WO2023275422A1 - Compound comprising ruthenium(iii) and 2,2'-biimidazole (runat-bi) and the therapeutic use thereof - Google Patents

Compound comprising ruthenium(iii) and 2,2'-biimidazole (runat-bi) and the therapeutic use thereof Download PDF

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WO2023275422A1
WO2023275422A1 PCT/ES2022/070415 ES2022070415W WO2023275422A1 WO 2023275422 A1 WO2023275422 A1 WO 2023275422A1 ES 2022070415 W ES2022070415 W ES 2022070415W WO 2023275422 A1 WO2023275422 A1 WO 2023275422A1
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runat
compound
cell
pharmaceutical composition
cancer
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PCT/ES2022/070415
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Spanish (es)
French (fr)
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Gloria RIBAS DESPUIG
Francisco José MARTÍNEZ LILLO
Marta ORTS ARROYO
Marta ALBANELL FERNÁNDEZ
Isabel CASTRO BLEDA
Elena JIMÉNEZ MARTÍ
Sara OLTRA SANCHÍS
Maider IBARROLA VILLAVA
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Universitat De València
Fundación Incliva
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/90—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • 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
    • A61K31/28—Compounds containing heavy metals
    • A61K31/295—Iron group metal compounds
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table

Definitions

  • the present invention belongs to the field of ruthenium(III) complexes and their use as therapeutic agents, especially as selective anticancer agents.
  • Chemotherapy is widely used to treat tumors, however, the drugs used often have high toxicities in healthy tissues.
  • platinum (II)-based anticancer drugs are being used for the treatment of cancer, although they have important side effects that limit their effectiveness and their activity against some tumors is sometimes limited. Furthermore, platinum(II)-based compounds have limited water solubility and short half-life.
  • Patent application CN101967164 discloses a Ru (II) complex, with the formula cis-[Ru(biim) 2 Cl 2 ] 2H 2 O, prepared from RuCl 3 nH 2 O, bilmidazole and lithium chloride, dissolved in dimethylformamide (DMF), and heating at reflux for 8 h under argon. A 72% yield of purple-black crystals was obtained after cooling to room temperature, adding acetone and cooling, precipitation and filtration.
  • this complex is an intermediate compound in the synthesis of others that are prepared later and its in vitro toxicity has not been evaluated in a panel of cancer cell lines.
  • the new Ru(III) compound, RUNAT-BI has not only been shown to be a potential antitumor agent but also to show a selective effect between tumor and non-tumor human cell lines.
  • the anticancer activity of the RUNAT-BI agent a racemic mixture of two isomers
  • AMD adult women
  • CMMJ young women's breast
  • HCT116 colon cancer cell line
  • AGS gastric cancer cell line
  • the MCF10A line from a non-tumoral human mammary gland was used. Of all of them, the results obtained in breast, colon, gastric cancer and a non-tumoral mammary gland cancer stand out. Ruthenium(III)-based complexes with 2,2'-biimidazole may have base pair interactions that further facilitate their mechanism of action with cancer cell DNA. Likewise, the fact that these compounds contain ruthenium (III) allows them to be even more robust and inert when it comes to reacting in redox processes or in ligand substitution reactions, thus increasing their stability and, therefore, extending their life. useful of the compound in the physiological environment.
  • a first aspect of the present invention relates to a mononuclear compound of Ru(III) with two molecules of 2,2'-biimidazole.
  • a second aspect of the invention refers to a process for the preparation of the Ru(III) compound - RUNAT-BI - defined above, comprising:
  • the temperature ramp comprises reaching a temperature between 85°C and 95°C, and more preferably 90°C.
  • the temperature ramp comprises reaching a temperature between 85°C and 95°C
  • temperature comprises reaching a temperature between 85°C and 95°C, and more preferably 90°C and maintaining said temperature for a period of time. time between 20 and 21 hours, preferably 20.5 h.
  • the time invested until reaching the final temperature is from 1 to 6 hours, preferably 5 hours.
  • the concentration of RuCl 3 H 2 O in the synthesis mixture is from 0.01 to 0.05 M, preferably 0.012 M.
  • the concentration of 2,2-biimidazole in the synthesis mixture is from 0.01 to 0.05 M, preferably 0.036 M.
  • the hydrochloric acid that can be used is commercial, and can have a concentration in the synthesis mixture of 1 to 6 M, preferably 3 M.
  • the cooling stage can last between 10 and 21 hours, preferably the cooling stage to room temperature is 20.5 hours.
  • a second aspect of the invention refers to the RUNAT-BI compound for therapeutic use, preferably for any disease characterized by a higher rate of cell growth with respect to the cells of healthy tissues.
  • a third aspect of the invention relates to the RUNAT-BI complex for its use as an anticancer agent.
  • the RUNAT-BI compound has antitumor activity. Specifically, the compound RUNAT-BI (racemic) has been shown to considerably reduce the proliferation, migration and expression of the HCT116 (colon), AGS (gastric) and HCC1806 (breast) tumor lines.
  • the selectivity of the compound of the invention between tumor and non-tumor cell lines may be due to the altered molecular mechanisms that these cells present. The latter, for example, the presence of mutations in oncogenes and other genes that tumor cells present and are not found in non-tumor cells. It may also be due to the high rate of replication of tumor cells. As the RUNAT-BI compound acts on DNA, it will affect more cells that have a higher replication rate, such as tumor cells.
  • the cancer is selected from among breast cancer, gastric cancer, and colon cancer.
  • the breast cancer is of the TNA and/or luminal B subtype.
  • the breast cancer (BC) is breast cancer in young women (JMMC).
  • the RUNAT-BI compound is stable for at least two weeks in solution in an organic solvent such as polyethylene glycol (PEG) and protected from exposure to light, at room temperature, that is, between 18 °C and 33 °C. , preferably 21 °C and 26 °C, more preferably 25 °C.
  • the RUNAT-BI compound as an isolated solid is stable for at least one month in contact with air at room temperature between 18 °C and 33 °C, preferably 21 °C and 26 °C, more preferably 25 °C. Therefore it is not necessary to prepare it immediately before use. No significant differences were found between the effect of the compound when it was prepared fresh (freshly prepared for use) or when it was prepared one to two weeks before use.
  • • RUNAT-BI is an agent whose action target is found in DNA, among other possible ones, since it affects cancer cell lines with greater proliferation capacity more.
  • the concentration of RUNAT-BI is between 0.01 ⁇ M and 1000 ⁇ M, preferably 1 ⁇ M and 100 ⁇ M, more preferably between 0.1 ⁇ M and 50 ⁇ M. Depending on the type of cancer, a lower or higher concentration may be used.
  • An additional object of the invention refers to a pharmaceutical composition comprising RUNAT-BI and at least one pharmaceutically acceptable carrier.
  • a “pharmaceutically acceptable carrier”, after administration, does not cause undesirable physiological effects.
  • the carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the RUNAT-BI, or active compound, and may be capable of stabilizing it.
  • One or more solubilizing agents may be used as pharmaceutical carriers or excipients for delivery of the active compound.
  • a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form.
  • examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow 10.
  • composition of the invention can be administered parenterally, orally, nasally, rectally, topically, or buccally.
  • parenteral refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.
  • the pharmaceutical composition of the invention may be administered as a sterile injectable which may be a solution or suspension in a non-toxic diluent or solvent acceptable for parenteral administration, such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • a sterile injectable which may be a solution or suspension in a non-toxic diluent or solvent acceptable for parenteral administration, such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • fixed oils are conventionally employed as a solvent or suspending medium (eg, synthetic mono- or diglycerides).
  • fatty acids such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are pharmaceutically acceptable natural oils, such as olive oil or castilla oil, in their polyoxye
  • oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents.
  • a long chain alcohol diluent or dispersant such as carboxymethyl cellulose or similar dispersing agents.
  • Other commonly used surfactants such as, Tweens or Spans or other emulsifying agents or similar bioavailability enhancers, which are commonly used in the manufacture of solid, liquid or other acceptable dosage forms may also be used for the purpose of formulation.
  • a composition for oral administration can be any orally acceptable dosage form, including capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
  • commonly used carriers include, among others, lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also often added.
  • useful diluents include lactose and dry corn starch.
  • the RUNAT-BI or the pharmaceutical composition can be administered in vivo or ex vivo, alone or in a composition together with other drugs or therapies, ie, a therapeutic cocktail.
  • the agents can be used alone or in combination with, for example, chemotherapeutic, radiotherapeutic, stroke, antiangiogenic and/or immunotoxin or coagulant agents.
  • the administration of a pharmaceutical composition of two or more agents/therapies is concurrent.
  • a first agent/therapy is administered before a second agent/therapy. It is understood by those skilled in the art that the formulations and/or routes of administration of the various agents/therapies used may vary.
  • the RUNAT-BI or the pharmaceutical composition can be administered as a treatment after or simultaneously with at least one treatment selected from among: surgery, radiotherapy, chemotherapy and immunotherapy.
  • the pharmaceutical composition includes the administration to the person of an additional therapeutic agent.
  • additional therapeutic agent include one or more selected from the group consisting of cis-platinum, 5-fluorouracil, oxaliplatin, irinotecan, capecitabine, gemcitabine, cetuximab, paclitaxel, docetaxel, bevacizumab, regorafenib, aflibercept, trastuzumab, pertuzumab, nab- paclitaxel, trastuzumab-emtansine, atezolizumab, and carboplatin.
  • the RUNAT-BI or the pharmaceutical composition of the invention can be used as treatment after or simultaneously with at least one treatment selected from among: surgery, chemotherapy, immunotherapy or pharmaceutical treatment.
  • the RUNAT-BI or the pharmaceutical composition can be administered with a variable administration interval, such as 1 to 7 times per week, preferably 2 to 6 times per week, more preferably 3 to 5 times per week. week.
  • RUNAT-BI is administered with an interval of 15, 21 or 28 days and in the form of cycles, for example, 4, 6, 8 or 12 cycles.
  • An expert in the field would know how to determine the best administration schedule for this compound. using common general knowledge.
  • the effective dose of RUNAT-BI and the pharmaceutical composition that comprises it depends on the nature of the conditions, the form of execution and the pharmaceutical formulation, and will be defined by the experience of a physician through the application of the usual investigations for a dose increase.
  • the effective dose can be considered to be 0.0001 to about 1000 mg/kg of body weight per day, or 0.01 to about 500 mg/kg of body weight per day, or 0.01 to 100 mg/ kg of body weight per day, or from 0.05 to about 10 mg/kg of body weight per day.
  • the daily dose for an adult person of about 70 kg body weight will be in the range of 1 mg to 1000 mg, or 5 mg to 500 mg, and can be accomplished by single or multiple, daily or non-daily doses.
  • Figure 1 Example of a temperature ramp for the synthesis of RUNAT-BI.
  • Figure 2 Molecular structure of the two cationic isomers that form the active racemic mixture RUNAT-BI. Hydrogen atoms, chloride anions, and H 2 O molecules in the formula have been omitted for clarity. The different connectivity of the atoms for the two isomers is shown in the Figure.
  • Figure 3 Percentage of viability of eight of the cell lines treated with fresh RUNAT-BI at 24, 48 and 72 h.
  • the MC lines MCF-7, HCC1937, MDA-MB-231, BT474, HCC1806, HCC1500, the gastric cancer line: AGS and the colon cancer line: HCT116. All cell lines were treated with fresh RUNAT-BI (0 to 42 ⁇ M) for 24 hours (A); 48 hours (B) and 72 hours (C). Cell proliferation was determined with the MTT assay. Points indicate the mean of three independent experiments.
  • Figure 4 Percentage of viability of the non-tumor cell line treated with fresh RUNAT-BI at 72 h.
  • the MCF10A mammary gland cell line has been used as a model to verify the selectivity of RUNAT-BI between tumor and non-tumor lines. This cell line has been treated with fresh RUNAT-BI (0 to 42 ⁇ M) for 72 hours. Cell proliferation was determined with the MTT assay. Points indicate the mean of three independent experiments.
  • RUNAT-BI not fresh at 24, 48 and 72 h.
  • FIG. 6 Comparison of cell migration in the "wound closure” assay in the cell lines studied after 48 hours of treatment. The cell migration of the cell lines was measured with the "wound closure” assay after being treated with the RUNAT-BI agent (21 ⁇ M) for 48 hours. Three independent experiments were performed and the most significant results are shown. Columns express the mean ⁇ SD of the percent closure in three independent experiments. Light gray color represents PEG controls while dark gray is RUNAT-BI treated cells. *P s 0.1, **P s 0.05, ***P s 0.01 statistically significant.
  • Figure 7 Comparison of cell migration in the "wound closure” assay in the cell lines studied after 72 hours of treatment. The cell migration of the cell lines was measured with the "wound closure” assay after being treated with the RUNAT-BI agent (21 ⁇ M) for 72 hours. Three independent experiments were performed and the most significant results are shown. Columns express the mean ⁇ SD of the percent closure in three independent experiments. Light gray color represents PEG controls while dark gray is RUNAT-BI treated cells. *P s 0.1, **P s 0.05, ***P s 0.01 statistically significant.
  • Figure 8 Cell growth of cell lines treated with RUNAT-BI. Representation of cell growth for 10 days to study the relationship between the effectiveness of RUNAT-BI and the speed of cell growth. Growth was measured with the Nuebauer chamber using the "Automated Cell Counter TC10TM". Points indicate the mean of two independent experiments.
