• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Use of a series of indazole compounds and their derivatives in the preparation of a drug for administration, either alone or in combination with another active substance suitable for the treatment of monoclonal gammopathies, and more specifically in multiple myeloma (mm). (Machine-translation by Google Translate, not legally binding)
    公开号:ES2644216A1
    申请号:ES201630539
    申请日:2016-04-27
    公开日:2017-11-28
    发明作者:José Antonio PÉREZ SIMÓN；María Victoria Barbado González；Maite MEDRANO DOMÍNGUEZ；Nuria Eugenia Campillo Martín；Juan Antonio PÁEZ PROSPER；Pedro José GONZÁLEZ NARANJO
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Servicio Andaluz de Salud；
    IPC主号:
    专利说明:

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    data represent the mean ± s.d. of three experiments in triplicate. Statistical significance *: p = 0.05 with respect to control conditions (untreated, CNT). See table 2 for IC50 values. (D) Cell viability after treatment with WIN-55 in the long term, 48 and 72 hours, in the most resistant and most sensitive myeloma cell lines, U266 and RPMI. On the left, cell viability determined by MTT assay at 48 hours (upper graph) and at 72 hours (lower graph). To the right of the panel, cell viability analyzed by flow cytometry at 48 hours (upper graph) and at 72 hours (lower graph). The data represent the mean ± s.d. of three independent experiments in triplicate.
    Figure 2. Selective effect of WIN-55 on the myelomatous cells of patients, while normal cells of healthy individuals are not affected. (A) Isolated bone marrow cells (BM cells) obtained from 6 MM patients were treated with WIN55 (0-50 µM) for 18 h. The different populations of BM were stained with 7AAD and an appropriate combination of antibodies to identify granulomonocitic (CD64 +), lymphocytic (CD45 +) and myelomatous (CD38 +) cells. The upper panel shows the plot of cytometric points (DotPlot) representative of a patient that corresponds to the control conditions (CNT, left) and BM cells treated with 50 µM of WIN-55 (W50, right). The lower panel shows the graph corresponding to the data obtained from the quantitative analysis of BM cells of all patients with MM (n = 6). (B) Peripheral blood cells (PB cells) from healthy donors were immunomagnetically classified using CD34 +, CD3 + and CD19 + microbeads for the isolation of hematopoietic stem cells, T cells and B cells, respectively. The upper panel shows the cell viability of hematopoietic stem cells (CD34 +), T lymphocytes (CD3 +) and B cells (CD19 +) after treatment with WIN-55 (0-50 µM) for 18 h analyzed by MTT assay. The lower panel shows cell viability in T lymphocytes (LT, CD3 +) and B cells (LB, CD19 +) after treatment with two indazole compounds of the PNG family, PGN-6 and -17. Data represent the mean ±
    s.d. of three experiments in triplicate. Statistical significance *: p = 0.05 with respect to control conditions (CNT, untreated).
    Figure 3. The anti-proliferative effect of WIN-55 is mediated primarily by caspase-dependent apoptosis mechanisms and by the Akt transduction signaling pathway. U266, the most resistant cell line, was treated with 50 µM of WIN-55 at the indicated times. (A) Western-blot of PARP forms (complete FULL and CL cleaved) and Casp-3, -9, 2 and -8 integers (PRO, pro-form) and cleaved (CL, cleaved). (B) Western-blot of the proteins of the family Bcl-2, Bak, Bax, Bcl-xL and Mcl-1. (C) Cell viability of cell lines
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    U266 and RPMI after incubation with the pan-caspase inhibitor ZVAD-FMK (PC) and / or WIN-55 (W) for 18 hours at the indicated concentrations, analyzed by MTT assay. The data represent the mean ± s.d. of three independent experiments in triplicate. Statistical significance *: p = 0.05 with respect to cells treated with 20 µM of WIN-55 for U266 and 10 µM for the RPMI cell line. (D) Effect of the compound of the invention on the Akt, -Erk, -JNK and -p38 signaling pathways evaluated by Western blot of U266 cell extracts incubated with 50 µM of WIN-55 at the indicated times. Tubulin was used as load control.
