Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

• the present invention relates in particular to pharmaceutical compositions containing derivatives of ⁇ -carboline, an alkaloid, corresponding in particular to those extracted from Peganum harmala, such as harmine, harmane and harmalacidin, and their use in the context cancer treatment.
• Peganum harmala is used in traditional medicine in many conditions.
• Peganum alkaloids are known for their antibacterial, antifungal, antiviral, hypothermic properties and especially their hallucinogenic effects.
• a previous study demonstrated an anti-tumor activity of extracts of P. harmala seeds on rat and mouse tumors .
• the administration of a crude alkaloid extract of P. harmala seeds, at a dose of 50 mg / kg / day, orally caused grafted mouse tumors (subcutaneous) to disappear in 80% of the mice treated (Lamchouri et al.
• the present invention aims to provide pharmaceutical compositions comprising compounds derived from / 3-carboline for the preparation of medicaments intended for the treatment of cancers.
• Another object of the present invention is to provide another compound capable of entering into synergy with compounds derived from / 3-carboline for the preparation of medicaments intended for the treatment of cancers.
• - Ri represents H, OH, or an alkoxyl group of 1 to 12 carbon atoms
• - R 2 represents H, an alkoxycarbonyl group of 1 to 12 carbon atoms, in particular the tert-butoxycarbonyl group, or an alkyl group from 1 to 12 carbon atoms
• - R 3 represents O or CH, provided that when R 3 represents O, then a represents a double bond, b and c represent a single bond and Rt represents H, and that when R 3 represents CH 3 then a represents a single bond, b and c represent a double bond and P does not represent any group; or its pharmaceutically acceptable salts, for the preparation of a medicament intended for the treatment of cancers, such as colon cancers, leukemias, myelomas, breast cancers, neuroblastomas, hepatocarcinomas, lung cancers, prostate cancer, ovarian cancer, testicular cancer, gastric cancer, pancreatic cancer, or retinoblastoma.
• cancers
• At least one compound of general formula (1) is associated with at least one compound which inhibits DNA replication.
• “Compound inhibiting DNA replication” means any compound capable of inhibiting a stage of DNA replication, whether by inhibiting the activity of the enzymes involved in replication, such as DNA polymerases, topoisomerases, helicases, primases, ligases, or by binding or modifying DNA, for example by binding to two strands of DNA (such as alkylating agents), or by preventing the synthesis of thymidine.
• the association of a compound of general formula (1) with a compound which inhibits DNA replication has synergistic effects in the treatment of tumors.
• the compound of general formula (1) is advantageously used as an adjuvant intended to increase the effects of compounds inhibiting DNA replication in the context of the preparation of anti-tumor drugs.
• association means that the compound of general formula (1) and the DNA replication inhibiting compound are both present structurally independently in a drug or pharmaceutical composition according to the invention and that they are not linked together by strong chemical bonds of the covalent or coordination type.
• the pharmaceutically acceptable salts of the compounds of general formula (1) the hydrochloride salts are particularly preferred.
• the compound of general formula (1) corresponds: - to the compounds of formula (2) below, - or to the compounds of formula (3) below,
• the compound of general formula (1) corresponds to: - the harmony (4):
• harmine as well as harmalacidin has an anti-tumor action in vivo, in particular on human tumors. This action is enhanced when harmine and harmalacidin are administered in combination with a compound that inhibits DNA replication.
• the pharmaceutically acceptable salts of the compounds of formulas (4), (5) and (6) above can also be used according to the invention, and in particular harmine hydrochloride, harmane hydrochloride and hydrochloride d 'harmalacidine.
• ha ⁇ nane whose chemical structure is close to that of harmine, has a common cell target with harmine (Sobhani et al. (2002) J. Pharm. Pharmaceut. Sci. 5: 19-23).
• the compound inhibiting DNA replication is chosen from the group comprising: - an alkylating agent, such as cyclophosphamide, mitomycin C or thiotepa; - an antimetabolite, such as 5-fluorouracil, macaw C or methotrexate; - a platinum coordination complex, such as carboplatin or cisplatin; - or an agent inhibiting topoisomerase II, such as doxorubicin, mitoxantrone or amsacrine.
• alkylating agent such as cyclophosphamide, mitomycin C or thiotepa
• an antimetabolite such as 5-fluorouracil, macaw C or methotrexate
• - a platinum coordination complex such as carboplatin or cisplatin
• - or an agent inhibiting topoisomerase II such as doxorubicin, mitoxantrone or amsacrine.
• an alkylating agent acts by preventing the separation of the two DNA strands of the same fragment by carrying out a solid covalent bridging.
• An antimetabolite prevents either by taking the place of the bases (5-fluorouracil or ara-C), or by inhibiting the enzymatic synthesis of thymidine (5-fluorouracil, methotrexate).