  • the compound obtained, RUNAT-BI has the following infrared bands (IR/ cm -1 ): 3278 (m), 3147 (m), 3129 (m), 3010 (m), 2923 (m), 2765 (m ), 1638(s), 1526(s), 1417(m), 1394(m), 1319(w), 1252(w), 1177(m), 1129(m), 1078(m), 1008(w) ), 922 (w), 870 (w), 811 (w), 754 (s), 682 (m).
  • the temperature ramp (shown in figure 1) comprises a heating stage for 5 hours until reaching a temperature of 90 °C, maintaining this temperature for 20.5 hours, and a cooling stage to room temperature for 20.5 hours. .
  • MTT cell proliferation assays The protocol used was an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide) assay in which 3000 cells were grown in 96-well plates. Subsequently, the cells were treated with the specific agent and the MTT assay was performed at 24, 48 and 72 hours after treatment. MTT is a colorimetric assay indicating cell survival and proliferation.
  • % Viability (OD treated cells x 100) / OD control cells
  • the "wound healing assay” assay aims to study cell migration. It is an easy, cheap and widely used assay by researchers. It is based on the observation of the behavior of a confluent monolayer of cells that has previously been breached or "wounded". The cells at the edge of the gap will move towards the opening until establishing new cell-cell contacts, thus closing the "wound”. By capturing images at the beginning and at regular time intervals during cell migration near the gap, it is possible to compare and quantify cell migration. In the study, 6-well plates were plated at a density of 4 x 10 5 cells/well and incubated overnight until 70% confluent.
  • a pipette tip was used and then the cells were treated with RUNAT-BI (21 ⁇ M). Each experiment was performed in triplicate and repeated at least 2 times. times. Images were obtained at 0, 24, 48, and 72 h in the same position, and the percentage of cell migration was evaluated using the Imaged program, a program for processing digital images.
  • the cell lines of MC both those of CMMJ cancer: HCC1500 and HCC1937, those of CMMA: MCF-7, BT474, MDA-MB-231, HCC1806. Table 1 shows information on these cell lines:
  • the non-tumor mammary gland cell line (MCF10A), the gastric cancer cell line (AGS) and the colon cancer cell line (HCT116) have been used to study cell growth rate and determine cell doubling time. (CDT).
  • Cell counts were made at regular intervals. On day 0, cells were plated in 6-well plates, with an initial concentration of 1 x 10 5 cells per well, and each cell line was cultured in duplicate. Duplicate counts were made in a Neubauer chamber using “Automated Cell Counter TC10TM” (BioRad), every 24 h for 10 days after staining the cells with trypan blue. From the data of the cell counts, during the time studied, the cell growth curve was constructed and the CDT was calculated using the online tool http://www.doubling-time.com/compute.php
  • RUNAT-BI is prepared fresh before performing each experiment.
  • the MC lines belonging to the breast cancer group in adult women CMMA: HCC1806, MCF-7, BT474 and MDAMB-231 and those belonging to the breast cancer group in young women CMMJ: HCC1937 and HCC1500, the breast cancer cell line colon (HCT116), gastric cancer cell line (AGS) and mammary gland line of non-tumor human origin (MCF10A) were subjected to increasing concentrations of RUNAT-Bl (0, 5.25, 10.5, 21 and 42 ⁇ M) for 24, 48 and 72 hours. Said concentrations have been used since the prepared mother compound had an initial concentration of 42 mM (2 mg of product in 100 pL of PEG). RUNAT-BI was prepared before each experiment.
  • the MDA-MB-231 line reduced its viability by almost 30% at 48h and 40% at 72h ( Figure 3 B and C).
  • the BT474 line reduced 30% at 48h and 50% the viability in 72h.
  • the CMMJ line: HCC1937 was not one of the most affected, remaining at three times (24, 48 and 72 h) over 80% viability and the HCC1500 line did not present changes in its viability at any time, staying above 90% at 72 h.
  • Tables 3 and 4 show the viability results at 48 and 72 h.
  • Treatment with RUNAT-BI shows a relationship between the reduction of cell viability and growth speed (cell doubling time), observing that the lines that grow the fastest are the most affected by RUNAT-BI. This relationship is shown in more detail in the results of the cell doubling time calculation study.
  • MDA-MB-231 continues to be the line that reduces its viability the most, staying at around 60%, as was the case with RUNAT-BI fresh, as can be seen in graph C of Figure 3. The same This occurs with MCF-7 and HCC1937, they also show a greater reduction at 72 h, remaining around 75-80% in both cases, as occurs with fresh RUNAT-BI.
  • the MC lines belonging to the CMMA group: MCF7, MDA-MB-231, HCC1806 and BT474, those belonging to the CMMJ group: HCC1937 and HCC1500, the gastric cancer line: AGS, the colon cancer line: HCT116 and the from mammary gland of non-tumoral human origin: MCF10A were treated with 21 ⁇ M RUNAT-BI or 21 ⁇ M PEG (control).
  • a gap (wound) was made at 0 h and the percentage closure of said wound was measured using images from 0 to 72 h.
  • Figure 6 shows the results at 48 h and Figure 7 at 72 h. Table 6 shows the results at both times.
  • the "wound closure" assay demonstrated that the antitumor agent RUNAT-BI significantly reduces cell migration of these cell lines. More specifically, the HCC1806 line treated with RUNAT-BI showed a migration reduction of 47.1% at 48 h and 65.7% at 72 h compared to the HCC1806 line treated with PEG. AGS treated with RUNAT-BI also showed a very significant reduction, especially at 72 h, reaching 54.3% compared to the AGS line treated with PEG. HCT116 treated with RUNAT-BI did not show significant differences in the reduction of cell migration between 48 and 72 h, remaining in both cases between 21-22% closure compared to the line HCT116 treated with PEG.
  • AGS and HCT116 are cell lines with a very high growth rate, in the case of HCT116 in In the treatment with RUNAT-BI, a significant reduction in cell growth rate was observed in comparison with the control treatment (treated with PEG), while AGS maintained its high growth rate and cell doubling time, but showed a greater reduction in cell growth rate. cell migration. Migration values are obtained from the difference in wound area between 0 and 48 h or 72 h for each condition. These results are consistent with those previously observed in MTT assays, as these three lines showed a significant reduction in cell viability with RUNAT-BI treatment.
  • the BT474 line at 48 h in the treatment with RUNAT-BI showed a 14.1% reduction in cell migration and at 72 h reduced migration to 23.1% compared to the BT474 line treated with PEG. However, it should be noted that this cell line showed clear apoptosis with the RUNAT-BI treatment at 72 h, as it is a line that grows very slowly.
  • HCC1937 and HOC 1500 were among the lines that showed the least reduction in viability and similarity occurred with cell migration, HCC1937 treated with RUNAT-BI reduced its migration by 14.5 % at 48 h. 19.0% at 72 h with respect to the line HCC1937 treated with PEG. It should be noted that the samples of this cell line treated with RUNAT-BI considerably reduced their growth rate, as occurred in BT474.
  • HCC1500 hardly showed migration with the control treatment (PEG) at any time, probably due to the slow cell growth of this line and with the RUNAT-BI agent the opening of the gap was even increased at 48 and 72 h, observing a clear increase in dead cells between the images at 0 h and at 48 h and 72 h.
  • the non-tumor mammary gland line MCF10A did not show differences in migration between conditions, remaining practically the same in the treatment with RUNAT-BI as in the control, with PEG. Yes, differences were observed between 48 h and 72 h, since cell migration occurred at the latter time in both conditions. This line did not show apoptosis in the samples treated with RUNAT-BI, as shown in previous proliferation experiments. These results corroborate those obtained in the cell viability assay, once again demonstrating the selectivity of RUNAT-BI between tumor and non-tumor lines.
  • CDT Cell doubling time
  • the cell doubling time (CDT) of the cell lines HCC1500, HCC1937, MCF-7, BT474, MDA-MB-231, HCC1806, AGS, HCT 116 and MCF10A have been calculated. This value allows us to know the growth speed of each line, since the lower the CDT, the faster that line grows.
  • the relationship between the CDT and the effectiveness of the RUNAT-BI has been studied, since the main target of action is found in the DNA and we want to know the relationship between the speed of cell growth and the effectiveness of the agent.
  • RUNAT-BI has a greater effect on lines that divide faster, since, by replicating faster, the complex intercalates with the DNA that is being replicated, thus affecting cell viability.
  • Figure 8 shows the growth graph of each of the cell lines in 10 days
  • Table 7 shows the cell doubling time of the panel of cell lines in the experiment.
  • the lines HCT116, HCC1806 and AGS are the ones that show the greatest slope in the graph, therefore, the ones with the lowest CDT, as can be seen in the adjoining table, and they are also the ones in which RUNAT-BI has been most effective, see panel C of Figure 3.
  • the lines with the highest CDT and, therefore, grow slowest are HCC1500 and HCC1937.
  • lines BT474 and MDAMB-231 RUNAT-BI presented less effectiveness. This fact can be related to the fact that its growth rate is lower than HCT116, HCC1806 and AGS.
  • MCF-7 is a line that divides relatively quickly, however, RUNAT-BI does not have a very significant effect on it. Similarly, it occurs with MCF10A, which despite having a rapid RUNAT-BI division does not interfere with its growth.
  • the effect of RUNAT-BI is related to the speed of cell growth.

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Abstract

Disclosed are: a mononuclear Ru(III) compound comprising two molecules of 2,2'-biimidazole as ligands; a method for obtaining same; and the therapeutic use thereof, preferably as an anti-carcinogenic agent. The cancer may be breast cancer, gastric cancer or colon cancer, preferably triple negative A (TNA) and/or luminal B breast cancer.

Description

Compuesto de Rutenio (III) y 2,2'-biimidazol (RUNAT-BI) y su uso terapéutico Ruthenium (III) compound and 2,2'-biimidazole (RUNAT-BI) and its therapeutic use
La presente invención pertenece al campo de los complejos de rutenio (III) y su uso como agentes terapéuticos, especialmente como anticancerígenos selectivos. The present invention belongs to the field of ruthenium(III) complexes and their use as therapeutic agents, especially as selective anticancer agents.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
El cáncer es uno de los principales problemas en salud pública siendo la segunda causa de muerte a nivel mundial. La quimioterapia es ampliamente usada para tratar tumores, sin embargo, los fármacos empleados suelen presentar toxicidades elevadas en tejidos sanos. Cancer is one of the main public health problems, being the second cause of death worldwide. Chemotherapy is widely used to treat tumors, however, the drugs used often have high toxicities in healthy tissues.
Actualmente, para el tratamiento del cáncer se están utilizando fármacos anticancerígenos basados en platino (II) aunque tienen importantes efectos secundarios que limitan su efectividad y su actividad frente a algunos tumores se ve en ocasiones limitada. Además, los compuestos basados en platino (II) tienen limitada solubilidad en agua y corto tiempo de vida media. Currently, platinum (II)-based anticancer drugs are being used for the treatment of cancer, although they have important side effects that limit their effectiveness and their activity against some tumors is sometimes limited. Furthermore, platinum(II)-based compounds have limited water solubility and short half-life.
En la búsqueda de nuevos agentes antitumorales más eficaces y con menos efectos adversos, han destacado los basados en Rutenio, los más conocidos son NAMI-A y KP1019, que han logrado completar ensayos clínicos de fase I (Chem. Soc. Rev., 2018, 47, 909; Structure-activity relationships for ruthenium and osmium anticancer agents - towards clinical development; Samuel M. Meier-Menches, Christopher Gemer, Walter Berger, Christian G. Hartinger and Bernhard K. Keppler). In the search for new, more effective antitumor agents with fewer adverse effects, those based on Ruthenium have stood out, the best known being NAMI-A and KP1019, which have managed to complete phase I clinical trials (Chem. Soc. Rev., 2018 , 47, 909; Structure-activity relationships for ruthenium and osmium anticancer agents - towards clinical development; Samuel M. Meier-Menches, Christopher Gemer, Walter Berger, Christian G. Hartinger and Bernhard K. Keppler).
Actualmente, solo existen en la bibliografía sistemas de Ru (III) que contienen 1 o 3 moléculas de 2,2'-biimidazol (C. Tan et al. European Journal of Medicinal Chemistry 2011, 46, 1555-1563, DOI: https://doLorg/10. 1016/j.ejmech.2011.01.074), y solamente se han hecho estudios in vitro (líneas celulares HepG2, MCF-7, 95-D y HeLa) y con compuestos que presentan una sola molécula de 2,2'-biimidazol coordinada al Ru (III).Currently, only Ru(III) systems containing 1 or 3 2,2'-biimidazole molecules exist in the literature (C. Tan et al. European Journal of Medicinal Chemistry 2011, 46, 1555-1563, DOI: https: // doLorg/10. 1016/j.ejmech.2011.01.074), and studies have only been done in vitro (HepG2, MCF-7, 95-D and HeLa cell lines) and with compounds that present a single 2-molecule ,2'-biimidazole coordinated to Ru(III).
