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    • 专利摘要:
    Medical uses of apolipoprotein a and activators thereof. The present invention relates to a use of apolipoprotein A, a peptide mimetic thereof or an activator thereof for the preparation of a medicament for the treatment or prevention of cancer. The invention also relates to a method for determining the stage of a colorectal tumor in a patient comprising determining in a biofluid of said patient ApoA1 levels, wherein reduced levels of ApoA1 with respect to a reference value indicate a further stage of the tumor, as well as to a method for selecting the treatment of a patient with cancer which comprises determining in a biofluid of said patients ApoA1 levels, wherein reduced levels of ApoA1 with respect to a reference value indicate that the therapy of The choice is a therapy based on apolipoprotein A, on a mimetic peptide thereof or on an activator thereof. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2681124A1
    申请号:ES201730304
    申请日:2017-03-08
    公开日:2018-09-11
    发明作者:Cristina AGUIRRE PORTOLÉS；Guillermo REGLERO RADA；Ana RAMÍREZ DE MOLINA
    申请人:Fund Imdea Alimentacion；Fundacion Imdea Alimentacion；
    IPC主号:
    专利说明:

    Medical uses of apolipoprotein a and activators thereof 5 TECHNICAL FIELD OF THE INVENTION
    The present invention relates to a new medical use of apolipoprotein A, a mimetic peptide thereof, or an activator thereof in the treatment or prevention of cancer.
    BACKGROUND OF THE INVENTION
    The latest World Cancer Report showed that during 2012 there were about 14 million new cases and there were 8.2 million deaths associated with this disease (International Agency for Research on Cancer, 2014). According to forecasts, in the next 20 years the number of new cases will increase by approximately 70%. To date, most of the steps that lead to the formation of a tumor have been studied in detail, however, our knowledge of metastatic processes can be considered shallow. Primary tumors are the cause of 10% of deaths due to
    20 cancer while metastasis is responsible for the death of the patient in 90% of cases. Taking these premises into account, the need to find new mechanisms to slow down colonization of tissues distant from the primary tumor becomes apparent.
    25 In 2014, Teodoro Vargas et al. Demonstrated that the activation of the ABCA1, ACSL1, AGPAT1 and SCD genes was the main factor in malignant progression in patients with stage 11 colorectal cancer (U-CRC) (T Vargas, J Moreno- Rubio, J Herranz, et al. Genes associated with metabolic syndrome predict disease-free sUlVival in stage 11 colorectal cancer patients: A novel link between metabolic dysregulation and
    30 colorectal cancer. Mol Oncol. December 2014; 8 (8): 1469-81). Among them, ABCA1 (ATPbinding cassette transporter A 1) is a transporter protein whose main function is to extract from the cell the cholesterol that will be captured by apolipoprotein A-1 (ApoA1) to give rise to high density lipoproteins (HDL). In 2013, Mohelnikova-Duchonova and his collaborators reported increases in ABeA1 expression levels in one of the
    35 cancers more resistant to therapies, pancreatic cancer (Mohelnikova-Duchonova B BV, Oliverius M, Honsova E, et al. Differences in transcript levels of ABC transporters between

    pancreatic adenocarcinoma and nonneoplastic tissues. Pancreas 2013; 42: 707-716). However, Angela H. Ting's laboratory shows decreased levels of ABCA 1 in patients with malignant prostate cancer as opposed to those with benign tumors (Lee BH, Taylor MG, Robinet P, et al. Dysregulation of cholesterol homeostasis in human prostate cancer through loss of ABCA 1. Cancer Res 2013; 73: 12118). In the case of ovarian cancer, elevated levels of ABCA 1 have been linked to poor prognosis of the disease (Hedditch EL, Gao B, Russell AJ, et al. ABCA transporter gene expression and poor outcome in epithelial ovarian cancer. J Natl. Cancer Inst. 2014; 106 (7): 766--76.); However, the decrease in expression levels through hypermethylation of its promoter has also been associated with poor prognosis (Chou JL, Huang RL, Shay J, et al. Hypermethylation of the TGF-¡3 target, ASCA 1 is associated with poor prognosis in ovarian cancer patients Clin Epigenetics 2015 January 14; 7 (1) 1).
    Therefore, there is a need in the art for identifying new treatments for cancer patients, as well as methods for identifying patients who are candidates for presenting a good response to said treatment.
    SUMMARY OF THE INVENTION
    The authors of the present invention have observed that, surprisingly, apolipoprotein A, a mimetic peptide thereof or an activator thereof are capable of inhibiting the proliferation, migration and invasion of cancer cells, in particular cancer cells. that have overexpression of ASCA 1. The inventors have also discovered that patients diagnosed with tumors in advanced stages of the disease have reduced levels of ApoA 1 expression in a tumor sample with respect to patients diagnosed with tumors in earlier stages. Likewise, the inventors have discovered that patients suffering from metastases have reduced plasma ApoA1 levels compared to patients diagnosed with a primary tumor.
    Thus, in a first aspect, the invention relates to a use of apolipoprotein A, a mimetic peptide thereof or an activator thereof for the preparation of a medicament for the treatment or prevention of cancer.
     03-08-201 7
    In a second aspect, the invention relates to a method for determining the stage of a colorectal tumor in a patient comprising determining in a biofluid of said patient the levels of ApoA 1, where reduced levels of ApoA 1 with respect to a value of reference indicate a more advanced stage of the tumor.
    In a third aspect, the invention relates to a method for selecting the treatment of a cancer patient comprising determining in a biofluid of said patients the levels of ApoA 1, where reduced levels of ApoA 1 with respect to a reference value indicate that the therapy of choice is a therapy based on apolipoprotein A, a mimetic peptide thereof or an activator thereof.
    DESCRIPTION OF THE FIGURES
    FIGURE 1. A) Levels of expression of ApoA1 messenger RNA analyzed by quantitative PCR (** p value = 0.0020). B) Proliferation curve in a feasibility test carried out using XCELLigence technology (ACEA Biosciences, Inc.). The graph shows the growth of DLD1 control cells against DLD1 cells (epithelial cells derived from colon cancer) that overexpress ApoA1 e) Invasion assays carried out on Corning® BioCoaFM Matrigel plates (# 354480). Each point on the graph represents an analyzed insert. The results are shown normalized with respect to the control and represent the area invaded in the lower part of the matrix through which the cells migrate (* p value = 0.0218).
    FIGURE 2. A) Levels of ApoA1 mRNA in patients of the University Hospital of La Paz. CTR samples correspond to healthy tissue of a certain patient. Each Sil or SIII sample corresponds to a tumor sample from the same patient whose healthy tissue was also analyzed (** p value = 0.0141; **. P value = 0.0005). B) ApoA1 levels
    analyzed by an ELlSA test on plasmas derived from patients at the University Hospital of La Paz diagnosed with primary colon tumor versus metastatic patients.
    FIGURE 3. A) Proliferation curve in a feasibility test carried out using XCELUgence technology (ACEA Biosciences, Inc.). The graph shows the growth of control DLD1 cells against DLD1 cells (epithelial cells derived from colon cancer) in the absence and presence of ApoA1 (40J..lg / ml for 96 hours prior to the experiment and 20J..lg / ml for data acquisition). B) Migration and invasion tests led to