    Figure 4. WIN-55 induces the synthesis of ceramides in MM cells. (A) Immunohistochemical detection of ceramide in untreated U266 cells (CNT, left) and after treatment with 50 µM of WIN-55 for 6 hours (WIN, right). (B) Western blot of SPT, the speed-limiting enzyme of ceramide synthesis, in U266 cells treated with 50µm of WIN-55 during the indicated times. (C) PARP expression levels evaluated by Western blotting in cells treated with WIN-55 (WIN) and with / without 50 µM of Fumonisin B1 (FB1). (D) Analysis of cell viability using the MTT assay in U266 (left) and RPMI (right) cells treated with WIN-55 (W), Fumonisin B1 (FB1) or a combination of both for 18 h, at doses indicated. The data represent the mean ± s.d. of three independent experiments in triplicate. Statistical significance *: p = 0.05 with respect to cells treated with 20 µM of WIN-55 for U266 and 10 µM for the RPMI cell line. Tubulin was used as load control.
    Figure 5. WIN-55 attenuates the stress response of the basal endoplasmic reticulum in U266 cells and promotes an early loss of mitochondrial membrane potential.
    (A) Western-blot of the proteins involved in the response to deployed proteins (Unfolded Protein Response, UPR), such as CHOP, ATF-4, p-IRE1 and XBP-1s and XBP-1u after treatment with 50 µM of WIN -55 at the specified time points. (B) Loss of mitochondrial membrane potential in U266 cells after treatment with 50 µM of WIN-55 at the indicated times, as a control (CNT) incubation medium with DMSO <0.15% was used and as a positive loss control Potential CCCP was used. The data represent the mean ± s.d. of three independent experiments in triplicate. (C) Cell viability determined by MTT assay after treatment with WIN-55 and / or CB2-specific cannabinoid antagonists: PGN-8, PGN-37 and PGN-70 at 100µM for U266 and 50µM for RPMI. The data represent the mean ± s.d. of three independent experiments in triplicate. Statistical significance *: p = 0.05 with respect to cells treated with 20 µM of WIN-55 for U266 (W20) and 10 µM for the RPMI cell line (W10). (D) CB2 receptor expression pattern in several lines


    Cells and primary cells of healthy individuals (hematopoietic stem cells, T lymphocytes and B cells) determined by Western blot. The 40 kDa band corresponds to the complete monomeric form of the CB2 receptor and the 30 kDa band to the truncated form. Tubulin was used as load control.
    Figure 6. WIN-55 synergizes with other anti-myeloma agents. Determination of cell viability in U266, U266-LR7, RPMI and RPMI-LR5 using the MTT assay, after treatment with a fixed dose of WIN-55 (W; 20µM for U266, U266-LR7 and RPMI-LR5, and 10µM for RPMI), which was below the IC50 corresponding to each cell line tested according to Table 2, in combination with increasing concentrations of dexamethasone (DEX, between 5µM and 20µM) (panel A) or melphalan (MPH, between 1µM and 4µM for lines U266 and U266-LR7; and between 0.05 µM and 0.5 µM for lines RPMI and RPMI-LR5) (panel B). The asterisks indicate the values of the Combination Index (CI) corresponding to the combination and shown below each graph.
    Figure 7. WIN-55 considerably inhibits tumor growth in NGS mice in vivo. 5x106 U266 cells were inoculated subcutaneously in the interscapular flank and the mice were randomly distributed in three groups (n = 10) to receive 5 mg / kg i.p. of WIN-55 every 24 hours, every 48 hours, and the vehicle (<0.15% of DMSO in the middle) as a positive control group. The diameter of the tumors was measured every two days and the volume was estimated as the volume of the ellipse. The graph shows the evolution of the tumor volume for the indicated days. Statistical significance was defined as p≤0.05 and the asterisk indicates the first day on which the differences were statistically significant for each dose, day 13 for the group treated every 24 hours and day 16 for the group treated every 48 hours. The mice of the control group (CNT) were sacrificed on day 19 for ethical reasons. The data represent the mean ± s.d. of volume of all mice in each group. DETAILED DESCRIPTION OF THE INVENTION
    The authors of the present invention describe for the first time the use of the indazole compounds of the present invention for the prevention, relief, improvement and / or treatment of monoclonal gammopathies, and more specifically, of multiple myeloma. Multiple myeloma (MM) is a neoplasm that is characterized by the clonal proliferation of malignant plasma cells in the bone marrow and is associated with the presence of a monoclonal component or M protein in blood and / or serum.