El artículo Journal of Biological Inorganic Chemistry (2019) 24:591-606; Agnieszka Gilewska, Barbara Barszcz, Joanna Mastemak, Katarzyna Kazimierczuk, Jerzy Sitkowski, Joanna Wietrzyk,- Eliza Turlej, describe un compuesto de Ru (II) con una sola molécula de 2,2’-biimidazol, por lo tanto, distinto del complejo de la invención. La toxicidad de este complejo se probó en líneas celulares de cáncer colorrectal, mama y leucemia y en fibroblastos murinos como control de línea celular no tumoral. En la Tabla 3 del artículo se observa que los complejos testados no son efectivos en la línea celular de cáncer de mama y en la de colon (además, en este caso el complejo más efectivo tiene iridio en vez de rutenio). En ambos casos el tiempo de incubación es de 72h.The article Journal of Biological Inorganic Chemistry (2019) 24:591-606; Agnieszka Gilewska, Barbara Barszcz, Joanna Mastemak, Katarzyna Kazimierczuk, Jerzy Sitkowski, Joanna Wietrzyk,- Eliza Turlej, describes a Ru(II) compound with a single 2,2'-biimidazole molecule, therefore, distinct from the complex of the invention. The toxicity of this complex was tested in colorectal, breast and leukemia cancer cell lines and in murine fibroblasts as a non-tumor cell line control. Table 3 of the article shows that the tested complexes are not effective in the breast and colon cancer cell lines (in addition, in this case the most effective complex has iridium instead of ruthenium). In both cases the incubation time is 72h.
La solicitud de patente CN101967164 divulga un complejo de Ru (II), de fórmula cis- [Ru(biim)2Cl2] 2H2O, preparado a partir de RuCl3 nH2O, bilmidazol y cloruro de litio, disuelto en dimetilformamida (DMF), y calentamiento a reflujo durante 8 h bajo argón. Se obtuvo, después de enfriar a temperatura ambiente, añadir acetona y enfriar, precipitación y filtrado, un rendimiento del 72% de cristales negro-púrpura. Patent application CN101967164 discloses a Ru (II) complex, with the formula cis-[Ru(biim) 2 Cl 2 ] 2H 2 O, prepared from RuCl 3 nH 2 O, bilmidazole and lithium chloride, dissolved in dimethylformamide (DMF), and heating at reflux for 8 h under argon. A 72% yield of purple-black crystals was obtained after cooling to room temperature, adding acetone and cooling, precipitation and filtration.
Sin embargo, este complejo es un compuesto intermedio en la síntesis de otros que se preparan posteriormente y no se ha evaluado su toxicidad in vitro en un panel de líneas celulares de cáncer. However, this complex is an intermediate compound in the synthesis of others that are prepared later and its in vitro toxicity has not been evaluated in a panel of cancer cell lines.
El nuevo compuesto de Ru (III), RUNAT-BI, no solo ha mostrado que se trata de un potencial agente antitumoral sino también que muestra un efecto selectivo entre líneas celulares humanas tumorales y no tumorales. The new Ru(III) compound, RUNAT-BI, has not only been shown to be a potential antitumor agent but also to show a selective effect between tumor and non-tumor human cell lines.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
De acuerdo con la presente invención se ha estudiado la actividad anticancerígena del agente RUNAT-BI, mezcla racémica de dos isómeros, y se ha analizado su actividad en nueve líneas celulares. Concretamente, se ha experimentado en seis líneas de cáncer de mama: cuatro de ellas pertenecientes al grupo de cáncer de mama en mujeres adultas (CMMA): MCF-7, BT474, HCC1806, y MDA-MB-231 y dos líneas celulares de cáncer de mama de mujeres jóvenes (CMMJ): HCC1937 y HCC1500, en una línea celular de cáncer de colon (HCT116) y en una línea celular de cáncer gástrico (AGS) para comprobar su actividad antitumoral. Además, para comparar si RUNAT-BI tiene efecto selectivo entre las líneas tumorales y las no tumorales se utilizó la línea MCF10A procedente de glándula mamaria de origen humano no tumoral. De todas ellas destacan los resultados obtenidos en cáncer de mama, colon, gástrico y una no tumoral de glándula mamaria. Los complejos basados en rutenio (III) con 2,2'-biimidazol pueden tener interacciones entre pares de bases que facilitan aún más su mecanismo de acción con el ADN de las células cancerosas. Asimismo, el hecho de que estos compuestos contengan rutenio (III) permite que sean aún más robustos e inertes a la hora de reaccionar en procesos redox o en reacciones de sustitución de ligando, permitiendo así aumentar la estabilidad y, por tanto, alargar la vida útil del compuesto en el medio fisiológico. In accordance with the present invention, the anticancer activity of the RUNAT-BI agent, a racemic mixture of two isomers, has been studied and its activity has been analyzed in nine cell lines. Specifically, six breast cancer lines have been experimented on: four of them belonging to the group of breast cancer in adult women (AMBC): MCF-7, BT474, HCC1806, and MDA-MB-231 and two cancer cell lines. of young women's breast (CMMJ): HCC1937 and HCC1500, in a colon cancer cell line (HCT116) and in a gastric cancer cell line (AGS) to check their antitumor activity. In addition, to compare whether RUNAT-BI has a selective effect between tumor and non-tumor lines, the MCF10A line from a non-tumoral human mammary gland was used. Of all of them, the results obtained in breast, colon, gastric cancer and a non-tumoral mammary gland cancer stand out. Ruthenium(III)-based complexes with 2,2'-biimidazole may have base pair interactions that further facilitate their mechanism of action with cancer cell DNA. Likewise, the fact that these compounds contain ruthenium (III) allows them to be even more robust and inert when it comes to reacting in redox processes or in ligand substitution reactions, thus increasing their stability and, therefore, extending their life. useful of the compound in the physiological environment.
En la presente memoria la denominación RUNAT-BI se refiere a la mezcla racémica de los isómeros del compuesto de Ru(lll): {cis-[RuCl2(H2biim)2]CI}2-4H2O (H2biim = 2,2'- biimidazol). Por lo tanto, salvo que se indique otra cosa, el término “compuesto” o “complejo” a lo largo de la memoria se refiere a la mezcla racémica. In the present specification, the name RUNAT-BI refers to the racemic mixture of the isomers of the Ru(lll) compound: {cis-[RuCl 2 (H 2 biim)2]CI} 2 -4H 2 O (H 2 biim = 2,2'-biimidazole). Therefore, unless otherwise indicated, the term "compound" or "complex" throughout the specification refers to the racemic mixture.
El término compuesto y complejo se usan indistintamente en esta memoria. The terms compound and complex are used interchangeably herein.
Un primer aspecto de la presente invención se refiere a un compuesto mononuclear de Ru (III) con dos moléculas de 2,2’-biimidazol. A first aspect of the present invention relates to a mononuclear compound of Ru(III) with two molecules of 2,2'-biimidazole.
El primer objeto de la presente invención se refiere a un compuesto de Ru (III) (RUNAT- BI), que tiene la fórmula {cis-[RuCl2(H2biim)2]CI}2-4H2O (H2biim = 2,2'-biimidazol).The first object of the present invention refers to a compound of Ru (III) (RUNAT- BI), which has the formula {cis-[RuCl 2 (H 2 biim)2]CI} 2 -4H 2 O (H 2 biim = 2,2'-biimidazole).
La difracción de rayos X sobre monocristal permitió dilucidar que en realidad el compuesto RUNAT-BI está formado por dos isómeros (figura 2). X-ray diffraction on a single crystal made it possible to elucidate that the RUNAT-BI compound is actually made up of two isomers (figure 2).
Un segundo aspecto de la invención se refiere a un procedimiento para la preparación del compuesto de Ru (III) - RUNAT-BI - definido anteriormente, que comprende: A second aspect of the invention refers to a process for the preparation of the Ru(III) compound - RUNAT-BI - defined above, comprising:
- realizar una síntesis solvotermal mediante una rampa de temperatura del compuesto de rutenio RUCl3 H2O, con 2,2-biimidazol en ácido clorhídrico - carry out a solvothermal synthesis by means of a temperature ramp of the ruthenium compound RUCl 3 H 2 O, with 2,2-biimidazole in hydrochloric acid
- una etapa de enfriamiento hasta la temperatura ambiente (considerada entre 20-25 °C). - a cooling stage up to room temperature (considered between 20-25 °C).
- el aislamiento del sólido RUNAT-BI obtenido en la mezcla de reacción a través de su cristalización. - the isolation of the RUNAT-BI solid obtained in the reaction mixture through its crystallization.
Se puede definir como 'solvotermal' porque el disolvente no es agua pura, sino agua y ácido clorhídrico. Esta mezcla tiene un punto de ebullición por debajo del agua pura, que también se ve modificado por la presión que ejerce el recipiente cerrado. La rampa de temperatura comprende alcanzar una temperatura de entre 85°C y 95°C, y más preferentemente de 90 °C. It can be defined as 'solvothermal' because the solvent is not pure water, but water and hydrochloric acid. This mixture has a boiling point below that of pure water, which is also modified by the pressure exerted by the closed container. The temperature ramp comprises reaching a temperature between 85°C and 95°C, and more preferably 90°C.
Según realizaciones particulares, la rampa de temperatura comprende alcanzar una temperatura de entre 85°C y 95°C, temperatura comprende alcanzar una temperatura de entre 85°C y 95°C, y más preferentemente de 90 °C y mantener dicha temperatura durante un tiempo entre 20 y 21 horas, preferentemente 20,5 h. According to particular embodiments, the temperature ramp comprises reaching a temperature between 85°C and 95°C, temperature comprises reaching a temperature between 85°C and 95°C, and more preferably 90°C and maintaining said temperature for a period of time. time between 20 and 21 hours, preferably 20.5 h.
El tiempo invertido hasta alcanzar la temperatura final es de 1 a 6 horas, preferentemente de 5 horas. The time invested until reaching the final temperature is from 1 to 6 hours, preferably 5 hours.
La concentración de RuCl3 H2O en la mezcla de síntesis es desde 0,01 a 0,05 M, preferentemente 0,012 M. The concentration of RuCl 3 H 2 O in the synthesis mixture is from 0.01 to 0.05 M, preferably 0.012 M.
La concentración de 2,2-biimidazol en la mezcla de síntesis es desde 0,01 a 0,05 M, preferentemente 0,036 M. The concentration of 2,2-biimidazole in the synthesis mixture is from 0.01 to 0.05 M, preferably 0.036 M.
El ácido clorhídrico que se puede utilizar es comercial, y puede tener una concentración en la mezcla de síntesis de 1 a 6 M, preferentemente, de 3 M. The hydrochloric acid that can be used is commercial, and can have a concentration in the synthesis mixture of 1 to 6 M, preferably 3 M.
La etapa de enfriamiento puede durar entre 10 y 21 horas, de forma preferente la etapa de enfriamiento hasta temperatura ambiente es de 20,5 h. The cooling stage can last between 10 and 21 hours, preferably the cooling stage to room temperature is 20.5 hours.
Un segundo aspecto de la invención se refiere al compuesto RUNAT-BI para uso terapéutico, preferentemente para cualquier enfermedad caracterizada por una mayor tasa de crecimiento celular respecto a las células de tejidos sanos. A second aspect of the invention refers to the RUNAT-BI compound for therapeutic use, preferably for any disease characterized by a higher rate of cell growth with respect to the cells of healthy tissues.
Un tercer aspecto de la invención se refiere al complejo RUNAT-BI para su uso como agente anticancerígeno. A third aspect of the invention relates to the RUNAT-BI complex for its use as an anticancer agent.
Existe, además, un efecto selectivo entre líneas tumorales y no tumorales, como se demuestra con la falta de efecto en la línea de glándula mamaria MCF10A. There is also a selective effect between tumor and non-tumor lines, as demonstrated by the lack of effect in the MCF10A mammary gland line.
El compuesto RUNAT-BI presenta actividad antitumoral. Concretamente, el compuesto RUNAT-BI (racé mico) ha demostrado reducir considerablemente la proliferación, migración y expresión de las líneas tumorales HCT116 (colon), AGS (gástrico) y HCC1806 (mama). The RUNAT-BI compound has antitumor activity. Specifically, the compound RUNAT-BI (racemic) has been shown to considerably reduce the proliferation, migration and expression of the HCT116 (colon), AGS (gastric) and HCC1806 (breast) tumor lines.
La selectividad del compuesto de la invención entre líneas celulares tumorales y no tumorales puede deberse a los mecanismos moleculares alterados que presentan estas últimas, por ejemplo, presencia de mutaciones en oncogenes y otros genes que presentan las células tumorales y no se encuentran en las no tumorales. También puede deberse a la alta tasa de replication de las células tumorales, al actuar el compuesto de RUNAT-BI sobre el ADN, afectará más a las células que tengan una tasa de replication más alta como es el caso de las células tumorales. The selectivity of the compound of the invention between tumor and non-tumor cell lines may be due to the altered molecular mechanisms that these cells present. The latter, for example, the presence of mutations in oncogenes and other genes that tumor cells present and are not found in non-tumor cells. It may also be due to the high rate of replication of tumor cells. As the RUNAT-BI compound acts on DNA, it will affect more cells that have a higher replication rate, such as tumor cells.
En una realización particular el cáncer se selecciona entre cáncer de mama, cáncer gástrico y cáncer de colon. In a particular embodiment, the cancer is selected from among breast cancer, gastric cancer, and colon cancer.