    Corning® BioCoaFM Control (# 354578) and Corning® BioCoat ™ Matrigel (# 354480) respectively. Each point on the graph represents an analyzed insert. The tests were carried out in the presence of ApoA 1 40¡.Jg / ml 72 hours prior to the experiment. The tests were performed in the presence of ApoA 1 20¡.Jg / ml. The results are shown normalized with respect to the control and represent the area occupied
    or invaded in the lower part of the matrix through which the cells migrate. (**** p value = 0.0001; • p value = 0.0386).
    FIGURE 4. A) Proliferation curve in a feasibility test carried out using XCELUgence technology. Growth of DLD1 cells treated with DMSO as a drug vehicle and with Rvx-208 (30¡JM). The right panel shows the significant differences that exist in the slope of the graph and, therefore, in the growth dynamics of the treated cells (** p value = 0.0087). B) Expression levels of E-cadherin and Vimentin proteins in the presence and absence of Rvx-208. The analysis performed by quantitative PCR. Relative normalized expression levels versus untreated control are shown.
    FIGURE 5. A) ABCA1 messenger RNA levels in the DLD1 cell line generated for its stable overexpression (** p value = 0.093). B) Proliferation curve in a feasibility test carried out using XCELUgence technology. The normalized cell index for the control cell line (NoORF, No Qpen Reading Frame) and the ABCA1 overexpressing line are shown. C) Migration and invasion tests. Data were collected and processed 72 hours after starting the experiment. (**** p value <0.0001; *** p value = 0.0009).
    FIGURE 6. A) Proliferation curve in a feasibility test carried out using XCELUgence technology. The results are shown for the stable ABCA 1 overexpression cell line in the absence and presence of ApoA 1. The cells were treated during the 72 hours prior to the experiment with 40¡Jg / ml of ApoA 1 and with a concentration of 20¡ .Jg / ml during acquisition. B) Migration and invasion tests. The procedure followed was the same as that described in Figure 1. (** p value = 0.003; **** p value <0.0001).
    FIGURE 7. A) Proliferation curve in a feasibility test carried out using XCELUgence technology. Growth of DLD1 cells treated with DMSO as a drug vehicle and with Rvx-208 (30¡JM). In the right panel the significant differences that exist in the slope of the graph and, therefore, in the dynamics of

    growth of treated cells (**** p value <0.0001). B) Migration and invasion tests. The procedure followed was the same as that described in Figure 2 (* p value = 0.0407; **** p value <0.0001). e) Expression levels of E-cadherin and Vimentin proteins in the presence and absence of Rvx-208. The analysis performed by quantitative PCR.
    5 Normalized relative expression levels are shown versus untreated control.Western blot immunodetection of ABCA1, Caveolina-1 and a-Tubulin. The latter asload control. The results of cells treated with Rvx-208 (30 IJM) or withthe DMSO vehicle.
    10 FIGURE 8: A) Proliferation curve in a feasibility test carried out using XCELUgence technology. Growth of DLD1 cells treated with DMSO as a drug vehicle and with Rvx-208 (30 IJM). The control cells (scramble, SCR) are shown in black. In gray, cells transfected with short hairpin against ApoA1. In the right panels the significant differences that exist in the values of the
    15 slope of the graphs on the left and, therefore, in the growth dynamics of the treated cells (. P value = 0.0471; •• p value = 0.0067; •••• p value <0.0001).
    Detailed description of the invention
    20 Medical use of apolipoprotein A of a mimetic peptide thereof or of an activator thereof
    The inventors have discovered that apolipoprotein A, a mimetic peptide thereof or an activator thereof are capable of inhibiting the proliferation, migration and invasion of 25 cancer cells, in particular cancer cells that exhibit ABCA1 overexpression.
    Therefore, in a first aspect the invention relates to a use of apolipoprotein A, a mimetic peptide thereof or an activator thereof for the preparation of a medicament for the treatment or prevention of cancer.
    In the context of the present invention, apolipoprotein A is selected from ApoA 1, ApoA2, ApoA4 and ApoA5. In a particular embodiment, apolipoprotein A is ApoA 1.
    In the context of the present invention, "apolipoprotein" is understood as a protein that contains and transports lipids in the blood. An apolipoprotein is a heteroprotein
     03-08-201 7
    amphipathic with a lipid prosthetic group that is part of lipoproteins. The apo-prefix of the term apolipoprotein means that it is the fundamental and protein part of lipoproteins, but apolipoprotein should not be confused with its apoprotein, which is the protein part. Either way, the term apolipoprotein used in the
    The context of the present invention refers interchangeably to apolipoprotein andcorresponding apoprotein.
    In the context of the present invention, "ApoA 1" means a gene encoding apolipoprotein A 1. The human gene is shown in the Ensembl database with the registration number ENSG00000118137.
    In the context of the present invention, "ApoA2" is understood as a gene encoding apolipoprotein A2. The human gene is shown in the Ensembl database with the registration number ENSG00000158874.
    In the context of the present invention, "ApoA4" is understood as a gene encoding apolipoprotein A4. The human gene is shown in the Ensembl database with the registration number ENSG0000011 0244.
    In the context of the present invention, "ApoA5", also known as APOA-V, RAP3, APOAV, is understood as a gene encoding apolipoprotein A5. The human gene is shown in the Ensembl database with the registration number ENSG00000110243.
    In the context of the present invention the terms "cancer" and "tumor" refer to the
    25 physiological condition in mammals characterized by uncontrolled cell growth, in which the balance between multiplication and cell death is broken. The cancer to be treated in the context of the present invention can be any type of cancer or tumor. These tumors or cancer include, and are not limited to, hematological cancers (for example leukemia or lymphomas), neurological tumors (for example astrocytomas or glioblastomas),
    30 mela noma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors (for example stomach cancer, pancreas or colon), liver cancer (for example hepatocellular carcinoma), renal cell cancer, genitourinary tumors ( for example ovarian cancer, vaginal cancer, cervical cancer, bladder cancer, testicular cancer, prostate cancer), bone tumors and vascular tumors.