• the platinum coordination complexes in particular carry out a solid bridging between the two DNA strands of the same fragment.
• Topoisomerase II has the activity of cutting and re-soldering the two DNA strands of the same fragment in the context of the relaxation of supercoiled DNA.
• this relates to the use as defined above, in which the molar amount of compound of general formula (1) is greater than the molar amount of the compound inhibiting the replication of the DNA associated with it.
• the molar quantity of compound of general formula (1) is less than 20% greater than the molar quantity of the compound which inhibits the replication of the DNA which is associated with it.
• it relates to the use as defined above of harmine or harmalacidin, and of cyclophosphamide.
• it relates to the use as defined above of harmine or harmalacidin, and of 5-fluorouracil.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising as active substance at least one compound of general formula (1), or one of its pharmaceutically acceptable salts, in combination with at least one compound inhibiting DNA replication and a vehicle pharmaceutically acceptable.
• the pharmaceutical composition is such that the compound of general formula (1) corresponds to: - the harmine (4):
• the pharmaceutical composition is such that the compound inhibiting DNA replication is chosen from the group comprising: - an alkylating agent, such as cyclophosphamide, mitomycin C or thiotepa ; - an antimetabolite, such as 5-fluorouracil, macaw C or methotrexate; - a platinum coordination complex, such as carboplatin or cisplatin; - or an agent inhibiting topoisomerase II, such as doxorubicin, mitoxantrone or amsacrine.
• the pharmaceutical composition according to the invention is suitable for administration by oral or intravenous route.
• the pharmaceutical composition according to the invention comprises, as active substance, harmine or harmalacidin, in combination with cyclophosphamide and a pharmaceutically acceptable vehicle.
• the abovementioned pharmaceutical composition is suitable for administration by the oral route: from approximately 1 to approximately 10 mg / kg / d of harmine, or from approximately 1 to approximately 5 mg / kg / d d of harmalacidin, and from about 1 to about 5 mg / kg / d of cyclophosphamide.
• the abovementioned pharmaceutical composition is suitable for administration by the intravenous route: from approximately 1 to approximately 3 mg / kg / d of harmonic acid, or from approximately 1 to approximately 3 mg / kg / d of harmonalacidin, and from about 1 to about 5 mg / kg / d of cyclophosphamide.
• the pharmaceutical composition according to the invention comprises, as active substance, harmine or harmalacidin, in combination with 5-fluorouracil and a pharmaceutically acceptable vehicle.
• the abovementioned pharmaceutical composition is suitable for administration by the oral route: from approximately 1 to approximately 10 mg / kg / d of harmine, or from approximately 1 to approximately 5 mg / kg / d d of harmonalacidin, and from approximately 1 to approximately 10 mg kg / d of 5-fluorouracil.
• the abovementioned pharmaceutical composition is suitable for administration by the intravenous route: from approximately 1 to approximately 3 mg / kg / d of harmonic acid, or from approximately 1 to approximately 3 mg / kg / d of harmonalacidin, and from about 1 to about 5 mg / kg / d of 5-fluorouracil.
• the pharmaceutical composition as defined above is suitable for the administration of a molar quantity of compound of general formula (1) greater than the molar quantity of the compound inhibiting the replication of the DNA which it is associated.
• the pharmaceutical composition is suitable for the administration of a molar quantity of compound of general formula (1) at least 20% greater than the molar quantity of the compound inhibiting the replication of the DNA which is associated with it.
• the present invention also relates to products containing - at least one compound of general formula (1), or one of its pharmaceutically acceptable salts, and - at least one compound which inhibits DNA replication, as combination products for simultaneous, separate or spread over time in the treatment of cancers, such as colon cancer, leukemia, myeloma, breast cancer, neuroblastoma, hepatocarcinoma, cancer lung, prostate cancer, ovarian cancer, testicular cancer, gastric cancer, pancreatic cancer, or retinoblastoma.
• cancers such as colon cancer, leukemia, myeloma, breast cancer, neuroblastoma, hepatocarcinoma, cancer lung, prostate cancer, ovarian cancer, testicular cancer, gastric cancer, pancreatic cancer, or retinoblastoma.
• the term "combination” means that the compound of general formula (1) and the DNA replication inhibiting compound are both structurally independent in the products according to the invention and that they are not linked to each other by strong chemical bonds of
• the combination products are such that the compound inhibiting DNA replication is chosen from the group comprising: - an alkylating agent, such as cyclophosphamide, mitomycin C or thiotepa; - an antimetabolite, such as 5-fluorouracil, macaw C or methotrexate; - a platinum coordination complex, such as carboplatin or cisplatin; - or an agent inhibiting topoisomerase II, such as doxorubicin, mitoxantrone or amsacrine.