En los ejemplos de realización se muestra que las líneas celulares de CMMA (Cáncer de mama en mujeres adultas) que pertenecen al subtipo triple negativo (TNA) (HCC1806, MDA-MB-231 HCC1937 y HCC1500) y luminal B (BT474) presentan una mayor afectación por el tratamiento con RUNAT-BI, esto es muy relevante ya que los tumores de este perfil tienen un peor pronóstico y menos opciones de tratamiento.In the exemplary embodiments, it is shown that the CMMA (Breast Cancer in Adult Women) cell lines belonging to the triple negative (TNA) subtype (HCC1806, MDA-MB-231 HCC1937 and HCC1500) and luminal B (BT474) present a greater affectation by treatment with RUNAT-BI, this is highly relevant since tumors with this profile have a worse prognosis and fewer treatment options.
En otra realización particular el cáncer de mama es subtipo TNA y/o luminal B. In another particular embodiment, the breast cancer is of the TNA and/or luminal B subtype.
En otra realización particular el cáncer de mama (CM) es cáncer de mama en mujeres jóvenes (CMMJ). In another particular embodiment, the breast cancer (BC) is breast cancer in young women (JMMC).
En la presente memoria, mujeres jóvenes son aquellas con una edad igual o inferior a 35 años. Esta distinción se aplica también a las líneas celulares CMMA y CMMJ. In the present report, young women are those with an age equal to or less than 35 years. This distinction also applies to the CMMA and CMMJ cell lines.
Las ventajas de la presente invención son entre otras, las siguientes: The advantages of the present invention are, among others, the following:
• El compuesto RUNAT-BI es estable durante al menos dos semanas en disolución en un solvente orgánico como el polietilenglicol (PEG) y protegido de la exposición a la luz, a temperatura ambiente, esto es, de entre 18 °C y 33 °C, preferentemente 21 °C y 26 °C, más preferentemente 25 °C. El compuesto RUNAT-BI como sólido aislado, es estable durante al menos un mes en contacto con el aire a temperatura ambiente de entre 18 °C y 33 °C, preferentemente 21 °C y 26 °C, más preferentemente 25 °C. Por lo tanto no es necesario prepararlo inmediatamente antes de su uso. No se encontraron diferencias significativas entre el efecto de compuesto cuando está preparado en fresco (recién preparado para su uso) o cuando estaba preparado entre una o dos semanas antes de su uso. • RUNAT-BI se trata de un agente cuya diana de actuación se encuentra en el ADN, entre otras posibles, ya que afecta más a las líneas celulares cancerígenas con mayor capacidad de proliferación. • The RUNAT-BI compound is stable for at least two weeks in solution in an organic solvent such as polyethylene glycol (PEG) and protected from exposure to light, at room temperature, that is, between 18 °C and 33 °C. , preferably 21 °C and 26 °C, more preferably 25 °C. The RUNAT-BI compound as an isolated solid is stable for at least one month in contact with air at room temperature between 18 °C and 33 °C, preferably 21 °C and 26 °C, more preferably 25 °C. Therefore it is not necessary to prepare it immediately before use. No significant differences were found between the effect of the compound when it was prepared fresh (freshly prepared for use) or when it was prepared one to two weeks before use. • RUNAT-BI is an agent whose action target is found in DNA, among other possible ones, since it affects cancer cell lines with greater proliferation capacity more.
En una realización particular la concentración de RUNAT-BI es de entre 0,01 μM y 1000 μM, preferentemente, 1 μM y 100 μM, más preferentemente entre 0,1 μM y 50 μM. Dependiendo del tipo de cáncer se podrá emplear una concentración más reducida o elevada. In a particular embodiment, the concentration of RUNAT-BI is between 0.01 µM and 1000 µM, preferably 1 µM and 100 µM, more preferably between 0.1 µM and 50 µM. Depending on the type of cancer, a lower or higher concentration may be used.
Un objeto adicional de la invención se refiere a una composición farmacéutica que comprende RUNAT-BI y al menos un portador farmacéuticamente aceptable. An additional object of the invention refers to a pharmaceutical composition comprising RUNAT-BI and at least one pharmaceutically acceptable carrier.
Un "portador farmacéuticamente aceptable", después de ser administrado, no causa efectos fisiológicos indeseables. El portador en la composición farmacéutica debe ser "aceptable" también en el sentido de que es compatible con el RUNAT-BI, o compuesto activo, y puede ser capaz de estabilizarlo. Uno o más agentes solubilizantes pueden ser utilizados como portadores o excipientes farmacéuticos para la entrega del compuesto activo. Los ejemplos de un portador farmacéuticamente aceptable incluyen, pero no se limitan a, vehículos biocompatibles, adyuvantes, aditivos y diluyentes para lograr una composición utilizable como forma de dosificación. Entre los ejemplos de otros portadores se incluyen el óxido de silicio coloidal, el estearato de magnesio, la celulosa, el laurilsulfato de sodio y el D&C Yellow 10. A "pharmaceutically acceptable carrier", after administration, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the RUNAT-BI, or active compound, and may be capable of stabilizing it. One or more solubilizing agents may be used as pharmaceutical carriers or excipients for delivery of the active compound. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow 10.
La composición farmacéutica de la invención puede administrarse por vía parenteral, oral, nasal, rectal, tópica o bucal. The pharmaceutical composition of the invention can be administered parenterally, orally, nasally, rectally, topically, or buccally.
El término "parenteral", tal como se utiliza aquí, se refiere a la inyección subcutánea, intracutánea, intravenosa, intramuscular, intraarticular, intraarterial, intrasinovial, intraesternal, intratecal, intralesional o intracraneal, así como a cualquier técnica de infusión adecuada. The term "parenteral" as used herein refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.
La composición farmacéutica de la invención puede administrarse como un inyectable estéril que puede ser una solución o suspensión en un diluyente o disolvente no tóxico aceptable para la administración parenteral, tales soluciones incluyen, pero no se limitan a, 1,3-butanediol, manitol, agua, solución de Ringer y solución ¡sotónica de cloruro de sodio. Además, se emplean convencionalmente aceites fijos como disolvente o medio de suspensión (por ejemplo, mono o diglicéridos sintéticos). Los ácidos grasos, como el ácido oleico y sus derivados glicéridos, son útiles en la preparación de inyectables, al igual que los aceites naturales farmacéuticamente aceptables, como el aceite de oliva o el aceite de castilla, en sus versiones polioxietiladas. Estas soluciones o suspensiones de aceite también pueden contener un diluyante o dispersante de alcohol de cadena larga, como la carboximetilcelulosa o agentes dispersantes similares. Otros tensioactivos comúnmente utilizados, tales como, Tweens o Spans u otros agentes emulsionantes o potenciadores de la biodisponibilidad similares, que se utilizan comúnmente en la fabricación de formas farmacéuticas sólidas, líquidas u otras formas de dosificación aceptables también pueden ser utilizados para el propósito de la formulación. The pharmaceutical composition of the invention may be administered as a sterile injectable which may be a solution or suspension in a non-toxic diluent or solvent acceptable for parenteral administration, such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (eg, synthetic mono- or diglycerides). fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are pharmaceutically acceptable natural oils, such as olive oil or castilla oil, in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants, such as, Tweens or Spans or other emulsifying agents or similar bioavailability enhancers, which are commonly used in the manufacture of solid, liquid or other acceptable dosage forms may also be used for the purpose of formulation.
Una composición para administración oral puede ser cualquier forma de dosificación oralmente aceptable, incluyendo cápsulas, comprimidos, emulsiones y suspensiones acuosas, dispersiones y soluciones. En el caso de los comprimidos, los portadores comúnmente utilizados incluyen, entre otros, la lactosa y el almidón de maíz. También se suelen añadir agentes lubricantes, como el estearato de magnesio. Para la administración oral en forma de cápsula, los diluyentes útiles incluyen la lactosa y el almidón de maíz seco. Cuando las suspensiones o emulsiones acuosas se administran por vía oral, el ingrediente activo puede suspenderse o disolverse en una fase oleosa combinada con agentes emulsionantes o suspensores. Si se desea, pueden añadirse ciertos agentes edulcorantes, aromatizantes o colorantes. A composition for oral administration can be any orally acceptable dosage form, including capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets, commonly used carriers include, among others, lactose and corn starch. Lubricating agents, such as magnesium stearate, are also often added. For oral administration in capsule form, useful diluents include lactose and dry corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oil phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring or coloring agents may be added.
El RUNAT-BI o la composición farmacéutica puede administrarse in vivo o ex vivo, solo o en una composición junto con otros fármacos o terapias, es decir, un cóctel terapéutico. Por ejemplo, en el tratamiento de tumores, particularmente de tumores malignos, los agentes pueden utilizarse solos o en combinación con, por ejemplo, agentes quimioterapéuticos, radioterapéuticos, apopléticos, antiangiogénicos y/o inmunotoxinas o coagulantes. En algunas realizaciones, la administración de una composición farmacéutica de dos o más agentes/terapias es concurrente. En otras realizaciones, un primer agente/terapia se administra antes de un segundo agente/terapia. Los expertos en la materia entienden que las formulaciones y/o las vías de administración de los diversos agentes/terapias utilizados pueden variar. En una realización particular el RUNAT-BI o la composición farmacéutica puede administrarse como tratamiento posterior o simultáneamente a al menos un tratamiento seleccionado entre: cirugía, radioterapia, quimioterapia e inmunoterapia. The RUNAT-BI or the pharmaceutical composition can be administered in vivo or ex vivo, alone or in a composition together with other drugs or therapies, ie, a therapeutic cocktail. For example, in the treatment of tumors, particularly malignant tumors, the agents can be used alone or in combination with, for example, chemotherapeutic, radiotherapeutic, stroke, antiangiogenic and/or immunotoxin or coagulant agents. In some embodiments, the administration of a pharmaceutical composition of two or more agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered before a second agent/therapy. It is understood by those skilled in the art that the formulations and/or routes of administration of the various agents/therapies used may vary. In a particular embodiment, the RUNAT-BI or the pharmaceutical composition can be administered as a treatment after or simultaneously with at least one treatment selected from among: surgery, radiotherapy, chemotherapy and immunotherapy.
En una realización particular, la composición farmacéutica incluye la administración a la persona de un agente terapéutico adicional. Los ejemplos del agente terapéutico adicional incluyen uno o más seleccionados del grupo que consiste en cis-platino, 5- fluorouracilo, oxaliplatino, irinotecan, capecitabina, gemcitabina, cetuximab, paclitaxel, docetaxel, bevacizumab, regorafenib, aflibercept, trastuzumab, pertuzumab, nab- paclitaxel, trastuzumab-emtansina, atezolizumab y carboplatino. In a particular embodiment, the pharmaceutical composition includes the administration to the person of an additional therapeutic agent. Examples of the additional therapeutic agent include one or more selected from the group consisting of cis-platinum, 5-fluorouracil, oxaliplatin, irinotecan, capecitabine, gemcitabine, cetuximab, paclitaxel, docetaxel, bevacizumab, regorafenib, aflibercept, trastuzumab, pertuzumab, nab- paclitaxel, trastuzumab-emtansine, atezolizumab, and carboplatin.
El RUNAT-BI o la composición farmacéutica de la invención puede utilizarse como tratamiento posterior o simultáneamente a al menos un tratamiento seleccionado entre: cirugía, quimioterapia, inmunoterapia o tratamiento farmacéutico. The RUNAT-BI or the pharmaceutical composition of the invention can be used as treatment after or simultaneously with at least one treatment selected from among: surgery, chemotherapy, immunotherapy or pharmaceutical treatment.
En otra realización particular, el RUNAT-BI o la composición farmacéutica puede administrarse con un intervalo de administración variable, como por ejemplo de 1 a 7 veces por semana, preferiblemente de 2 a 6 veces por semana, más preferiblemente de 3 a 5 veces por semana. In another particular embodiment, the RUNAT-BI or the pharmaceutical composition can be administered with a variable administration interval, such as 1 to 7 times per week, preferably 2 to 6 times per week, more preferably 3 to 5 times per week. week.
Preferentemente el RUNAT-BI se administra con un intervalo de 15, 21 o 28 días y en forma de ciclos, por ejemplo, 4, 6, 8 o 12 ciclos, un experto en la materia sabría determinar la mejor pauta de administración de este compuesto empleando el conocimiento general común. Preferably RUNAT-BI is administered with an interval of 15, 21 or 28 days and in the form of cycles, for example, 4, 6, 8 or 12 cycles. An expert in the field would know how to determine the best administration schedule for this compound. using common general knowledge.