    In a preferred embodiment, the cancer is a primary tumor. In another preferred embodiment, cancer is a metastasis. In another preferred embodiment, the cancer is a colorectal cancer.
    The term "colorectal cancer" (RCC), as used herein, includes any type of
    5 neoplasms of the colon, rectum and appendix and refers to both early and late adenomas and carcinoma as well as hereditary, familial or sporadic cancer. In staging systems for the classification of colorectal cancer, the colon and rectum are treated as a single organ. The invention contemplates the treatment of colorectal cancer in its different stages such as stages A, B, C1, C2 and D according to the classification of
    10 Dukes, stages A, B 1, B2, B3, CI, C2, C3 and D according to the Astler-Coller classification, stages T1, T2, T3, NO, N1, N2, MO and M1 according to the TNM system as well as stages 0,1, 11, 111 and IV according to the classification of the AJCC (American Joint Committee on Cancer). According to the tumor / node / metastasis (TNM) staging system of the American Joint Committee on Caneer (AJCC) (Greene et al. (Eds.), AJeC Staging Manual cancer. 6th ed.
    15 New York, NY: Springer, 2002), the different stages of colorectal cancer are defined as follows: Tumor: T1: the tumor invades the submucosa, T2: the tumor invades the muscularis propria, T3: the tumor invades the muscularis propria in the subserosa, or pericolic or perirectal tissues; T4: The tumor directly invades other organs or structures, and / or
    20 drills Node: NO: There is no metastasis to regional lymph nodes; N 1: metastasis in 1 to 3 regional lymph nodes, N2: Metastasis in 4 or more regional lymph nodes. Metastasis: MO: Distant metastasis not present; M1: distant metastasis present.
    25 Thus, stages O, 1, 11, 111 and IV would have the following characteristics: S-O: Tisl NOI MO (Tis = carcinoma in situ); S-I: T1 1NOI MO or T21NOI MO; S-II: T31 NOI MO or T41NOI MO; S-III: Any TI N11 MO or any TI N21 MO
    30 S-IV: Any T / any NI M1
    In the context of the invention, "cancer treatment" is understood as the administration of apolipoprotein A according to the invention to prevent or delay the onset of symptoms, complications or biochemical indications of the cancer or tumor, to alleviate its
    35 symptoms or to stop or inhibit their development and progression such as, for example, the appearance of metastases. The treatment can be a prophylactic treatment to delay the

    appearance of the disease or to prevent the manifestation of its clinical or subclinical symptoms or a therapeutic treatment to eliminate or alleviate the symptoms after the manifestation of the disease or in relation to its surgical treatment or with radiotherapy. The administration of apolipoprotein A according to the invention can be performed simultaneously or consecutively to any other treatment already known in the treatment of cancer.
    In a preferred embodiment, the cancer is characterized by being in a patient who has a high level of expression of the ASCA 1 gene with respect to a reference value, by presenting a high level of a meta bolite resulting from the activity of ASCA 1 with regarding a reference value and / or for presenting a high level of a parameter associated with the activity of ASCA 1.
    In the context of the present invention, "ASCA1", also known as CERP, HDLDT1, ABC1, ABC-1, TGD, is understood as a gene that encodes the cholesterol carrier "ATP binding cassette subfamily A member 1". The human gene is shown in the Ensembl database with the registration number ENSG00000165029.
    The quantification of the expression levels of the ABCA 1 gene can be performed from the RNA resulting from the transcription of said gene (mRNA) or, alternatively, from the complementary DNA (cDNA) of said gene. Therefore, in a particular embodiment of the invention, the quantification of the expression levels of the ASeA1 gene comprises the quantification of the messenger RNA of the ABeA1 gene, a fragment of said mRNA, complementary DNA of the TFF 3 gene, a fragment of said cDNA, or their mixtures. Additionally, the method of the invention may include performing an extraction step in order to obtain the total RNA, which can be performed by conventional techniques (Chomczynski et al., Anal. Siochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532).
    Virtually any conventional method can be used within the framework of the invention to detect and quantify mRNA levels encoded by the ASCA1 gene or its corresponding cDNA. By way of illustration, not limitation, the levels of mRNA encoded by said gene can be quantified by the use of conventional methods, for example, methods comprising amplification of mRNA and quantification of the product of amplification of said mRNA, such as electrophoresis Y

