutions: linked to a general cytotoxicity, assays were carried out on an LoVo cancer which was resistant to the cytopathic effect to the parvovirus H1. These assays show that the LoVo cancer is totally resistant to the antihistamines. Control no product 1:100 dilution 100 ul of product in 9.9 ml of 1. Use of a compound which inhibits the expression of the growth medium 1:1000 dilution 10 ul of product in 10.0 ml of TPT1 gene, or of the products which it controls, for pro growth medium ducing a drug which is intended for treating cancer. 1:2 000 dilution 5 ul of product in 10.0 ml of growth 2. Use as claimed in claim 1, characterized in that the medium compound inhibiting the expression of the TPT1 gene, or of 1:5 000 dilution 2 ul of product in 10.0 ml of growth the products which it controls, is an antihistamine. medium. 3. Use as claimed in claim 2, characterized in that the antihistamine belongs to the chemical group of the pipera Zines and the . 0.054 The product is regarded as being active when the 4. Use as claimed in claim 3, characterized in that the percentage of Surviving cells is less than 30%. product is Selected from hydroxy Zine and promethazine. 0.055 The enclosed results show that, when compounds A 5. Use as claimed in one of claims 1 to 4, characterized in and C were used at dilutions of from 1:100 to 1:1000, all the that the cancer being treated is a leukemia or a cancer of the cancerous cells were destroyed, particularly in the case of breast. K562, U937, Jurkat, T47-D, MCF7, BT20 and 184B5, either within 48 hours or, for the most part, within 144 hours.