En otra realización particular, la dosis efectiva de RUNAT-BI y de la composición farmacéutica que lo comprende depende de la naturaleza de las condiciones, de la forma de ejecución y de la formulación farmacéutica, y será definida por la experiencia de un médico mediante la aplicación de las investigaciones habituales para un aumento de la dosis. Se puede considerar que la dosis efectiva es de 0,0001 a aproximadamente 1000 mg/kg de peso corporal por día, o de 0,01 a aproximadamente 500 mg/kg de peso corporal por día, o de 0,01 a 100 mg/kg de peso corporal por día, o de 0,05 a aproximadamente 10 mg/kg de peso corporal por día. Por ejemplo, la dosis diaria para una persona adulta de aproximadamente 70 kg de peso corporal estará en el intervalo de 1 mg a 1000 mg, o entre 5 mg y 500 mg, y puede realizarse mediante dosis únicas o múltiples, diarias o no. A lo largo de la descripción y de las reivindicaciones, la palabra "comprende" y sus variaciones no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Además, la palabra "comprende" y sus variaciones engloban el término "que consiste en". Otros objetos, ventajas y características de la invención resultarán evidentes para los expertos en la materia tras el examen de la descripción o podrán conocerse mediante la práctica de la invención. Los siguientes ejemplos se proporcionan a modo de ilustración y no pretenden ser limitativos de la presente invención. Además, la presente invención abarca todas las posibles combinaciones de las realizaciones particulares y preferidas descritas en el presente documento. In another particular embodiment, the effective dose of RUNAT-BI and the pharmaceutical composition that comprises it depends on the nature of the conditions, the form of execution and the pharmaceutical formulation, and will be defined by the experience of a physician through the application of the usual investigations for a dose increase. The effective dose can be considered to be 0.0001 to about 1000 mg/kg of body weight per day, or 0.01 to about 500 mg/kg of body weight per day, or 0.01 to 100 mg/ kg of body weight per day, or from 0.05 to about 10 mg/kg of body weight per day. For example, the daily dose for an adult person of about 70 kg body weight will be in the range of 1 mg to 1000 mg, or 5 mg to 500 mg, and can be accomplished by single or multiple, daily or non-daily doses. Throughout the description and claims, the word "comprises" and its variations are not intended to exclude other technical characteristics, additives, components or steps. Furthermore, the word "comprises" and its variations encompass the term "consisting of". Other objects, advantages, and features of the invention will be apparent to those skilled in the art upon examination of the description or may be learned through practice of the invention. The following examples are provided by way of illustration and are not intended to be limiting of the present invention. Furthermore, the present invention encompasses all possible combinations of the particular and preferred embodiments described herein.
Breve descripción de las figuras Brief description of the figures
Figura 1 : Ejemplo de una rampa de temperatura para la síntesis de RUNAT-BI.Figure 1: Example of a temperature ramp for the synthesis of RUNAT-BI.
Figura 2: Estructura molecular de los dos isómeros catiónicos que forman la mezcla racémica activa RUNAT-BI. Los átomos de hidrógeno, los aniones cloruro y las moléculas de H2O de la fórmula han sido omitidos para una mayor claridad. La diferente conectividad de los átomos para los dos isómeros se muestra en la Figura. Figure 2: Molecular structure of the two cationic isomers that form the active racemic mixture RUNAT-BI. Hydrogen atoms, chloride anions, and H 2 O molecules in the formula have been omitted for clarity. The different connectivity of the atoms for the two isomers is shown in the Figure.
Figura 3: Porcentaje de viabilidad de ocho de las líneas celulares tratadas con RUNAT-BI fresco a las 24, 48 y 72 h. Las líneas de CM: MCF-7, HCC1937, MDA-MB- 231 , BT474, HCC1806, HCC1500, la línea de cáncer gástrico: AGS y la línea de cáncer de colon: HCT116. Todas las líneas celulares fueron tratadas con RUNAT-BI fresco (0 a 42 μM) durante 24 horas (A); 48 horas (B) y 72 horas (C). La proliferación celular se determinó con el ensayo MTT. Los puntos indican la media de tres experimentos independientes. Figure 3: Percentage of viability of eight of the cell lines treated with fresh RUNAT-BI at 24, 48 and 72 h. The MC lines: MCF-7, HCC1937, MDA-MB-231, BT474, HCC1806, HCC1500, the gastric cancer line: AGS and the colon cancer line: HCT116. All cell lines were treated with fresh RUNAT-BI (0 to 42 μM) for 24 hours (A); 48 hours (B) and 72 hours (C). Cell proliferation was determined with the MTT assay. Points indicate the mean of three independent experiments.
Figura 4: Porcentaje de viabilidad de la línea celular no tumoral tratada con RUNAT-BI fresco a las 72 h. La línea celular de glándula mamaria MCF10A se ha empleado como modelo para comprobar la selectividad de RUNAT-BI entre líneas tumorales y no tumorales. Esta línea celular ha sido tratada con RUNAT-BI fresco (0 a 42 μM) durante 72 horas. La proliferación celular se determinó con el ensayo MTT. Los puntos indican la media de tres experimentos independientes. Figure 4: Percentage of viability of the non-tumor cell line treated with fresh RUNAT-BI at 72 h. The MCF10A mammary gland cell line has been used as a model to verify the selectivity of RUNAT-BI between tumor and non-tumor lines. This cell line has been treated with fresh RUNAT-BI (0 to 42 μM) for 72 hours. Cell proliferation was determined with the MTT assay. Points indicate the mean of three independent experiments.
Figura 5. Porcentaje de viabilidad de tres de las líneas celulares tratadas conFigure 5. Viability percentage of three of the cell lines treated with
RUNAT-BI no fresco a las 24, 48 y 72 h. Las líneas de CM perteneciente al grupo cáncer medular de mama atípico (CMMA): MCF-7 y MDA-MB-231 y la perteneciente al grupo cáncer de mama de mujeres jóvenes (CMMJ), fueron tratadas con RUNAT-BI no fresco (0 a 42 μM) durante 24 horas (A); 48 horas (B) y 72 horas (C). La proliferación celular se determinó con el ensayo MTT. Los puntos indican la media de dos experimentos independientes. RUNAT-BI not fresh at 24, 48 and 72 h. The CM lines belonging to the group atypical medullary breast cancer (AMBC): MCF-7 and MDA-MB-231 and the one belonging to the young women's breast cancer group (JMMC), were treated with non-fresh RUNAT-BI (0 to 42 μM) for 24 hours (A); 48 hours (B) and 72 hours (C). Cell proliferation was determined with the MTT assay. Points indicate the mean of two independent experiments.
Figura 6. Comparación de la migración celular del ensayo “cierre de la herida” en las líneas celulares estudiadas a las 48 h de tratamiento. La migración celular de las líneas celulares se midió con el ensayo “cierre de la herida” después de haber sido tratadas con el agente RUNAT-BI (21 μM) durante 48 horas. Tres experimentos independientes se realizaron y se muestran los resultados más significativos. Las columnas expresan la media ±SD del porcentaje de cierre en tres experimentos independientes. El color gris claro representa los controles con PEG mientras que el gris oscuro son las células tratadas con RUNAT-BI. *P s 0.1, **P s 0.05, ***P s 0.01 estadísticamente significativo. Figure 6. Comparison of cell migration in the "wound closure" assay in the cell lines studied after 48 hours of treatment. The cell migration of the cell lines was measured with the "wound closure" assay after being treated with the RUNAT-BI agent (21 μM) for 48 hours. Three independent experiments were performed and the most significant results are shown. Columns express the mean ± SD of the percent closure in three independent experiments. Light gray color represents PEG controls while dark gray is RUNAT-BI treated cells. *P s 0.1, **P s 0.05, ***P s 0.01 statistically significant.
Figura 7: Comparación de la migración celular del ensayo “cierre de la herida” en las líneas celulares estudiadas a las 72 h de tratamiento. La migración celular de las líneas celulares se midió con el ensayo “cierre de la herida” después de haber sido tratadas con el agente RUNAT-BI (21 μM) durante 72 horas. Tres experimentos independientes se realizaron y se muestran los resultados más significativos. Las columnas expresan la media ±SD del porcentaje de cierre en tres experimentos independientes. El color gris claro representa los controles con PEG mientras que el gris oscuro son las células tratadas con RUNAT-BI. *P s 0.1, **P s 0.05, ***P s 0.01 estadísticamente significativo. Figure 7: Comparison of cell migration in the "wound closure" assay in the cell lines studied after 72 hours of treatment. The cell migration of the cell lines was measured with the "wound closure" assay after being treated with the RUNAT-BI agent (21 μM) for 72 hours. Three independent experiments were performed and the most significant results are shown. Columns express the mean ± SD of the percent closure in three independent experiments. Light gray color represents PEG controls while dark gray is RUNAT-BI treated cells. *P s 0.1, **P s 0.05, ***P s 0.01 statistically significant.
Figura 8: Crecimiento celular de las líneas celulares tratadas con RUNAT-BI. Representación del crecimiento celular durante 10 días para estudiar la relación entre la efectividad de RUNAT-BI y la velocidad de crecimiento celular. El crecimiento se midió con la cámara de Nuebauer empleando “Automated Cell Counter TC10TM”. Los puntos indican la media de dos experimentos independientes. Figure 8: Cell growth of cell lines treated with RUNAT-BI. Representation of cell growth for 10 days to study the relationship between the effectiveness of RUNAT-BI and the speed of cell growth. Growth was measured with the Nuebauer chamber using the "Automated Cell Counter TC10TM". Points indicate the mean of two independent experiments.
Ejemplos examples
Ejemplo 1 : Síntesis del compuesto RUNAT-BI: Example 1 : Synthesis of the RUNAT-BI compound:
Mediante la rampa de temperatura de síntesis solvotermal mostrada en la figura 1 , se hizo reaccionar RUCl3 H2O (6,6 mg, 0,03 mmol) y 2,2-biimidazol (12,1 mg, 0,09 mmol) en MCI (2,5 mL, 3 M) a 90 °C durante 20,5 h. Seguidamente, la mezcla de reacción se sometió a un proceso de enfriamiento durante 20,5 h hasta alcanzar la temperatura ambiente (25 °C). En la disolución azul oscuro generada aparecen cristales de color verde azulado. Rendimiento: 25-30%. El compuesto obtenido, RUNAT-BI, presenta las siguientes bandas de infrarrojo (IR/ cm-1): 3278 (m), 3147 (m), 3129 (m), 3010 (m), 2923 (m), 2765 (m), 1638 (s), 1526 (s), 1417 (m), 1394 (m), 1319 (w), 1252 (w), 1177 (m), 1129 (m), 1078 (m), 1008(w), 922 (w), 870 (w), 811 (w), 754 (s), 682 (m). Using the solvothermal synthesis temperature ramp shown in Figure 1, RUCl 3 H 2 O (6.6 mg, 0.03 mmol) and 2,2-biimidazole (12.1 mg, 0.09 mmol) were reacted. in MCI (2.5 mL, 3 M) at 90 °C for 20.5 h. Next, the reaction mixture was subjected to a cooling process for 20.5 h until it reached room temperature (25 °C). Blue-green crystals appear in the dark blue solution generated. Yield: 25-30%. The compound obtained, RUNAT-BI, has the following infrared bands (IR/ cm -1 ): 3278 (m), 3147 (m), 3129 (m), 3010 (m), 2923 (m), 2765 (m ), 1638(s), 1526(s), 1417(m), 1394(m), 1319(w), 1252(w), 1177(m), 1129(m), 1078(m), 1008(w) ), 922 (w), 870 (w), 811 (w), 754 (s), 682 (m).
La rampa de temperatura (mostrada en la figura 1) comprende una etapa de calentamiento durante 5 horas hasta alcanzar una temperatura de 90 °C, mantenimiento de esta temperatura durante 20,5 horas y una etapa de enfriamiento hasta temperatura ambiente de 20,5 horas. The temperature ramp (shown in figure 1) comprises a heating stage for 5 hours until reaching a temperature of 90 °C, maintaining this temperature for 20.5 hours, and a cooling stage to room temperature for 20.5 hours. .
Métodos methods
Cultivo de las líneas celulares Culture of cell lines
Las células se crecieron en cultivo siguiendo las condiciones recomendadas por el proveedor. El medio de cultivo utilizado fue Roswell Park Memorial Institute Medium (RPMI), y para las líneas celulares BT474 y MCF10A Dulbecco's Modified Eagle Medium (DMEM), en ambos casos suplementario con 1% L-Glutamina y 10% Suero Fetal Bovino FBS y Penincilina/Estreptomicina al 1%, en unas condiciones de 37 °C al 5% de COa. Cuando los cultivos llegaron a una confluencia del 80-90%, las células fueron tripsinizadas con tripsina al 0,05% y se dividieron en una ratio 1:2 en medio fresco para permitir la expansión de los cultivos. Para la recogida del pellet, se esperó hasta que las células habían alcanzado una confluencia del 95% en el cultivo y se recogieron con PBS en frío mediante raspado con Cell Scraper. A continuación, las células recogidas se centrifugaron a 1500 rpm a 4 °C durante 10 minutos y se guardaron a -80 °C hasta próximo uso. Cells were grown in culture following the conditions recommended by the supplier. The culture medium used was Roswell Park Memorial Institute Medium (RPMI), and for the cell lines BT474 and MCF10A Dulbecco's Modified Eagle Medium (DMEM), in both cases supplemented with 1% L-Glutamine and 10% Fetal Bovine Serum FBS and Penicillin. /Streptomycin 1%, under conditions of 37 °C at 5% COa. When cultures reached 80-90% confluence, cells were trypsinized with 0.05% trypsin and split in a 1:2 ratio in fresh medium to allow expansion of cultures. For the collection of the pellet, it was waited until the cells had reached 95% confluence in the culture and were collected with cold PBS by scraping with Cell Scraper. The collected cells were then centrifuged at 1500 rpm at 4 °C for 10 minutes and stored at -80 °C until further use.