    The therapeutic improvements presented by immunomodulatory drugs such as lenalidomide, and proteasome inhibitors such as bortezomib among other agents, have allowed remarkable progress in the control of this disease. However, it is still considered incurable. In order to achieve an effective therapy against this disease, the authors of the present invention have developed and tested compounds of an indazole nature of synthetic origin that exert an antitumor effect.
    Specifically, it is demonstrated that the different compounds studied induce selective apoptosis in myeloma cell lines and in the first stage of malignant plasma cells of patients with MM, without affecting the viability of normal cells of healthy donors, including hematopoietic stem cells. This antiproliferative effect is mediated by the activation of caspases, mainly caspase 2, and is partially avoided by a pan-caspase inhibitor. Apoptosis induced by compounds of an indazole nature was correlated with an increase in Bax and Bak expression and a decrease in Bcl-xL and Mcl-1. In addition, treatment with indazole compounds induced a biphasic Akt / PKB response and significantly increased ceramide levels in MM cells. Surprisingly, blocking ceramide synthesis prevented apoptosis induced by indazole compounds, indicating that ceramides play a key role in the pro-apoptotic effect of said compounds on MM cells. In addition, CB2 cannabinoid receptor blockade also inhibited apoptosis by indazole compounds. The compound WIN-55 increased the anti-myeloma activity of dexamethasone and melphalan by overcoming cellular resistance to melphalan in vitro. Finally, administration of the WIN-55 cannabinoid to plasmacytoma model mice significantly suppressed tumor growth in vivo.
    Together, the data suggest that these indazole compounds can be considered as therapeutic agents in the treatment of a monoclonal gammopathy, and in particular multiple myeloma.
    Thus, the present invention relates to the use of indazole derivatives in the preparation of a medicament for the prevention, relief, improvement and / or treatment of a monoclonal gammopathy, preferably multiple myeloma.
    MEDICAL USE OF THE COMPOUND OF THE INVENTION
    Therefore, a first aspect of the invention relates to the use of a compound, hereafter compound of the invention, of general formula (I):


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    (I)
    where
    -R1 and R4 are members of the group consisting of hydrogen, halogen, nitro or amino.
    5-R2 is a member of the group consisting of propyl, butyl, pentyl, cyclohexylmethyl, phenethyl, naphthylmethyl, heterocycloalkyl, secondary or tertiary primary amine, or substituted benzyl wherein the phenyl group may contain 1 or 2 substituents of the group consisting of alkyl, hydroxy, methoxy, nitro, amino or halogen.
    -R3 is a member of the group consisting of methyl, ethyl, propyl, pentyl
    10 cycloalkylmethyl, cycloalkylethyl, dialkylaminoethyl, heterocycloalkylethyl, cycloalkylcarbonyl (carbonyl group attached to cycloalkyl), heteroarylcarbonyl (carbonyl group attached to heteroaryl), arylcarbonyl (carbonyl group attached to aryl) optionally substituted, or aralkylcarbonyl (optionally substituted carbonyl group);
    15 or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof, hereinafter compound of the invention, in the preparation of a medicine for the prevention, relief, improvement and / or treatment of a monoclonal gammopathy. Alternatively,
    20 refers to the compound of the invention for use in the prevention, relief, improvement and / or treatment of a monoclonal gammopathy.
    In a preferred embodiment of this aspect of the invention:
    -R1 is a member of the group consisting of hydrogen or amino;
    -R2 is a member of the group consisting of 4-methoxybenzyl, 1-naphthylmethyl, 2naphthylmethyl, heterocycloalkyl, diisopropylamino, dimethylamino, diethylamino, piperidinyl, morpholinyl, or pyrrodinyl;
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    1- (2-morpholinoethyl) -3- (2-naphthylmethoxy) -5-nitroindazole,
    3- (3,4-dimethylbenzyloxy) -1-methyl-5-nitroindazole,
    3- (1-Naphthylmethoxy) -1- (2- (1-pyrrolidinyl) ethyl) -5-nitroindazole,
    1- (cyclohexylmethyl) -3- (3,4-dimethylbenzyloxy) -5-nitroindazole,
    5-Bromo-3- (2-naphthylmethoxy) -1- (2-piperidinoethyl) indazole,
    1- (2- (diisopropylamino) ethyl) -3- (4-methoxybenzyloxy) indazole,
    5-amino-3- (2-naphthylmethoxy) -1- (2-piperidinoethyl) indazole,
    3- (4-methoxybenzyloxy) -5-nitro-1-pentylindazole,
    3- (2-Naphthylmethoxy) -5-nitro-1-propylindazole,
    or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof.