• an alkylating agent such as cyclophosphamide, mitomycin C or thiotepa
• an antimetabolite such as 5-fluorouracil, macaw C or methotrexate
• - a platinum coordination complex such as carboplatin or cisplatin
• - or an agent inhibiting topoisomerase II such as doxorubicin, mitoxantrone or amsacrine.
• the invention relates to products as defined above, containing - harmine or harmalacidin, and - 5-fluorouracil, as combination products for simultaneous, separate use or spread over time as part of the treatment of cancers, such as colon cancer, breast cancer, hepatocarcinoma, lung cancer, prostate cancer, ovarian cancer, gastric cancer, or pancreatic cancers.
• cancers such as colon cancer, breast cancer, hepatocarcinoma, lung cancer, prostate cancer, ovarian cancer, gastric cancer, or pancreatic cancers.
• the invention relates to products as defined above, containing: - harmine or harmalacidin, and - cyclophosphamide, as combination products for simultaneous, separate or spread over time as part of the treatment of cancers, such as colon cancer, leukemia, myeloma, breast cancer, neuroblastoma, hepatocarcinoma, lung cancer, ovarian cancer, cancer of the testicle, or retinoblastomas.
• the products as defined above comprise a molar amount of compound of general formula (1) greater than the molar amount of the DNA replication inhibiting compound with which it is in combination.
• the products comprise a molar quantity of compound of general formula (1) at least 20% greater than the molar quantity of the DNA replication inhibiting compound with which it is combined.
• Figure 1 represents the evolution of the size of HT29 tumors (in cm 3 , ordinate axis) grafted on untreated NOD-SCID mice (diamonds), or treated with harmine at 100 mg / kg / day (cross), 125 mg / kg / day (squares), 150 mg / kg / day (triangles) or 175 mg / kg / day (circles) depending on time (in days, abscissa axis).
• Figure 2A represents the evolution of the size of HT29 tumors (in cm 3 , ordinate axis) grafted on untreated NOD-SCID mice (cross), or treated with cyclophosphamide at 50 mg / kg / day (triangles ), 100 mg / kg / day (squares), or by a mixture of harmine 100 mg / kg / day + cyclophosphamide 100 mg / kg / day (circles) or harmine 150 mg / kg / day + cyclophosphamide 50 mg / kg / day (diamonds) as a function of time (in days, x-axis).
• Figure 2B represents the evolution of the size of HT29 tumors (in cm 3 , ordinate axis) grafted on untreated NOD-SCID mice (diamonds), or treated with harmine at 150 mg / kg / day (triangles), cyclophosphamide at 50 mg / kg / day (cross), or by a harmine mixture at 150 mg / kg / day + cyclophosphamide at 50 mg / kg / day (circles) as a function of time (in days, axis abscissa).
• Figure 3A represents the evolution of the size of HT29 tumors (in cm 3 , ordinate axis) grafted on untreated NOD-SCID mice (circles), or treated with 5-fluorouracil at 3 mg / kg / day (triangles), 6 mg / kg / day (squares), 9 mg / kg / day (cross), 12 mg / kg / day (diamonds) or 24 mg / kg / day (dashes) as a function of time (in days, abscissa axis).
• Figure 3B represents the evolution of the size of HT29 tumors (in cm 3 , ordinate axis) grafted on untreated NOD-SCLD mice (triangles), or treated with harmine at 150 mg / kg / day (cross), 5-fluorouracil at 12 mg / kg / day (squares), or by a harmine mixture at 150 mg / kg / day + 5-fluorouracil at 12 mg / kg / day (circles) as a function of time (in days, abscissa axis).
• Figure 4 represents the evolution of the size of HT29 tumors (in cm 3 , ordinate axis) grafted on untreated (square) NOD-SCID mice, or treated with harmalacidin at 25 mg / kg / day (triangles) or at 50 mg / kg / day (cross), with cyclophosphamide at 50 mg / kg / day (diamonds), or by a harmalacidin mixture 25 mg / kg / day + cyclophosphamide 50 mg / kg / day (dashes ) or harmalacidin 50 mg / kg / day + cyclophosphamide 50 mg / kg / day (circles), as a function of time (in days, abscissa axis).
• Harmalacidin (C) the molecular mass of the hydrochloride dihydrate of harmonalacidin: M 288.
• cytotoxic effects of the various alkaloid extracts of P. harmala have been studied on several cell lines: - human leukemic cell lines: K562 and Jurkat (leukemia), U937 (myeloma), - human solid tumor cell lines: KB (nasopharyngeal epithelioma) and HT29 (colon), - immortalized endothelial cell lines of human bone marrow: HBMEC.