Ensayos de proliferación celular MTT El protocolo usado fue un ensayo de MTT (Bromuro de 3-(4,5-dimetiltiazol-2-ilo)-2,5- difeniltetrazol) en el que se cultivaron 3000 células en placas de 96 pocilios. Posteriormente las células fueron tratadas con el agente específico y se realizó el ensayo de MTT a las 24, 48 y 72 horas del tratamiento. MTT se trata de un ensayo colorimétrico indicador de la supervivencia y proliferación celular. Se basa en la reducción metabólica del Bromuro de 3-(4,5-dimetiltiazol-2-¡lo)-2,5-d¡feniltetrazol realizada por la enzima mitocondrial Succinato-deshidrogenasa, en unas condiciones definidas, a un compuesto coloreado de color morado (formazan), permitiendo determinar la funcionabilidad mitocondrial de las células tratadas. MTT, se trata de un tetrazol amarillo que se reduce a morado en las células vivas. Una solución de DMSO se añade para solubilizar el compuesto y convertir el insoluble formazan morado en una solución coloreada, de la cual se mide posteriormente su absorbancia a 560 nm en el espectrofotómetro. Un incremento en la absorbancia se corresponde con un incremento en la proliferación celular, mientras que una disminución de la absorbancia supone una disminución de la proliferación celular. Cada valor se repite por triplicado y se calcula la media. Cada absorbancia del compuesto ensayado se compara con su respectivo control no tratado. La viabilidad se calcula de acuerdo con la siguiente ecuación: MTT cell proliferation assays The protocol used was an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide) assay in which 3000 cells were grown in 96-well plates. Subsequently, the cells were treated with the specific agent and the MTT assay was performed at 24, 48 and 72 hours after treatment. MTT is a colorimetric assay indicating cell survival and proliferation. It is based on the metabolic reduction of 3-(4,5-dimethylthiazol-2-¡l)-2,5-d¡phenyltetrazole bromide carried out by the mitochondrial enzyme Succinate-dehydrogenase, under defined conditions, to a colored compound of purple color (formazan), allowing to determine the mitochondrial functionality of the treated cells. MTT is a yellow tetrazole that is reduced to purple in living cells. A DMSO solution is added to solubilize the compound and convert the insoluble purple formazan to a colored solution, which is subsequently measured for absorbance at 560 nm in the spectrophotometer. An increase in absorbance corresponds to an increase in cell proliferation, while a decrease in absorbance corresponds to a decrease in cell proliferation. Each value is repeated in triplicate and the mean is calculated. Each absorbance of the tested compound is compared to its respective untreated control. Viability is calculated according to the following equation:
% Viabilidad = (DO células tratadas x 100) / DO células control % Viability = (OD treated cells x 100) / OD control cells
DO: Densidad óptica DO: Optical density
Ensayos de migración celular: “cierre de la herida” Cell migration assays: “wound closure”
El ensayo “cierre de herida” (Wound healing assay) tiene como objetivo el estudio de la migración celular. Es un ensayo fácil, barato y ampliamente utilizado por los investigadores. Se basa en la observación del comportamiento de una monocapa confluente de células a la que previamente se le ha realizado una brecha o “herida”. Las células en el borde de la brecha se moverán hacia la abertura hasta establecer nuevos contactos célula-célula, cerrando así la “herida”. Captando las imágenes al principio y en intervalos de tiempo regulares durante la migración celular cerca de la brecha, se logra comparar y cuantificar la migración celular. En el estudio se cultivaron placas de 6 pocilios con una densidad de 4 x 105 células/pocillo y se incubaron durante la noche hasta que alcanzaron una confluencia del 70%. Para crear la brecha en la capa celular se usó la punta de una pipeta y posteriormente las células se trataron con RUNAT-BI (21 μM). Cada experimento se realizó por triplicado y se repitió al menos 2 veces. Las imágenes se obtuvieron a las 0, 24, 48 y 72 h en la misma posición y el porcentaje de migración celular se evaluó usando el programa Imaged, un programa para procesar imágenes digitales. The "wound healing assay" assay aims to study cell migration. It is an easy, cheap and widely used assay by researchers. It is based on the observation of the behavior of a confluent monolayer of cells that has previously been breached or "wounded". The cells at the edge of the gap will move towards the opening until establishing new cell-cell contacts, thus closing the "wound". By capturing images at the beginning and at regular time intervals during cell migration near the gap, it is possible to compare and quantify cell migration. In the study, 6-well plates were plated at a density of 4 x 10 5 cells/well and incubated overnight until 70% confluent. To create the gap in the cell layer, a pipette tip was used and then the cells were treated with RUNAT-BI (21 μM). Each experiment was performed in triplicate and repeated at least 2 times. times. Images were obtained at 0, 24, 48, and 72 h in the same position, and the percentage of cell migration was evaluated using the Imaged program, a program for processing digital images.
Cálculo del tiempo de doblaje celular Cell doubling time calculation
Las líneas celulares de CM, tanto las de cáncer de CMMJ: HCC1500 y HCC1937, las de CMMA: MCF-7, BT474, MDA-MB-231, HCC1806. En la tabla 1 se muestra información de estas líneas celulares: The cell lines of MC, both those of CMMJ cancer: HCC1500 and HCC1937, those of CMMA: MCF-7, BT474, MDA-MB-231, HCC1806. Table 1 shows information on these cell lines:
TABLA1: cateo orización, información v condiciones de cultivo de las líneas celulares de cáncer de mama empleadas
Figure imgf000016_0001
TABLE 1: Screening, information, and culture conditions of the breast cancer cell lines used
Figure imgf000016_0001
La línea celular de glándula mamaria no tumoral (MCF10A), la línea celular de cáncer gástrico (AGS) y la línea celular de cáncer de colon (HCT116), se han usado para estudiar la velocidad de crecimiento celular y determinar el tiempo de doblaje celular (CDT). Se hicieron recuentos de células a intervalos regulares. En el día 0 las células se plaquearon en placas de 6 pocilios, con una concentración inicial de 1 x 105 células por pocilio y cada línea celular se cultivó por duplicado. Se hicieron recuentos por duplicado en cámara de Neubauer empleando “Automated Cell Counter TC10TM” (BioRad), cada 24 h durante 10 días después de teñir las células con azul de trypan. A partir de los datos de los recuentos de células, durante el tiempo estudiado se construyó la curva de crecimiento celular y se calculó el CDT mediante la herramienta online http://www.doubling-time.com/compute.php The non-tumor mammary gland cell line (MCF10A), the gastric cancer cell line (AGS) and the colon cancer cell line (HCT116) have been used to study cell growth rate and determine cell doubling time. (CDT). Cell counts were made at regular intervals. On day 0, cells were plated in 6-well plates, with an initial concentration of 1 x 10 5 cells per well, and each cell line was cultured in duplicate. Duplicate counts were made in a Neubauer chamber using “Automated Cell Counter TC10TM” (BioRad), every 24 h for 10 days after staining the cells with trypan blue. From the data of the cell counts, during the time studied, the cell growth curve was constructed and the CDT was calculated using the online tool http://www.doubling-time.com/compute.php
Resultados Results
Salvo que se indique lo contrario RUNAT-BI se prepara fresco antes de realizar cada experimento. Ensayo de proliferación celular tras el tratamiento con RUNAT-BI en líneas celulares tumorales Unless otherwise indicated, RUNAT-BI is prepared fresh before performing each experiment. Cell proliferation assay after treatment with RUNAT-BI in tumor cell lines
Las líneas de CM pertenecientes al grupo de cáncer de mama en mujeres adultas CMMA: HCC1806, MCF-7, BT474 y MDAMB-231 y las pertenecientes al grupo Cáncer de mama en mujeres jóvenes CMMJ: HCC1937 y HCC1500, la línea celular de cáncer de colon (HCT116), la línea celular de cáncer gástrico (AGS) y la línea de glándula mamaria de origen humano no tumoral (MCF10A) se sometieron a concentraciones crecientes de RUNAT- Bl (0, 5,25, 10,5, 21 y 42 μM) durante 24, 48 y 72 horas. Se han empleado dichas concentraciones ya que el compuesto madre elaborado tenía una concentración inicial de 42 mM (2 mg de producto en 100 pL de PEG). RUNAT-BI se preparó antes de cada experimento. The MC lines belonging to the breast cancer group in adult women CMMA: HCC1806, MCF-7, BT474 and MDAMB-231 and those belonging to the breast cancer group in young women CMMJ: HCC1937 and HCC1500, the breast cancer cell line colon (HCT116), gastric cancer cell line (AGS) and mammary gland line of non-tumor human origin (MCF10A) were subjected to increasing concentrations of RUNAT-Bl (0, 5.25, 10.5, 21 and 42 μM) for 24, 48 and 72 hours. Said concentrations have been used since the prepared mother compound had an initial concentration of 42 mM (2 mg of product in 100 pL of PEG). RUNAT-BI was prepared before each experiment.
Tal y como muestra la Figura 3 se observó en casi todas las líneas una reducción en la proliferación celular, dosis y tiempo dependiente. A las 24 h no se observaron resultados destacables en ninguna de las líneas (Figura 3 A) y tabla 2: As shown in Figure 3, a dose- and time-dependent reduction in cell proliferation was observed in almost all lines. At 24 h, no notable results were observed in any of the lines (Figure 3 A) and table 2:
TABLA 2: % de viabilidad celular a 24 h de tratamiento con distintas concentraciones de RUNAT-BI (μM)
Figure imgf000017_0001
aunque en varias de ellas: AGS, HCC 1806 y HCT116 la viabilidad ya se redujo en torno a un 30% a 42 μM. A las 48 h (Figura 3 B) ya se observó una reducción muy consistente de la viabilidad de las líneas HCT116 y HCC1806, cuyos resultados se corroboran con los obtenidos a las 72 h llegando a un 25% y 35% de viabilidad, respectivamente. La línea de cáncer gástrico AGS mostró también una reducción de la viabilidad bastante importante llegando casi al 40%, con notables diferencias entre 48 h y 72 h. Las líneas de CMMA: MDAMB- 231 y BT474 mostraron una reducción de la viabilidad en torno al 30% a las 48 h. La línea MDA-MB-231 redujo casi un 30% su viabilidad a las 48h y un 40% a las 72h (Figura 3 B y C). La línea BT474 redujo un 30% a las 48h y un 50% la viabilidad en 72h. La línea de CMMJ: HCC1937, no fue de las más afectadas, quedándose a los tres tiempos (24, 48 y 72 h) sobre el 80% de viabilidad y la línea HCC1500 no presentó cambios en su viabilidad a ningún tiempo, quedándose por encima del 90% a las 72 h. Ocurrió algo semejante con MCF-7, perteneciente al grupo CMMA, que no mostró reducciones significativas, quedándose en tomo al 80% a las 72 h, al igual que HCC1937. En las tablas 3 y 4 se pueden observar los resultados de viabilidad a 48 y 72 h. TABLE 2: % cell viability after 24 h of treatment with different concentrations of RUNAT-BI (μM)
Figure imgf000017_0001
although in several of them: AGS, HCC 1806 and HCT116 the viability was already reduced by around 30% at 42 μM. At 48 h (Figure 3 B) a very consistent reduction in the viability of the lines HCT116 and HCC1806 was already observed, the results of which are corroborated with those obtained at 72 h, reaching 25% and 35% viability, respectively. The AGS gastric cancer line also showed a quite significant reduction in viability, reaching almost 40%, with notable differences between 48 and 72 h. The CMMA lines: MDAMB-231 and BT474 showed a viability reduction of around 30% at 48 h. The MDA-MB-231 line reduced its viability by almost 30% at 48h and 40% at 72h (Figure 3 B and C). The BT474 line reduced 30% at 48h and 50% the viability in 72h. The CMMJ line: HCC1937, was not one of the most affected, remaining at three times (24, 48 and 72 h) over 80% viability and the HCC1500 line did not present changes in its viability at any time, staying above 90% at 72 h. Something similar occurred with MCF-7, belonging to the CMMA group, which did not show significant reductions, remaining around 80% at 72 h, the same as HCC1937. Tables 3 and 4 show the viability results at 48 and 72 h.
TABLA 3: % de viabilidad celular a 48 h de tratamiento con distintas concentraciones de RUNAT-BI (uM)
Figure imgf000018_0001
TABLE 3: % cell viability after 48 h of treatment with different concentrations of RUNAT-BI (uM)
Figure imgf000018_0001
TABLA 4: % de viabilidad celular a 72 h de tratamiento con distintas concentraciones de RUNAT-BI íuM)
Figure imgf000018_0002
TABLE 4: % cell viability after 72 h of treatment with different concentrations of RUNAT-BI iuM)
Figure imgf000018_0002
El tratamiento con RUNAT-BI muestra una relación entre la reducción de la viabilidad celular y la velocidad de crecimiento (tiempo de doblaje celular), observando que las líneas que más rápido crecen son las más afectadas por RUNAT-BI. Esta relación se muestra con más detalle en los resultados del estudio del cálculo del tiempo de doblaje celular. Treatment with RUNAT-BI shows a relationship between the reduction of cell viability and growth speed (cell doubling time), observing that the lines that grow the fastest are the most affected by RUNAT-BI. This relationship is shown in more detail in the results of the cell doubling time calculation study.