    In another preferred embodiment of the first aspect of the invention, monoclonal gammopathy is selected from the list consisting of multiple myeloma, plasma cell leukemia, Waldeströn macroglobulinemia, amyloidosis, or any combination thereof. In a more preferred embodiment, monoclonal gammopathy is multiple myeloma.
    Here, when reference is made to the term "pharmaceutically and / or physiologically acceptable salts or solvates" refers to any pharmaceutically acceptable salt, ester, solvate, or any other compound that, in its administration, is capable of providing (direct or indirectly) a compound such as those described herein. However, it will be noted that pharmaceutically unacceptable salts also fall within the scope of the invention, since these may be useful for the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs and derivatives can be carried out by methods known in the state of the art.
    For example, pharmaceutically acceptable salts of the compounds provided herein are synthesized from the compound of the invention, by conventional chemical methods. In general, such salts are prepared, for example, by reacting the free acidic or basic forms of these compounds with a stoichiometric amount of the appropriate base or acid in water, or in an organic solvent, or in a mixture of both. In general, non-aqueous media such as ether, acetate are preferred.
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    The aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
    The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.
    The administration of the compounds, compositions or pharmaceutical forms of the present invention can be performed by any suitable method, such as intravenous infusion and oral, topical or parenteral routes. Oral administration is preferred for the convenience of patients and for the chronic nature of the diseases to be treated.
    The amount administered of a compound of the present invention will depend on the relative efficacy of the compound chosen, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg / kg / day. It is important to keep in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration.
    The compounds and compositions of the present invention can be used together with other medicaments in combination therapies. The other drugs may be part of the same composition or of a different composition, for administration at the same time
    or at different times.
    COMBINED PREPARATION OF THE INVENTION AND USES
    A third aspect of the invention relates to a combined preparation, hereafter combined preparation of the invention, which comprises or consists of:
    a) A component A that is a compound (compound of the invention) or a
    composition (composition of the invention) as defined herein
    invention, and
    b) A component B that is an active substance that is selected from the list that
    It consists of prednisone, dexamethasone, doxorubicin, plerixafor, cyclophosphamide,
    granulocyte colony stimulating factor, melphalan, thalidomide, lenalidomide,
    pomalidomide, bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab,


    MOR202, elotuzumab, autologous stem cells (sASCT), allogeneic stem cells,
    or any of its combinations.
    In an even more preferred embodiment the active ingredient of (b) is dexamethasone. In another even more preferred embodiment the active ingredient of (b) is melphalan. In another preferred embodiment, the other active ingredient is bortezomib. In another preferred embodiment, the other active ingredient is lenalinomide or thalidomide.
    In another preferred embodiment the combined preparation of the invention further comprises pharmaceutically acceptable excipients. In another preferred embodiment, the combined preparation of the invention comprises, as active ingredients, only those mentioned above, although it may comprise other pharmaceutically acceptable excipients and carriers.
    A fourth aspect refers to the use of the combined preparation of the invention where components (a) and (b) are administered simultaneously, separately or sequentially for the prevention, relief, improvement and / or treatment of a monoclonal gammopathy. Alternatively it refers to the combined preparation of the invention for simultaneous, separate or sequential use in therapy.
    A preferred embodiment of this aspect refers to the use of the combined preparation of the invention in the preparation of a medicament for simultaneous, separate or sequential use in the treatment of a monoclonal gammopathy. Alternatively it refers to the combined preparation of the invention for simultaneous, separate or sequential use.