    staining, or alternatively, by Southern blot and use of appropriate probes, Northern blot and use of specific probes of the mRNA of the gene of interest (ABCA1) or its corresponding cDNA, mapping with the nuclease SI, RT-LCR, hybridization, microarrays, etc., preferably, by quantitative real-time PCR using an appropriate marker. Similarly, the levels of the cDNA corresponding to said mRNA encoded by the ASCA 1 gene can also be quantified using conventional techniques; in this case, the method of the invention includes a step of synthesis of the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA followed by amplification and quantification of the amplification product of said cDNA. Conventional methods of quantifying expression levels can be found, for example, in Sambrook, 2001. "Molecular cloning: a Laboratory Manual", 3 ed., Cold Spring Harbor Laboratory Press, N. Y., Vol. 1-3. In a particular embodiment, the quantification of the expression levels of the ABCA 1 gene is performed by a quantitative polymerase chain reaction (PCR).
    On the other hand, for the implementation of the invention, apart from quantifying the expression levels of the ASCA 1 gene, the expression levels of the protein encoded by said gene can also be quantified, that is, the ASCA1 protein, or any functionally equivalent variant of said ABCA1 protein. Thus, in a particular embodiment, the quantification of the levels of the protein encoded by the ABCA1 gene comprises the quantification of the ABeA1 protein.
    The expression level of the ASCA 1 protein can be quantified by any conventional method that allows detecting and quantifying said protein in a sample of a subject. By way of illustration, not limitation, the levels of said protein can be quantified, for example, by the use of antibodies capable of binding to ABCA1 (or fragments thereof containing an antigenic determinant) and the subsequent quantification of the complexes formed. The antibodies used in these assays may or may not be labeled. Illustrative examples of markers that can be used include radioactive isotopes, enzymes, fluorophores. Chemiluminescent reagents, enzymatic substrates or cofactors, inhibited enzymatic res, particles, dyes, etc. There is a wide variety of known assays that can be used in the present invention, which use unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibody); These techniques include Western blotting or Western blotting, ELlSA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELlSA

    (ELlSA sandwich with double antibody), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or microarrays of proteins that include specific antibodies or tests based on colloidal precipitation in formats such as dipsticks. Other ways to detect and quantify said ASCA1 protein, include affinity chromatography techniques, ligand binding assays, etc. On the market, there are commercial antibodies against ASeA1 that can be used in the context of the present invention. In a particular embodiment, the quantification of the protein levels encoded by the ASeA1 gene is performed by western blot, immunohistochemistry or ELlSA.
    In a preferred embodiment, the level of expression of ASeA1 in the primary tumor is at least twice the reference value.
    The term "reference value", as used herein in the context of the invention, refers to a laboratory value used as a reference for values / data obtained by laboratory tests of subjects or samples collected from subjects. The reference value or reference level may be an absolute value, a relative value, a value that has an upper and / or lower limit, a range of values, an average value, a medium value, an average value or a value in comparison with a particular baseline value control value. A reference value may be based on an individual sample value, such as, for example, a value obtained from a sample of the subject being evaluated, but at a time prior to the development of the disease or from a non-cancerous tissue. The reference value can be based on a large number of samples, for example, from the population of subjects of the matching chronological age group, or based on a group of samples that includes or excludes the sample to be tested. Several considerations are taken into account when determining the reference value. Among such considerations are age, weight, sex, general physical condition of the patient and the like. For example, equal amounts of a group of at least 2, at least 10, at least 100 and preferably more than 1000 subjects, preferably classified according to the above considerations, are taken as reference group, for example according to various age categories.
    The reference value for the level of expression of ASCA1 is preferably the average level of expression of ASeA1 in a group of non-tumor tissue samples. In a preferred embodiment, the non-tumor tissue is from a healthy subject that does not suffer from cancer. In a further preferred embodiment, the non-tumor tissue is from a subject suffering from cancer.

    different from the patient. In another preferred embodiment, the non-tumor tissue is from the patient himself.
    In another embodiment, the amount of the marker in a sample of a subject can be determined directly in relation to the reference value (for example, in terms of increase or decrease, or increase in number of times or decrease in number of times). Advantageously, this may allow comparing the amount of the marker in the subject's sample with the reference value (in other words, measuring the relative amount of the marker in the subject's sample against the subject with the reference value) without the need for first determine the respective absolute amounts of the marker. Once this reference value is established, the level of this marker expressed in tumor tissues of subjects can be compared with this reference value, and therefore a level of "increased" or "decreased" is assigned. For example, an increase in expression levels above the reference value of at least 1.1 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more compared to the reference value is considered as an "increased" level of expression. On the other hand, a decrease in expression levels below the reference value of at least 0.9 times, 0.75 times, 0.2 times, 0.1 times, 0.05 times, 0.025 times, 0, 02 times, 0.01 times, 0.005 -flexion or even less compared to the reference value is considered as a "diminished" level of expression.
    The term "patient" or "subject", as used herein, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, for example, humans, primates nonhumans, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a human male or female of any age or race.
    The metabolite resulting from the activity of ASCA 1 can be selected from phosphatidylcholine, phosphatidylserine, sphingomyelin, cholesterol, cholesterol esters and HDL. In a preferred embodiment of the invention, the metabolite resulting from ABCA1 activity is cholesterol.
    The determination of the metabolite resulting from the activity of ASCA1, preferably cholesterol, can be performed by any means known to the person skilled in the art in a biofluid sample.