Ensayo de proliferación celular tras el tratamiento con RUNAT-BI en una línea celular no tumoral Para comprobar la selectividad de actuación de RUNAT-BI entre líneas tumorales y no tumorales, se empleó la línea de glándula mamaria no tumoral MCF10A. Tal y como muestran los resultados de la Tabla 5, no se reduce su viabilidad a ningún tiempo ni a ninguna concentración. A pesar del rápido crecimiento de esta línea celular, ya que se encuentra inmortalizada y por tanto se divide muy rápido, el tratamiento con RUNAT-BI no le afecta. El incremento en la viabilidad durante el paso del tiempo se puede relacionar con la velocidad de crecimiento de dicha línea, ya que su tiempo de doblaje celular se encuentra en torno a 1,92 por lo que a los tres días debería doblar su población en torno al doble-triple, como se observa en la Tabla 5. El compuesto de la presente invención no afecta al crecimiento de líneas celulares no tumorales, como se observa con el ejemplo de la línea celular no tumoral MCF10A a las 72 h. Cell proliferation assay after treatment with RUNAT-BI in a non-tumor cell line To verify the selectivity of RUNAT-BI action between tumor and non-tumor lines, the MCF10A non-tumor mammary gland line was used. As the results in Table 5 show, their viability is not reduced at any time or at any concentration. Despite the rapid growth of this cell line, since it is immortalized and therefore divides very quickly, treatment with RUNAT-BI does not affect it. The increase in viability over time can be related to the speed of growth of said line, since its cell doubling time is around 1.92, so after three days its population should double around to double-triple, as observed in Table 5. The compound of the present invention does not affect the growth of non-tumor cell lines, as observed with the example of the non-tumor cell line MCF10A at 72 h.
TABLA 5: % de viabilidad celular de la línea no tumoral MCF10A a 24, 48 v 72 h de tratamiento con distintas concentraciones de RUNAT-BI (uM)
Figure imgf000019_0001
TABLE 5: % cell viability of the non-tumor line MCF10A at 24, 48 and 72 h of treatment with different concentrations of RUNAT-BI (uM)
Figure imgf000019_0001
Comparación entre RUNAT-BI fresco v no fresco en proliferación celular Comparison between fresh and non-fresh RUNAT-BI in cell proliferation
Otro de los experimentos llevado a cabo con RUNAT-BI fue comprobar si existían diferencias en la efectividad de RUNAT-BI en fresco o tras un máximo de 2 semanas desde su preparación, a temperatura ambiente y protegido de la luz. Para ello se sometieron a las líneas celulares de CM: MCF-7, HCC1937 y MDA-MB-231 a concentraciones crecientes de RUNAT-BI no fresco (0, 5,25, 10,5, 21 y 42 μM). Se seleccionaron estas líneas para valorar si el tiempo de preparación condicionaba la efectividad de RUNAT-BI y valorar si en estas líneas se obtenía mayor o menor reducción de la viabilidad, ya que en dos de ellas (MCF-7 y HCC1937) RUNAT-BI en fresco no mostró resultados muy efectivos y en MDA48 MB-231 la reducción de la viabilidad tampoco fue de las más notables, quedando al 60% a las 72 h de tratamiento. Como muestran los resultados de la Figura 5, a las 24 h del tratamiento con RUNAT-BI no fresco no se observan cambios muy notables en la viabilidad, datos que coinciden con los experimentos de RUNAT-BI en fresco. A las 48 h, la línea MDA-MB-231 es la que muestra una reducción más importante llegando a una viabilidad del 70%, sin embargo, las otras dos líneas se muestran menos afectadas por el tratamiento. A las 72 h, MDA-MB-231 sigue siendo la línea que más reduce su viabilidad quedándose en tomo al 60% como ocurría con RUNAT-BI en fresco, tal y como se observa en la gráfica C de la Figura 3. Lo mismo ocurre con MCF-7 y HCC1937, también muestran mayor reducción a las 72 h quedándose en tomo al 75-80% en ambos casos, como ocurre con RUNAT-BI fresco. Estos resultados confirman que no hay diferencias significativas entre el RUNAT-BI fresco, es decir preparado inmediatamente antes de usar, o no fresco, preparado con varias semanas de antelación. Another of the experiments carried out with RUNAT-BI was to check if there were differences in the effectiveness of RUNAT-BI fresh or after a maximum of 2 weeks from its preparation, at room temperature and protected from light. For this, the MC cell lines: MCF-7, HCC1937 and MDA-MB-231 were subjected to increasing concentrations of non-fresh RUNAT-BI (0, 5.25, 10.5, 21 and 42 μM). These lines were selected to assess whether the preparation time conditioned the effectiveness of RUNAT-BI and to assess whether a greater or lesser reduction in viability was obtained in these lines, since in two of them (MCF-7 and HCC1937) RUNAT-BI when fresh it did not show very effective results and in MDA48 MB-231 the reduction in viability was not the most notable either, remaining at 60% after 72 h of treatment. As the results in Figure 5 show, 24 h after treatment with non-fresh RUNAT-BI, no very notable changes in viability were observed, data that coincides with the experiments with fresh RUNAT-BI. At 48 h, the MDA-MB-231 line is the one that shows the most significant reduction, reaching a viability of 70%, however, the other two lines are less affected by the treatment. At 72 h, MDA-MB-231 continues to be the line that reduces its viability the most, staying at around 60%, as was the case with RUNAT-BI fresh, as can be seen in graph C of Figure 3. The same This occurs with MCF-7 and HCC1937, they also show a greater reduction at 72 h, remaining around 75-80% in both cases, as occurs with fresh RUNAT-BI. These results confirm that there are no significant differences between fresh RUNAT-BI, that is, prepared immediately before use, or not fresh, prepared several weeks in advance.
Reducción de la migración de las líneas celulares tratadas con RUNAT-BI, medidos con el ensayo “cierre de la herida” Reduction of migration of cell lines treated with RUNAT-BI, measured with the "wound closure" assay
Las líneas de CM pertenecientes al grupo CMMA: MCF7, MDA-MB-231, HCC1806 y BT474, las pertenecientes al grupo CMMJ: HCC1937 y HCC1500, la línea de cáncer gástrico: AGS, la línea de cáncer de colon: HCT116 y la línea de glándula mamaria de origen humano no tumoral: MCF10A fueron tratadas con 21 μM de RUNAT-BI o 21 μM de PEG (control). Se realizó una brecha (herida) a las 0 h y el porcentaje de cierre de dicha herida se midió usando imágenes de 0 a 72 h. En la Figura 6 se muestran los resultados a 48 h y en la Figura 7 a 72 h. En la tabla 6 se pueden ver los resultados en ambos tiempos. The MC lines belonging to the CMMA group: MCF7, MDA-MB-231, HCC1806 and BT474, those belonging to the CMMJ group: HCC1937 and HCC1500, the gastric cancer line: AGS, the colon cancer line: HCT116 and the from mammary gland of non-tumoral human origin: MCF10A were treated with 21 μM RUNAT-BI or 21 μM PEG (control). A gap (wound) was made at 0 h and the percentage closure of said wound was measured using images from 0 to 72 h. Figure 6 shows the results at 48 h and Figure 7 at 72 h. Table 6 shows the results at both times.
El ensayo “cierre de la herida” demostró que el agente antitumoral RUNAT-BI reduce significativamente la migración celular de estas líneas celulares. Más concretamente la línea HCC1806 tratada con RUNAT-BI mostró una reducción de la migración del 47,1% a las 48 h y del 65,7% a las 72 h respecto a la línea HCC1806 tratada con PEG. AGS tratada con RUNAT-BI mostró también una reducción muy significativa, sobre todo a las 72 h llegando al 54,3% respecto a la línea AGS tratada con PEG. HCT116 tratada con RUNAT-BI no mostró diferencias significativas en la reducción de la migración celular entre las 48 h y 72 h, quedándose en ambos casos entre 21-22% de cierre respecto a la línea HCT116 tratada con PEG. Cabe destacar que AGS y HCT116 son líneas celulares con una velocidad de crecimiento muy alta, en el caso de HCT116 en el tratamiento con RUNAT-BI se observó una reducción significativa en la velocidad de crecimiento celular en comparación con el tratamiento control (tratada con PEG) mientras que AGS mantuvo su elevada velocidad de crecimiento y su tiempo de doblaje celular, pero mostró mayor reducción en la migración celular. Los valores de migración se obtienen de la diferencia en el área de la herida entre las 0 h y las 48 h o 72 h para cada condición. Estos resultados concuerdan con los observados anteriormente en los ensayos de MTT, ya que estas tres líneas presentaron una reducción significativa de la viabilidad celular con el tratamiento con RUNAT-BI. La línea BT474 a las 48 h en el tratamiento con RUNAT-BI mostró una reducción de la migración celular del 14,1% y a las 72 h redujo la migración a 23,1% respecto a la línea BT474 tratada con PEG. Sin embargo, cabe destacar que esta línea celular mostró una clara apoptosis con el tratamiento RUNAT-BI a las 72 h, por ser una línea que crece muy lentamente. The "wound closure" assay demonstrated that the antitumor agent RUNAT-BI significantly reduces cell migration of these cell lines. More specifically, the HCC1806 line treated with RUNAT-BI showed a migration reduction of 47.1% at 48 h and 65.7% at 72 h compared to the HCC1806 line treated with PEG. AGS treated with RUNAT-BI also showed a very significant reduction, especially at 72 h, reaching 54.3% compared to the AGS line treated with PEG. HCT116 treated with RUNAT-BI did not show significant differences in the reduction of cell migration between 48 and 72 h, remaining in both cases between 21-22% closure compared to the line HCT116 treated with PEG. It should be noted that AGS and HCT116 are cell lines with a very high growth rate, in the case of HCT116 in In the treatment with RUNAT-BI, a significant reduction in cell growth rate was observed in comparison with the control treatment (treated with PEG), while AGS maintained its high growth rate and cell doubling time, but showed a greater reduction in cell growth rate. cell migration. Migration values are obtained from the difference in wound area between 0 and 48 h or 72 h for each condition. These results are consistent with those previously observed in MTT assays, as these three lines showed a significant reduction in cell viability with RUNAT-BI treatment. The BT474 line at 48 h in the treatment with RUNAT-BI showed a 14.1% reduction in cell migration and at 72 h reduced migration to 23.1% compared to the BT474 line treated with PEG. However, it should be noted that this cell line showed clear apoptosis with the RUNAT-BI treatment at 72 h, as it is a line that grows very slowly.
TABLA 6 Porcentaje de cierre de herida a 48 v 72 horas
Figure imgf000021_0001
TABLE 6 Percentage of wound closure at 48 v 72 hours
Figure imgf000021_0001
Las líneas celulares pertenecientes al grupo CMMJ: HCC1937 y HOC 1500, eran de las líneas que mostraron menor reducción de la viabilidad y ocurrió parecido con la migración celular, HCC1937 tratada con RUNAT-BI redujo su migración un 14,5 % a las 48 h y un 19,0% a las 72 h respecto a la línea HCC1937 tratada con PEG. Cabe destacar que las muestras de esta línea celular tratadas con RUNAT-BI redujeron considerablemente su velocidad de crecimiento, al igual que ocurrió en BT474. HCC1500 no mostró apenas migración con el tratamiento control (PEG) a ningún tiempo, probablemente, debido al lento crecimiento celular de esta línea y con el agente RUNAT-BI la apertura de la brecha incluso se aumentó a las 48 h y 72 h, observándose un claro incremento de células muertas entre las imágenes a las 0 h y a las 48 h y 72 h. The cell lines belonging to the CMMJ group: HCC1937 and HOC 1500, were among the lines that showed the least reduction in viability and similarity occurred with cell migration, HCC1937 treated with RUNAT-BI reduced its migration by 14.5 % at 48 h. 19.0% at 72 h with respect to the line HCC1937 treated with PEG. It should be noted that the samples of this cell line treated with RUNAT-BI considerably reduced their growth rate, as occurred in BT474. HCC1500 hardly showed migration with the control treatment (PEG) at any time, probably due to the slow cell growth of this line and with the RUNAT-BI agent the opening of the gap was even increased at 48 and 72 h, observing a clear increase in dead cells between the images at 0 h and at 48 h and 72 h.
La línea de glándula mamaria no tumoral MCF10A no mostró diferencias en la migración entre condiciones, quedándose prácticamente igual en el tratamiento con RUNAT-BI que, en el control, con PEG. Sí que se observaron diferencias entre las 48 h y 72 h, ya que a este último tiempo en las dos condiciones se produjo migración celular. Esta línea no mostró apoptosis en las muestras tratadas con RUNAT-BI, tal como se ha mostrado en los experimentos anteriores de proliferación. Estos resultados corroboran los obtenidos en el ensayo de viabilidad celular, demostrando de nuevo la selectividad de RUNAT-BI entre líneas tumorales y no tumorales. The non-tumor mammary gland line MCF10A did not show differences in migration between conditions, remaining practically the same in the treatment with RUNAT-BI as in the control, with PEG. Yes, differences were observed between 48 h and 72 h, since cell migration occurred at the latter time in both conditions. This line did not show apoptosis in the samples treated with RUNAT-BI, as shown in previous proliferation experiments. These results corroborate those obtained in the cell viability assay, once again demonstrating the selectivity of RUNAT-BI between tumor and non-tumor lines.
Los resultados obtenidos en el ensayo de migración celular se corroboran con los obtenidos en los ensayos de viabilidad, siendo claramente las líneas HCT116, AGS y HCC1806 en las que RUNAT-BI ejerce mayor efecto. La selectividad de actuación entre las líneas tumorales y la línea no tumoral (MCF10A) se vuelve a demostrar con los resultados obtenidos en el ensayo “cierre de la herida”, ya que a ninguno de los dos tiempos estudiados se observa diferencia en la migración entre las distintas condiciones. En la Figura 6 se muestran las diferencias de migración celular expresadas por línea celular a las 48 h, y en la Figura 7, a las 72 h. The results obtained in the cell migration assay are corroborated with those obtained in the viability assays, clearly being the lines HCT116, AGS and HCC1806 in which RUNAT-BI exerts the greatest effect. The selectivity of action between the tumor lines and the non-tumor line (MCF10A) is once again demonstrated with the results obtained in the "wound closure" assay, since at neither of the two times studied was a difference observed in the migration between the different conditions. Figure 6 shows the differences in cell migration expressed by cell line at 48 h, and Figure 7, at 72 h.