    In another preferred embodiment of this aspect, monoclonal gammopathy is selected from multiple myeloma, plasma cell leukemia, Waldeströn macroglobulinemia, amyloidosis, or any combination thereof. In a more preferred embodiment, monoclonal gammopathy is multiple myeloma.
    The term "treatment" as understood in the present invention refers to combating the effects caused as a result of a disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:
    (i)  inhibit the disease or pathological condition, that is, stop its development;
    (ii) alleviate the disease or the pathological condition, that is, cause the regression of the disease or the pathological condition or its symptomatology;
    (iii) stabilize the disease or pathological condition.
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    The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, or tolerance of the mammal. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of indazole agent or agents that produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said prodrugs, derivatives or analogs and the therapeutic effect to be achieved. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known to those skilled in the art.
    It should be emphasized that the term "combined preparation" or also called "juxtaposition", herein, means that the components of the combined preparation need not be present as a joint, for example in a composition, in order to be available for separate application or sequential. In this way, the expression "juxtaposed" implies that it is not necessarily a true combination, in view of the physical separation of the components.
    Another aspect relates to a method of treating a monoclonal gammopathy, which comprises the administration of a compound of the invention. or any of its pharmaceutically acceptable salts, esters, tautomers, solvates and hydrates, or any combination thereof, as defined above.
    In a preferred embodiment of this aspect of the invention, the composition further comprises one or more pharmaceutically acceptable excipients or vehicles. Preferably the composition of the invention is a pharmaceutical composition comprising as the only active ingredient a compound of the invention, although it may comprise one or more pharmaceutically acceptable excipients and / or carriers. In another preferred embodiment the composition further comprises another active ingredient. In a more preferred embodiment, the other active ingredient is selected from the list consisting of prednisone, dexamethasone, doxorubicin, plerixafor, cyclophosphamide, granulocyte colony stimulating factor, melphalan, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, daxazomuma, daxatuma isatuximab, MOR202, elotuzumab, autologous stem cells (sASCT), allogeneic stem cells, or any combination thereof. In an even more preferred embodiment, the other active ingredient is dexamethasone. In another even more preferred embodiment, the other active ingredient is melphalan. In another preferred embodiment, the other active ingredient is bortezomib. In another preferred embodiment, the other active ingredient is lenalinomide or thalidomide.
    In another preferred embodiment of this second aspect of the invention, monoclonal gammopathy is selected from multiple myeloma, plasma cell leukemia,
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    Waldeströn macroglobulinemia, amyloidosis, or any combination thereof. In a more preferred embodiment, monoclonal gammopathy is multiple myeloma.
    METHOD OF PREPARATION OF THE COMPOUNDS OF THE INVENTION
    The preparation of derivatives of indazole ethers to be used according to the invention is described in European Journal of Medicinal Chemistry 2014, 73, 56-72 (EJMC-2014) and in PCT / ES2010 / 000400.
    The compounds have been prepared in several stages according to the procedures described in EJMC-2014. The first is the protection of nitrogen from position 1 of indazole derivatives by the reaction of ethyl chloroformate. The second stage consists of the introduction of the R2 group. The third step consists in the deprotection of the nitrogen of position 1 and introduction of the substituent R3 by reaction with the corresponding halides, where R1, R2 and R3 have the aforementioned significance.
    In patent (PCT / ES2010 / 000400) these indazole derivatives are claimed for the treatment, prevention or improvement of glaucoma, bronchial asthma and chronic bronchitis, of allergies such as contact dermatitis or allergic conjunctivitis, of arthritis, pain, diseases associated with organ transplants, motor disorders associated with Tourette's syndrome, Parkinson's disease or Huntington's chorea, multiple sclerosis, emesis and other toxic effects or undesirable associated with cancer and appetite chemotherapy.
    In the article (EJMC-2014) these derivatives of indazole ethers are described as potential drugs for the treatment of Alzheimer's disease.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. EXAMPLES OF THE INVENTION
    The examples show that the compounds of the invention exert a pro-apoptotic effect on MM cells, without affecting the viability of healthy cells, interacting


    selectively with CB2 receptors, triggering pro-apoptotic activity through the caspase-2 pathway, increasing pro-apoptotic regulators and decreasing anti-apoptotics, increasing de novo synthesis of ceramide, and decreasing membrane potential mitochondrial
    In addition, these new compounds inhibit tumor growth in vivo, and increase susceptibility to anti-myeloma drugs such as dexamethasone and melphalan.