    In the present invention, the term "sample" or "biological sample" refers to the material
    Biological isolated from a subject. The biological sample can contain any material
    Biological suitable for detecting the desired marker and can comprise cells and!
    5 or non-cellular material of the subject. The sample can be isolated from any tissue or fluid
    suitable biological, such as blood, blood plasma, serum, urine, liquid
    Cerebrospinal (CSF) or stool. In the context of the second aspect of the invention,
    The samples are samples of biofluids. The terms '' biological fluid "and" biofluid "are
    used interchangeably in this document and refer to aqueous fluids of origin
    1 o biological. The biofluid can be obtained from any location (such as blood,
    plasma, serum, urine, bile, cerebrospinal fluid, vitreous or aqueous humor, or any
    body secretion), an exudate (such as fluid obtained from an abscess or any
    another site of infection or inflammation), or the fluid obtained from a joint (by
    For example, a normal joint or joint affected by a disease such as
    fifteen rheumatoid arthritis).
    In a preferred embodiment, the biofluid is a blood sample from the patient.
    The reference value for a metabolite resulting from ABCA1 activity is
    twenty preferably the average level of concentration of said metabolite resulting from the
    ABCA 1 activity in a group of biofluid samples in subjects who do not suffer from
    disease.
    The parameter associated with the ABCA 1 activity can be selected from the life
    25 mean of caveolin-1 (CAV1), plasma HDL level, and intracellular level of esters
    of cholesterol In a preferred embodiment of the invention, the parameter associated with the
    ABCA1 activity is the half-life of caveolina-1 (CAV1). In the context of the
    This invention is understood as "CAV1", also known as BSCL3, CAV, PPH3,
    LCCNS, CGL3, VIP21, MSTP085, a gene that encodes caveolin 1. The human gene is
    30 shows in the Ensembl database with the registration number ENSG0000010597 4. In
    In the context of the invention, the term "half-life of caveolin-1" refers to time
    required to degrade 50% of a specific amount of caveolina-1. Her et al.
    (Nam-Hu Her, et al., Cell Cycle (2013), 12:10, 1521-1535) demonstrate in a cell line
    of prostate cancer that ABCA1 depletion results in a drastic reduction in
    35 CAV1 protein due to its lower stability, while overexpression of
    ABCA1 results in greater stability of CAV1 due to an increase in membrane cholesterol levels.
    5 In preferred embodiments of the invention, the reference value is the level of ABCA1 expression in a non-tumor tissue, the level of the metabolite resulting from ABCA1 activity in a healthy subject who does not suffer from cancer or the half-life value. of caveolina-1 in a subject who does not suffer from cancer.
    1 o In the context of the first aspect of the present invention, "apolipoprotein A activator" is understood as any molecule capable of increasing the expression of apolipoprotein A messenger RNA, as well as increasing the concentration of apolipoprotein A, resulting in an increase in high density lipid protein (HDL).
    fifteen In a preferred embodiment, the apolipoprotein A activator is apabetalone (RVX-208). RVX-208 (2- (4- (2-hydroxyethoxy) -3,5-dimethyllenyl) -5,7-dimethoxyquinazolin-4 (3H) -one) is described in detail in Figure 20 of U8 patent 8,053,440. Thus, in a preferred embodiment, the apolipoprotein A activator has the following structure:
    ) (_ "",
    NH
    In another preferred embodiment, the apolipoprotein A activator is lecithin cholesterol acyl transferase (LCAl). In the context of the invention, "LCAl" refers to the gene encoding the lecithin cholesterol acyl transfera8. The human gene is shown in the Ensembl database with the registration number EN8G00000213398.
    In another preferred embodiment, the apolipoprotein A activator is (48,5R) -5- [3,5-bis (trilluoromethyl) lenyl) -3- ((2- [4-lluoro-2-methoxy-5 - (propan-2-yl) lenyl) -5- (trifluoromethyl) lenyl} methyl)