En casi todas líneas se observa mayor migración a las 72 h que a las 48 h y una clara diferencia entre las muestras control (tratadas con PEG) y las tratadas con RUNAT-BI. Generalmente son las líneas que más rápido crecen las que más logran cerrar la brecha en las muestras control, algunas de ellas son capaces de cerrar casi por completo la brecha, como por ejemplo la línea celular AGS a las 72 h. In almost all lines, greater migration is observed at 72 h than at 48 h and a clear difference between the control samples (treated with PEG) and those treated with RUNAT-BI. Generally, the lines that grow the fastest are the ones that most manage to close the gap in the control samples, some of them are capable of almost completely closing the gap, such as the AGS cell line at 72 h.
Tiempo del doblaje celular (CDT) de las líneas celulares empleadas. Cell doubling time (CDT) of the cell lines used.
Se ha calculado el tiempo de doblaje celular (CDT, Cell Doubling Time) de las líneas celulares HCC1500, HCC1937, MCF-7, BT474, MDA-MB-231 , HCC1806, AGS, HCT 116 y MCF10A. Este valor nos permite conocer la velocidad de crecimiento de cada línea, ya que cuanto menor sea el CDT más rápido crece esa línea. Se ha estudiado la relación entre el CDT y la efectividad del RUNAT-BI, ya que la principal diana de acción principal se encuentra en el ADN y se quiere conocer la relación entre la velocidad de crecimiento celular y la efectividad del agente. El RUNAT-BI tiene mayor efecto en las líneas que más rápido se dividen, ya que, al replicarse más rápido, el complejo se intercala con el ADN que se está replicando, afectando así a la viabilidad de la célula. En la Figura 8 se observa la gráfica de crecimiento de cada una de las líneas celulares en 10 días, en la Tabla 7 se puede observar el tiempo de doblaje celular del panel de líneas celulares del experimento. Son las líneas HCT116, HCC1806 y AGS las que mayor pendiente muestran en la gráfica, por tanto, las que menor CDT tienen, como se observa en la tabla contigua y además son en las que RUNAT-BI ha resultado más efectivo, ver panel C de la Figura 3. Las líneas con mayor CDT y que, por tanto, crecen más lento son HCC1500 y HCC1937. En las líneas BT474 y MDAMB-231 RUNAT-BI presentaba menor efectividad. Este hecho se puede relacionar con que su velocidad de crecimiento es inferior a HCT116, HCC1806 y AGS. Sin embargo, no existen una relación tan obvia ya que MCF-7 es una línea que se divide relativamente rápido, sin embargo, RUNAT-BI, no tiene un efecto muy significativo en ella. De forma similar ocurre con MCF10A, que a pesar de tener una división rápida RUNAT-BI no interfiere en su crecimiento. The cell doubling time (CDT) of the cell lines HCC1500, HCC1937, MCF-7, BT474, MDA-MB-231, HCC1806, AGS, HCT 116 and MCF10A have been calculated. This value allows us to know the growth speed of each line, since the lower the CDT, the faster that line grows. The relationship between the CDT and the effectiveness of the RUNAT-BI has been studied, since the main target of action is found in the DNA and we want to know the relationship between the speed of cell growth and the effectiveness of the agent. RUNAT-BI has a greater effect on lines that divide faster, since, by replicating faster, the complex intercalates with the DNA that is being replicated, thus affecting cell viability. Figure 8 shows the growth graph of each of the cell lines in 10 days, Table 7 shows the cell doubling time of the panel of cell lines in the experiment. The lines HCT116, HCC1806 and AGS are the ones that show the greatest slope in the graph, therefore, the ones with the lowest CDT, as can be seen in the adjoining table, and they are also the ones in which RUNAT-BI has been most effective, see panel C of Figure 3. The lines with the highest CDT and, therefore, grow slowest are HCC1500 and HCC1937. In lines BT474 and MDAMB-231 RUNAT-BI presented less effectiveness. This fact can be related to the fact that its growth rate is lower than HCT116, HCC1806 and AGS. However, there is not such an obvious relationship since MCF-7 is a line that divides relatively quickly, however, RUNAT-BI does not have a very significant effect on it. Similarly, it occurs with MCF10A, which despite having a rapid RUNAT-BI division does not interfere with its growth.
TABLA 7: Tiempo de doblaje celular de un panel de líneas celulares empleadas
Figure imgf000023_0001
TABLE 7: Cell doubling time of a panel of cell lines used
Figure imgf000023_0001
A la vista de los resultados, el efecto de RUNAT-BI se relaciona con la velocidad de crecimiento celular. In view of the results, the effect of RUNAT-BI is related to the speed of cell growth.

Claims

REIVINDICACIONES
1. Compuesto de rutenio (III) que tiene la fórmula {cis-[RuCl2(H2biim)2]CI}2-4H2O en la que H2biim significa 2,2'-biimidazol. 1. A ruthenium (III) compound having the formula {cis-[RuCl 2 (H 2 biim)2]CI} 2 -4H 2 O in which H 2 biim means 2,2'-biimidazole.
2. Compuesto según la reivindicación 1 , que está formado por dos isómeros. 2. Compound according to claim 1, which is formed by two isomers.
3. Procedimiento para la preparación de un compuesto de Ru (III) definido en una de las reivindicaciones 1 a 2 que comprende: Process for the preparation of a Ru(III) compound defined in one of claims 1 to 2 comprising:
- realizar una síntesis solvotermal mediante una rampa de temperatura de un compuesto de rutenio de partida, que es RUCl3 H2O, con 2,2'-biimidazol en ácido clorhídrico, - carry out a solvothermal synthesis by means of a temperature ramp of a starting ruthenium compound, which is RUCl 3 H 2 O, with 2,2'-biimidazole in hydrochloric acid,
- una etapa de enfriamiento hasta la temperatura ambiente. - a cooling stage to room temperature.
- aislamiento del sólido obtenido en la mezcla de reacción. - isolation of the solid obtained in the reaction mixture.
4. Procedimiento según la reivindicación anterior en el que la rampa de temperatura comprende elevar la temperatura hasta una temperatura entre 85 y 95°C. 4. Method according to the preceding claim, in which the temperature ramp comprises raising the temperature to a temperature between 85 and 95°C.
5. Procedimiento según una de las reivindicaciones 3 o 4, en el que el tiempo invertido hasta alcanzar la temperatura final es de 1 a 6 horas, preferentemente de 5 horas.Process according to one of Claims 3 or 4, in which the time taken to reach the final temperature is from 1 to 6 hours, preferably 5 hours.
6. Procedimiento según una de las reivindicaciones 3 a 5, en el que la concentración de RUCl3 H2O en la mezcla de síntesis es desde 0,01 a 0,05 M, preferentemente 0,012 M. Process according to one of claims 3 to 5, in which the concentration of RUCl 3 H 2 O in the synthesis mixture is from 0.01 to 0.05 M, preferably 0.012 M.
7. Procedimiento según una de las reivindicaciones 3 a 6, en el que la concentración de 2,2 -biimidazol en la mezcla de síntesis es desde 0,01 a 0,05 M, preferentemente 0,036 M. Process according to one of Claims 3 to 6, in which the concentration of 2,2-biimidazole in the synthesis mixture is from 0.01 to 0.05 M, preferably 0.036 M.
8. Procedimiento según una de las reivindicaciones 3 a 7, en el que el ácido clorhídrico tiene una concentración en la mezcla de síntesis de 1 a 6 M, preferentemente, de 3 M. Process according to one of Claims 3 to 7, in which the hydrochloric acid has a concentration in the synthesis mixture of 1 to 6 M, preferably 3 M.
9. Compuesto definido en una de las reivindicaciones 1 o 2, u obtenido de acuerdo con el procedimiento definido en una cualquiera de las reivindicaciones 3 a 8, para su uso terapéutico. 9. Compound defined in one of claims 1 or 2, or obtained according to the process defined in any one of claims 3 to 8, for therapeutic use.
10. Compuesto definido según la reivindicación anterior, para su uso como agente anticancerígeno. 10. Compound defined according to the preceding claim, for use as an anticancer agent.
11. Compuesto según la reivindicación anterior, en el que el cáncer se selecciona entre cáncer de mama, cáncer gástrico y cáncer de colon. 11. The compound according to the preceding claim, wherein the cancer is selected from breast cancer, gastric cancer and colon cancer.
12. Compuesto según una de las reivindicaciones 10 u 11, en el que el cáncer de mama es subtipo TNA y/o luminal B. Compound according to one of claims 10 or 11, in which the breast cancer is TNA and/or luminal B subtype.
13. Compuesto según una cualquiera de las reivindicaciones 10 a 12, en el que el cáncer de mama es cáncer de mama en mujeres jóvenes. The compound according to any one of claims 10 to 12, wherein the breast cancer is breast cancer in young women.
14. Una composición farmacéutica que comprende el compuesto definido en una cualquiera de las reivindicaciones anteriores 1 o 2, u obtenido de acuerdo con el procedimiento definido en una cualquiera de las reivindicaciones 3 a 8, que comprende al menos un portador farmacéuticamente aceptable. A pharmaceutical composition comprising the compound defined in any one of the preceding claims 1 or 2, or obtained according to the process defined in any one of claims 3 to 8, comprising at least one pharmaceutically acceptable carrier.
15. Una composición farmacéutica que comprende el compuesto definido en una cualquiera de las reivindicaciones anteriores 1 o 2, u obtenido de acuerdo con el procedimiento definido en una cualquiera de las reivindicaciones 3 a 8, que comprende al menos un portador farmacéuticamente aceptable para su uso como agente anticancerígeno, según una cualquiera de las reivindicaciones 10 a 13. 15. A pharmaceutical composition comprising the compound defined in any one of the preceding claims 1 or 2, or obtained according to the process defined in any one of claims 3 to 8, comprising at least one pharmaceutically acceptable carrier for use as an anticancer agent, according to any one of claims 10 to 13.
16. La composición farmacéutica según la reivindicación 15, que se administra a un paciente por vía parenteral, oral, nasal, rectal, tópica o bucal. The pharmaceutical composition according to claim 15, which is administered to a patient parenterally, orally, nasally, rectally, topically or buccally.
17. La composición farmacéutica según una cualquiera de las reivindicaciones anteriores 15 a 16, que se administra como tratamiento posterior o simultáneamente a al menos un tratamiento seleccionado entre: cirugía, radioterapia, quimioterapia e inmunoterapia. 17. The pharmaceutical composition according to any one of the preceding claims 15 to 16, which is administered as a treatment after or simultaneously with at least one treatment selected from among: surgery, radiotherapy, chemotherapy and immunotherapy.
18. La composición farmacéutica según la reivindicación 14 a 17, que comprende además un agente terapéutico adicional como cis-platino, 5-fluorouracilo, oxaliplatino, irinotecan, capecitabina, gemcitabina, cetuximab, paclitaxel, docetaxel, bevacizumab, regorafenib, aflibercept, trastuzumab, pertuzumab, nab-paclitaxel, trastuzumab- emtansina, atezolizumab, carboplatino. 18. The pharmaceutical composition according to claim 14 to 17, which further comprises an additional therapeutic agent such as cis-platinum, 5-fluorouracil, oxaliplatin, irinotecan, capecitabine, gemcitabine, cetuximab, paclitaxel, docetaxel, bevacizumab, regorafenib, aflibercept, trastuzumab, pertuzumab, nab-paclitaxel, trastuzumab-emtansine, atezolizumab, carboplatin.
19. La composición farmacéutica según cualquiera de las reivindicaciones 15 a 18 anteriores, en la que la dosis eficaz de cis-[RuCl2(H2biim)2]CI}2-4H2O en la que l-febiim significa 2,2 -biimidazol es de 0,0001 a aproximadamente 1000 mg/kg de peso corporal por día. 19. The pharmaceutical composition according to any of the preceding claims 15 to 18, wherein the effective dose of cis-[RuCl 2 (H 2 biim)2]CI} 2 -4H 2 O wherein l-febiim means 2, 2-Bimidazole is from 0.0001 to about 1000 mg/kg of body weight per day.
PCT/ES2022/070415 2021-07-02 2022-06-29 Compound comprising ruthenium(iii) and 2,2'-biimidazole (runat-bi) and the therapeutic use thereof WO2023275422A1 (en)

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Citations (1)

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CN102516309B (en) * 2011-10-25 2014-07-02 暨南大学 Ruthenium complex capable of inhibiting tumor angiogenesis and preparation method and application thereof

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CAIPING TAN; SHENG HU; JIE LIU; LIANGNIAN JI;: "Synthesis, characterization, antiproliferative and anti-metastatic properties of two rutheniumDMSO complexes containing 2,2-biimidazole", EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, vol. 46, no. 5, 31 January 2011 (2011-01-31), AMSTERDAM, NL , pages 1555 - 1563, XP028370921, ISSN: 0223-5234, DOI: 10.1016/j.ejmech.2011.01.074 *
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