    Therefore, this invention represents a very promising therapy against multiple myeloma and related diseases.
    Example 1. Materials and methods
    Statement of Ethics
    All research involving animal or human samples was approved by the Clinical Research Ethics Committee (CEIC) of the Virgen del Rocío University Hospital, and was carried out in accordance with the Declaration of Helsinki.
    Multiple myeloma cell cultures and patient cells.
    In vitro studies were carried out using six different human MM cell lines, U266, RPMI8226, MM1S, MM1R, U266-LR7 and RPMI-LR5. For ex vivo trials, human primary cells from healthy donors and patients with MM were used (Table 1). The human MM U266, RPMI8226 and MM1.S cell lines were purchased from ATCC and the U266-LR7, RPMI-LR5 and MM1.R lines were kindly provided by Dr. Enrique Ocio (University Hospital of Salamanca, Spain). Primary cells were obtained from bone marrow aspirates (BM) or peripheral blood samples (PB), and mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation and washed twice in phosphate buffered saline (PBS) It contains 1% BSA. Hematopoietic stem cells, and B and T lymphocytes were isolated from healthy PB donors by positive immunomagnetic separation using human CD34 +, CD19 + and CD3 + microbeads respectively. MM plasma cells were obtained from the bone marrow of patients (BM) with a cell infiltration of more than 30% (Table 1). MM plasma cells were identified using CD138 + and then distinguished from the other cell populations by flow cytometry using a suitable combination of antibodies: anti-human antibodies CD64-FITC, CD34-PE, CD56-APC, CD38-APC-H7 , and CD45-Pacific Blue (BD Biosciences, San Jose, CA). All cell lines are


    cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, as recommended by the supplier. For human primary cells, the concentration of FBS was up to 20%.
    Table 1. Clinical characteristics of the patients from whom the primary myeloma plasma cells were obtained. UPN: unique patient number; del = deletion; t = translocation; amp = amplification.
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    Medications and treatments.
    10 WIN-55 cannabinoid agonists, 212-2 mesylate (mesylate) were purchased from Tocris Bioscience. Indazole agonists PGN-6, -17, -34 and -72 and selective CB2 antagonists PGN-8, -37 and -70 were synthesized and kindly provided by Dr. Nuria Campillo of the Center for Biological Research, Madrid. Fumonisin B1 (FB1) was obtained from Enzo Life Sciences, Z-VAD (OMe) -FMK (pan-caspase inhibitor) from Abcam and TMRE
    15 (tetramethylrodamine methyl ester perchlorate), from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-myeloma agents dexamethasone and melphalan (melphalan) were provided by the pharmaceutical department of the Virgen del Rocío University Hospital.
    Western blot and antibodies.
    Western blot extracts were made after 0, 2, 6, 18 and 24 h. Cells were lysed according to Gilbert et al., 2002. (J Immunol Methods. 2002, 271: 185-201),
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    The evaluation of the synergy of the indazole compound WIN-55 with other anti-myeloma agents, such as dexamethasone and melphalan, was performed by evaluating cell viability by MTT assay. The potency of the combination was quantified with the Calcusyn software (Biosoft, Ferguson, MO), which is based on the Chou Talalay method and calculates a combination index (CI) with the following interpretation: CI> 1: antagonistic effect, CI = 1: additive effect and IC <1: synergistic effect.
    Analysis of mitochondrial transmembrane potential.
    U266 cell lines were exposed to 50 µM of WIN-55 for 15, 30, 45 and 60 min. The loss of mitochondrial membrane potential (Δψm) was calculated using TRME (tetramethylrhodamine-ethyl-ester-perchlorate) according to the manufacturer's instructions and CCCP (2- [2- (3-Chlorophenyl) hydrazinylyidene] propanedinitrile) was used as a control to induce the loss of Δψm.
    Immunofluorescence
    Cells treated for 6 h with indazole compounds were collected and placed on slides. Immunofluorescence staining was performed as previously described by Vielhaber et al. 2001 (Glycobiology. 2001.11: 451-7) using anti-ceramide as the primary antibody. The ceramide antibody was obtained from Sigma-Aldrich and the secondary conjugated with Alexa-488 from Abcam. As a control condition, the cells were treated with DMSO (<0.15%) in Gibco RPMI 1640 medium (Gaithersburg, MD).