• the cells are cultured in an incubator under a 5% CO 2 atmosphere and at 37 ° C., in RPMI1640 medium for K562, Jurkat, U937 and HT29 and DMEM for KB, supplemented with 10% fetal calf serum, penicillin-streptomycin 0.01 % and 2 mM L-glutamine and in EGM2 medium for HBMEC cells.
• the cytotoxicity test is carried out in a 96-well microplate in the presence of the extract to be tested at various concentrations of 40, 20, 10, 5, 1, 0.5 ⁇ g / ml and in the absence of product, after a 4 day incubation at 37 ° C. On Day 3, we add a neutral red solution which is absorbed by living cells.
• the optical density (OD) of dye released by lysed cells is measured at 540 nm by an Elisa plate reader.
• the toxicity (% growth inhibition) is inversely proportional to the optical density.
• the percentage inhibition is defined as being the difference between the OD without product and the OD in the presence of product compared to the OD without product.
• the concentration inhibiting at 50% the cell growth is obtained from the curve representative of the percentage of inhibition as a function of the logarithm of the concentration. All the results obtained are collated in the following Table 1.
• the IC 5 o vis-à-vis the cancerous and endothelial cells tested is as follows: 5 to 12 ⁇ g / ml for the crude alkaloid extract, 2.9 to 4.6 ⁇ g / ml (14 - 22 ⁇ M) for the harmine, 3 to 10 ⁇ g / ml (10.5 - 35 ⁇ M) for harmine hydrochloride, and 10.5 to 29 ⁇ g / ml (34.4 - 134 ⁇ M) for harmalacidin hydrochloride.
• NOD-SCID having received a xenograft of human tumor cells.
• NOD-SCLO mice male and female, over 3 months old, were raised in an environment of strict sterility, in an isolator ventilated by filtered and sterilized air, at 22 ° C and 40% humidity, under a day-12h / night-12h cycle.
• the cages, bottles and water have been sterilized in an autoclave at 120 ° C for 30 minutes and the food and the litter are treated by ⁇ irradiation. All the manipulations were carried out aseptically under a laminar flow hood.
• the mice were subjected to general anesthesia by ip injection of 0.3 to 0.4 ml of hypnomidate 2 mg / ml.
• the harmine-inhibiting action on tumor growth was manifested in a dose-dependent manner between 125 to 175 mg.
• the dose of 100 mg / kg / day has a very weak effect.
• the dose of 125 mg / kg / day begins to show a significant effect: the tumor volume reaches 55 to 67% of the tumor volume of the untreated group.
• the tumor volume represents only 42% of that of the control group until day 50, but reached 51% in the 60 th day.
• the dose of 200 mg / kg / day was not tolerated, the mice died after a few days of treatment.
• 150 mg of cyclophosphamide (Sigma) were dissolved in 8 ml of water and the solution obtained was sterilized by filtration on a membrane with a porosity of 0.22 ⁇ m.
• the mice received either 50, 100, or 150 mg / kg / day of cyclophosphamide alone, or a harmine mixture 150 mg / kg / day + cyclophosphamide 50 mg / kg / day or harmine 100 mg / kg / day + cyclophosphamide 100 mg / kg / day.
• Table 3 Treatment with cyclophosphamide, alone or in combination with harmine, of mice xenografted with human tumor cells HT29
• 50 mg of 5-fluorouracil (Sigma) were dissolved in 8 ml of water and the solution obtained was sterilized by filtration on a membrane with a porosity of 0.22 ⁇ m.
• the mice received either 3, 6, 9, 12, or 24 mg / kg / day of 5-fluorouracil alone, or a harmine mixture 150 mg / kg / day + 5-fluorouracil 12 mg / kg / day.
• the results obtained are presented in Table 4 and in Figures 3A and SB-
• mice xenografted with human tumor cells HT29 Dose Group
• Day 50 Day 60 1 0 0 100 100 100 100 100 100 2 0 3 43 56 66 61 86 3 0 6 37 53 60 64 70 4 0 9 41 41 59 52 59 5 0 12 73 66 54 47 59 6 0 24 17 54 54 7 150 12 25 26 22 21 23
• 5 -fluorouracil is delivered alone per os, at doses between 3 and
• Harmine 150 mg / kg / day was administered simultaneously in combination with increasing doses of 5-fluorouracil: 3, 6, 9 and 12 mg / kg / day respectively. Only the harmine 150 mg / kg / day + 5-fluorouracil 12 mg / kg / day association is given in Table 4 (harmine / 5-fluorouracil molar ratio of 7.7 / 1). This combination, administered in a 6-day treatment and 6-day rest cycle, has synergistic effects on the inhibition of tumor growth (Table 4, Figure 3B and was able to keep the tumor volume even less than 1.3 cm 3 on the 60 th day of treatment, with 23% T / C.

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