    4-methyl-1, 3-oxazolidin-2-one. Thus, in this apolipoprotein A it has the following structure:
    F
    F + - {
    F
    F F
    preferred embodiment, the activator of the
    /OR
    F F
    F F
    In another preferred embodiment, the apolipoprotein A activator is trans4 - ({(5S) 5 - [{(3,5-bis (trifluoromethyl) phenyl] methyl} (2-methyl-2H-tetrazol-5-yl) ) amino] -7,9-dimethyl-2,3,4,5-tetrahydro-1 H-benzazepin-1-yl} methyl) cyclohexanecarboxylic acid Thus, in this preferred embodiment, the apolipoprotein A activator has the following structure:
    or
    OH
    In the context of the first aspect of the present invention, the term "mimetic peptide" refers to a small chain similar to the protein designed to mimic a peptide. They typically arise from the modification of an existing peptide, or by the design of 15 similar systems that mimic peptides, such as peptoids and peptides. Regardless of the approach, the altered chemical structure is designed to advantageously adjust molecular properties such as stability or biological activity. This may have a role in the development of drug-like compounds from existing peptides. These modifications involve changes in the peptide that are not
     03-08-201 7
    they will produce naturally (such as an altered linear structure and the incorporation of unnatural amino acids).
    In a preferred embodiment of the invention, the ApoA1 mimetic peptide is a lipoprotein complex comprising a fraction of ApoA-1 apolipoprotein and a lipid fraction.
    In the context of the present invention, lipoproteins are macromolecular complexes that are composed of proteins and lipids. Lipoproteins transport fats throughout the body. They are spherical, water-soluble, formed by (a) a core of apolar lipids (esterified cholesterol and triglycerides) and (b) a polar outer layer formed by apoproteins, phospholipids and free cholesterol. Lipoproteins are classified into different groups according to their density, the higher the density the higher the protein content (the larger the diameter, the higher the lipid content): chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (lDL), lipoproteins low density (LDL), and high density lipoproteins (H DL). Each type of lipoprotein has a characteristic composition and proportion of apolipoproteins (ApoA, ApoB, ApoC, ApoE).
    The lipid fraction of the lipoprotein complex comprises phosphatidylcholine (Iecithin), fosfati d i leta nola m ina (cephalin), phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, ganglioside, cerebroside, or combinations thereof.
    The molar ratio of the lipid fraction to the ApoA-1 apolipoprotein fraction may be in the range of about 200: 1 to 2: 1; from about 180: 1 to 2: 1; from about 160: 1 to 2: 1; approximately
    140: 1 to 2: 1; from about 120: 1 to 2: 1; from about 100: 1 to 2: 1; from about 90: 1 to 2: 1; from about 80: 1 to 2: 1; from about 70: 1 to 2: 1; from about 60: 1 to 2: 1; from about 50: 1 to 2: 1; from about 40: 1 to 2: 1; from about 30: 1 to 2: 1; from about 20: 1 to 2: 1; from about 10: 1 to 2: 1; from about 5: 1 to 2: 1.
    In a preferred embodiment, the lipid fraction of the lipoprotein complex consists essentially of sphingomyelin and about 3% by weight of a negatively charged phospholipid, and the molar ratio of the lipid fraction to the ApoA-1 apolipoprotein fraction is in the range from about 200: 1 to 2: 1.
     03-08-201 7
    In an even more preferred embodiment, the ApoA 1 mimetic peptide is CER-001: (www.cerenis.com/en/our-therapies/cer-OO 1).
    CER-001 comprises ApoA 1, sphingomyelin and DPPG in a ratio of phospholipid weight to lipoprotein weight of 1: 2.7; And with a weight ratio of sphingomyelin versus DPPG weight of 97: 3. CER-001 is prepared according to the method described in Example 4 of WO 2012/109162.
    Method to determine the stage of a colorectal tumor in a patient
    The inventors have discovered that patients diagnosed with tumors in advanced stages of the disease have reduced levels of ApoA 1 expression in a tumor sample with respect to patients diagnosed with tumors in earlier stages. Likewise, the inventors have discovered that patients suffering from metastases have reduced plasma ApoA 1 levels compared to patients diagnosed with a primary tumor.
    Therefore, in a second aspect the invention relates to a method for determining the stage of a colorectal tumor in a patient comprising determining in a biofluid of said patient the levels of ApoA 1, where reduced levels of ApoA 1 with respect to at a reference value they indicate a more advanced stage of the tumor.
    The terms "cancer", "colorectal cancer" and "patient", have been described in detail above, and apply equally to the methods according to the method of this second aspect of the invention.
    In the present invention, the term "sample" or "biological sample" refers to the biological material isolated from a subject. The biological sample may contain any biological material suitable for detecting the desired marker and may comprise cells and!
    or non-cellular material of the subject. The sample can be isolated from any suitable biological tissue or fluid, such as blood, blood plasma, serum, urine, cerebrospinal fluid (CSF) or feces. In the context of the second aspect of the invention, the samples are samples of biofluids. The terms "biological fluid" and "biofluid" are used interchangeably herein and refer to aqueous fluids of biological origin. The biofluid can be obtained from any location (such as blood, plasma, serum, urine, bile, cerebrospinal fluid , vitreous or aqueous humor, or any

    body secretion), an exudate (such as fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or joint affected by a disease such as rheumatoid arthritis).
    In a preferred embodiment, the biofluid is a plasma sample from the patient.
    The level of ApoA1 protein expression can be quantified by any conventional method that allows detecting and quantifying said protein in a biofluid sample of a subject. By way of illustration, not limitation, the levels of said protein can be quantified, for example, by the use of antibodies capable of binding ApoA1 (or fragments thereof containing an antigenic determinant) and the subsequent quantification of the complexes formed. The antibodies used in these assays may or may not be labeled. Illustrative examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc. There is a wide variety of known assays that can be used in the present invention, which use unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibody); These techniques include Western-blot or Western blotting, ELlSA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELlSA (ELlSA sandwich with double antibody), immunocytochemical and immunohistochemical techniques , techniques based on the use of biochips or microarrays of proteins that include specific antibodies or tests based on colloidal precipitation in formats such as dipsticks. Other ways to detect and quantify said ApoA 1 protein, include affinity chromatography techniques, ligand binding assays, etc. On the market, there are commercial antibodies against ApoA 1 that can be used in the context of the present invention. In a particular embodiment, the quantification of the protein levels encoded by the ApoA 1 gene is performed by western blot, immunohistochemistry or ELlSA.
    The term "reference value", as used herein in the context of the invention, refers to a laboratory value used as a reference for values / data obtained by laboratory tests of subjects or samples collected from subjects. The reference value or reference level may be an absolute value, a relative value, a value that has an upper and / or lower limit, a range of values, an average value, a medium value,

    an average value or a value compared to a particular baseline value control value. A reference value may be based on an individual sample value, such as, for example, a value obtained from a sample of the subject being evaluated, but at a time prior to the development of the disease or from a non-cancerous tissue. The reference value can be based on a large number of samples, for example, from the population of subjects of the matching chronological age group, or based on a group of samples that includes or excludes the sample to be tested. Several considerations are taken into account when determining the reference value. Among such considerations are age, weight, sex, general physical condition of the patient and the like. For example, equal amounts of a group of at least 2, at least 10, at least 100 and preferably more than 1000 subjects, preferably classified according to the above considerations, are taken as reference group, for example according to various age categories.
    The reference value for the ApoA1 level is preferably the average plasma ApoA1 level of a group of subjects who do not suffer from cancer. In another preferred embodiment, the reference value is the average plasma ApoA 1 level of a group of subjects diagnosed with a primary tumor. In another preferred embodiment, the reference value is the average level of ApoA 1 in plasma of a group of subjects diagnosed with metastases.
    In another embodiment, the amount of the marker in a sample of a subject can be determined directly in relation to the reference value (for example, in terms of increase or decrease, or increase in number of times or decrease in number of times). Advantageously, this may allow comparing the amount of the marker in the subject's sample with the reference value (in other words, measuring the relative amount of the marker in the subject's sample against the subject with the reference value) without the need for first determine the respective absolute amounts of the marker. Once this reference value is established, the level of this marker expressed in tumor tissues of subjects can be compared with this reference value, and therefore a level of "increased" or "decreased" is assigned. For example, an increase in expression levels above the reference value of at least 1.1 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more compared to the reference value is considered as an "increased" level of expression. On the other hand, a decrease in expression levels below the reference value of at least 0.9 times, 0.75 times, 0.2 times, 0.1 times, 0.05 times, 0.025 times, 0, 02 times, 0.01 times, 0.005 -flexion or even less