    MM Xenografts.
    "NOD / scid / IL-2R gammae null" (NGS) mice were purchased from Charles River (France). Tumor xenografts were induced by subcutaneous injection of 5x106 U266 cells mixed with 100 µl of Matrigel (BD Biosciences) in 8-week-old mice. When the tumors became palpable (> 0.5 cm), the mice were randomized in the following groups (10 mice per group), which received ip: 1) 5 mg / kg WIN-55 every 24 hours, 2 ) 5 mg / kg WIN-55 every 48 hours, and 3) vehicle. Two groups were left tumor free and served as a negative control, receiving treatment every 24 or 48 hours, respectively. Tumor growth was assessed daily by measuring the two bisector diameters of the tumor with a caliper or digital king's foot. The volume was calculated using the following formula: volume = length x (width) e2 x 0.4 mm3. The animals were sacrificed when the length or width of the tumor reached 2 cm.
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    including granulomonocites (CD64 +) and lymphocytes (CD45 +), obtained from bone marrow of patients, was hardly affected. Only the highest dose tested, 50 µM, induced an antiproliferative effect on the lymphocytic population (CD45 +). For this reason, the two main lymphocytic populations were isolated, B cells (CD19 +) and T cells (CD3 +) 5 obtained from healthy individuals and then their viability was analyzed separately; As shown in Figure 2B, the antiproliferative effect observed in the lymphocytic population was mainly due to the effect on B cells (CD19 +). In addition, the effect of WIN-55 on hematopoietic stem cells (CD34 +) from healthy donors has been tested and, surprisingly, the viability of hematopoietic stem cells was unaffected.
    10 for treatment with the indazole compounds of the invention (Figure 2B). Finally, the effect of two of the PNG indazole compounds, PGN-6 and PGN-17, was also evaluated; these did not affect the viability of the lymphocytes at the doses at which an important antiproliferative effect on MM cell lines was observed.
    These results indicate that the indazole compounds of the invention have an effect.
    15 very selective pro-apoptotic on myelomatous cells, while the viability of healthy cells, including hematopoietic precursor cells, is not affected. Table 2. Inhibitory concentration values of tested indazole compounds.
    The 50 inhibitory concentration values (IC50) of different indazole compounds (listed above) tested on all myeloma 20 cell lines (listed on the left), calculated from the cell viability data obtained by the MTT assay After exposure to each of the indazole compounds in each cell line for 18 h at different doses (0-50 M). The IC50 values of the most effective indazole compounds for each cell line are highlighted in bold. At the bottom of the table the average IC50 values appear
    Corresponding to each of the indazole compounds.
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    权利要求:
    Claims (1)
    [1]
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    公开号 | 公开日
    EP3449922A4|2020-01-01|
    EP3449922A1|2019-03-06|
    US20200281897A1|2020-09-10|
    ES2644216B1|2018-09-11|
    WO2017186999A1|2017-11-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2007058626A1|2005-11-16|2007-05-24|SBio Pte Ltd|Indazole compounds|
    ES2378139B1|2009-10-01|2013-07-01|Universidad Rey Juan Carlos|FAMILY OF 3-INDAZOLIL ETERES WITH CANNABINOID AND / OR COLINERGICAL PROPERTIES.|
    GB2516436A|2013-07-20|2015-01-28|Pornthip Lattmann|Benzydamine, an anti-neoplastic agent, acting as cholecystokinin antagonist Gl and brain cancers|MX2019000006A|2016-06-23|2019-08-29|Servicio Andaluz De Salud|Compositions for the treatment of ischemic ulcers and stretch marks.|
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    EP17788869.0A| EP3449922A4|2016-04-27|2017-04-27|Indazole derivatives for cancer treatment|
    US16/097,149| US20200281897A1|2016-04-27|2017-04-27|Indazole derivatives for cancer treatment|
    PCT/ES2017/070261| WO2017186999A1|2016-04-27|2017-04-27|Indazole derivatives for cancer treatment|
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