    Compared to the reference value, it is considered a "diminished" level of expression.
    Method to select the treatment of a cancer patient
    The inventors have discovered that patients suffering from metastases have levels ofApoA 1 in plasma reduced compared to patients diagnosed with a primary tumor.
    Therefore, in a third aspect the invention also relates to a method for
    10 selecting the treatment of a cancer patient comprising determining in a biofluid of said patients the levels of ApoA 1, where reduced levels of ApoA 1 with respect to a reference value indicate that the therapy of choice is a therapy based on apolipoprotein A, in a mimetic peptide thereof or in an activator thereof.
    In a preferred embodiment, cancer is a primary tumor. In another preferred embodiment, cancer is a metastasis. In another preferred embodiment, the cancer is a colorectal cancer.
    20 The terms "cancer", "colorectal cancer" and "patient" have been described in detail
    above, and apply equally to the methods according to the method of this third aspect of the invention.
    In the context of the third aspect of the invention, "cancer treatment" is understood as
    The administration of apolipoprotein A according to the invention to prevent or delay the onset of symptoms, complications or biochemical indications of cancer or tumor, to alleviate its symptoms or to stop or inhibit its development and progression such as, for example, the appearance of metastasis. The treatment can be a prophylactic treatment to delay the onset of the disease or to prevent the manifestation of its symptoms
    30 clinical or subclinical or a therapeutic treatment to eliminate or relieve symptoms after the manifestation of the disease or in relation to its surgical treatment or radiotherapy. The administration of apolipoprotein A according to the invention can be performed simultaneously or consecutively to any other treatment already known in the treatment of cancer.
     03-08-201 7
    In the present invention, the term "sample" or "biological sample" refers to the biological material isolated from a subject. The biological sample may contain any suitable biological material to detect the desired marker and may comprise cells and I
    or non-cellular material of the subject. The sample can be isolated from any suitable biological tissue or fluid, such as blood, blood plasma, serum, urine, cerebrospinal fluid (CSF) or feces. In the context of the second aspect of the invention, the samples are samples of biofluids. The terms "biological fluid" and "biofluid" are used interchangeably herein and refer to aqueous fluids of biological origin. The biofluid can be obtained from any location (such as blood, plasma, serum, urine, bile, cerebrospinal fluid , vitreous or aqueous humor, or any body secretion), an exudate (such as fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by a disease such as rheumatoid arthritis).
    In a preferred embodiment, the biofluid is a plasma sample from the patient.
    The level of ApoA1 protein expression can be quantified by any conventional method that allows detecting and quantifying said protein in a biofluid sample of a subject. By way of illustration, not limitation, the levels of said protein can be quantified, for example, by the use of antibodies capable of binding ApoA1 (or fragments thereof containing an antigenic determinant) and the subsequent quantification of the complexes formed. The antibodies used in these assays may or may not be labeled. Illustrative examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc. There is a wide variety of known assays that can be used in the present invention, which use unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibody); These techniques include Western-blot or Western blotting, ELlSA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELlSA (ELlSA sandwich with double antibody), immunocytochemical and immunohistochemical techniques , techniques based on the use of biochips or microarrays of proteins that include specific antibodies or tests based on colloidal precipitation in formats such as dipsticks. Other ways to detect and quantify said ApoA 1 protein, include affinity chromatography techniques, ligand binding assays, etc. In 21 10

    On the market, there are commercial antibodies against ApoA 1 that can be used in the context of the present invention. In a particular embodiment, the quantification of the protein levels encoded by the ApoA 1 gene is performed by western blot, immunohistochemistry or ELlSA.
    The term "reference value", as used herein in the context of the invention, refers to a laboratory value used as a reference for values / data obtained by laboratory tests of subjects or samples collected from subjects. The reference value or reference level may be an absolute value, a relative value, a value that has an upper and / or lower limit, a range of values, an average value, a medium value, an average value or a value in comparison with a particular baseline value control value. A reference value may be based on an individual sample value, such as, for example, a value obtained from a sample of the subject being evaluated, but at a time prior to the development of the disease or from a non-cancerous tissue. The reference value can be based on a large number of samples, for example, from the population of subjects of the matching chronological age group, or based on a group of samples that includes or excludes the sample to be tested. Several considerations are taken into account when determining the reference value. Among such considerations are age, weight, sex, general physical condition of the patient and the like. For example, equal amounts of a group of at least 2, at least 10, at least 100 and preferably more than 1000 subjects, preferably classified according to the above considerations, are taken as reference group, for example according to various age categories.
    The reference value for the ApoA 1 level is preferably the average plasma ApoA 1 level of a group of subjects who do not suffer from cancer. In another preferred embodiment, the reference value is the average plasma ApoA1 level of a group of subjects diagnosed with a primary tumor. In another preferred embodiment, the reference value is the average level of ApoA 1 in plasma of a group of subjects diagnosed with metastases.
    In another embodiment, the amount of the marker in a sample of a subject can be determined directly in relation to the reference value (for example, in terms of increase or decrease, or increase in number of times or decrease in number of times). Advantageously, this may allow comparing the amount of the marker in the subject's sample with the reference value (in other words, measuring the relative amount of the marker in the subject's sample against the subject with the reference value) without the need for first determine the respective absolute amounts of the marker. A

    Once this reference value is established, the level of this marker expressed in tumor tissues of subjects can be compared with this reference value, and therefore a level of "increased" or "decreased" is assigned. For example, an increase in expression levels above the reference value of at least 1.1 times, 1.5 times, 2 times, 5 5 times, 10 times, 20 times, 30 times, 40 times, 50 times , 60 times, 70 times, 80 times, 90 times, 100 times or even more compared to the reference value is considered as an "increased" expression level. On the other hand, a decrease in expression levels below the reference value of at least 0.9 times, 0.75 times, 0.2 times, 0.1 times, 0.05 times, 0.025 times, 0, 02 times, 0.01 times, 0.005 -flexion or even less
    10 compared to the reference value is considered as a "decreased" level of expression.
    In the context of the present invention, apolipoprotein A is selected from ApoA 1, ApoA2, ApoA4 and ApoA5. In a particular embodiment, apolipoprotein A is ApoA 1.
    15 The terms "apolipoprotein A", "ApoA 1", "ApoA2", "ApoA4" and "ApoA5" have been described in detail above, and apply equally to the methods according to the method of this third aspect of the invention.
    In the context of the third aspect of the present invention, "apolipoprotein A activator" means any molecule capable of increasing the expression of apolipoprotein A messenger RNA, as well as increasing the concentration of apolipoprotein A, resulting in an increase in high density lipid protein (HOL).
    In a preferred embodiment, the apolipoprotein A activator is apabetalone (RVX-208). RVX-208 (2- (4- (2-hydroxyethoxy) -3,5-dimethylphenyl) -5,7-dimethoxyquinazolin-4 (3H) -one) is described in detail in Figure 20 of US Patent 8,053,440. Thus, in a preferred embodiment, the apolipoprotein A activator has the following structure:
     03-08-201 7
    NH
    or
    In another preferred embodiment, the apolipoprotein A activator is lecithin cholesterol acyl transferase.
    In another preferred embodiment, the apolipoprotein A activator is (48, 5R) -5- (3,5-bis (trilluoromethyl) lenyl) -3- ((2- [4-lluoro-2-methoxy-5- (propan-2-yl) lenyl) -5- (trifluoromethyl) lenyl) methyl) 4-methyl-1,3-oxazolidin-2-one. Thus, in this preferred embodiment, the apolipoprotein A activator has the following structure:
    /
    OR
    F F
    In another preferred embodiment, the apolipoprotein A activator is trans-4 - ({(5S) 5 - [{[3,5-bis (trifluoromethyl) phenyl] methyl} (2-methyl-2H-tetrazol-5) -yl) amino] -7,9-dimethyl-2,3,4,515 tetrahydro-1 H-benzazepin-1-yl} methyl) cyclohexanecarboxylic. Thus, in this preferred embodiment, the apolipoprotein A activator has the following structure:
     03-08-201 7
    or
    OH
    F
    F
    F
    F F
    In the context of the third aspect of the present invention, the term "mimetic uppeptide" refers to a small chain similar to the protein designed to mimic a peptide. 5 Typically they arise from the modification of an existing peptide, or by the design of similar systems that mimic peptides, such as peptoids and peptides! 3. Regardless of the approach, the altered chemical structure is designed to advantageously adjust molecular properties such as stability or biological activity.This may have a role in the development of drug-like compounds from from
    10 existing peptides. These modifications involve changes in the peptide that will not occur naturally (such as an altered linear structure and the incorporation of unnatural amino acids).
    In a preferred embodiment of the invention, the ApoA1 mimetic peptide is a lipoprotein complex comprising a fraction of ApoA-1 apolipoprotein and a lipid fraction.
    In the context of the present invention, lipoproteins are macromolecular complexes that are composed of proteins and lipids. Lipoproteins transport fats throughout the body. They are spherical, water-soluble, formed by (a) a core of apolar lipids (esterified cholesterol and triglycerides) and (b) a polar outer layer formed by apoproteins, phospholipids and free cholesterol. Lipoproteins are classified into different groups according to their density, the higher the density the higher the protein content (the larger the diameter, the higher the lipid content): chylomicrons, very low density lipoproteins
    25 (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Each type of lipoprotein has a characteristic composition and proportion of apolipoproteins (ApoA, ApoB, ApoC, ApoE).
     03-08-201 7
    The lipid fraction of the lipoprotein complex comprises phosphatidylcholine (Iecithin), phosphatidylethanolamine (cephalin), phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, ganglioside, cerebroside, or combinations thereof.
    The molar ratio of the lipid fraction to the ApoA-1 apolipoprotein fraction may be in the range of about 200: 1 to 2: 1; from about 180: 1 to 2: 1; from about 160: 1 to 2: 1; approximately
    140: 1 to 2: 1; from about 120: 1 to 2: 1; from about 100: 1 to 2: 1; from
    10 about 90: 1 to 2: 1; from about 80: 1 to 2: 1; from about 70: 1 to 2: 1; from about 60: 1 to 2: 1; from about 50: 1 to 2: 1; from about 40: 1 to 2: 1; from about 30: 1 to 2: 1; from about 20: 1 to 2: 1; from about 10: 1 to 2: 1; from about 5: 1 to 2: 1.
    In a preferred embodiment, the lipid fraction of the lipoprotein complex consists essentially of sphingomyelin and about 3% by weight of a negatively charged phospholipid, and the molar ratio of the lipid fraction to the ApoA-1 apolipoprotein fraction is in the range of approximately 200: 1 to 2: 1.
    In an even more preferred embodiment, the ApoA1 mimetic peptide is CER-001: (wvvw.cerenis.com/en/our-therapies/cer-OO 1).
    CER-001 comprises ApoA 1, sphingomyelin and DPPG in a ratio of phospholipid weight to lipoprotein weight of 1: 2.7; And with a weight ratio of sphingomyelin versus
    DPPG weight of 97: 3. CER-001 is prepared according to the method described in Example 4 of WO 2012/109162.
    The invention is detailed below by means of the following examples, which are merely illustrative and in no way intended to limit the scope of the invention.
    EXAMPLES
    Materials and methods

    Viability and cell proliferation assays have been carried out using XCELLigence technology (ACEA Biosciences, Inc.) following the manufacturer's instructions. Migration and invasion tests have been carried out on Corning® BioCoat ™ Control (# 354578) and Corning® SioCoat ™ Matrigel (# 354480) plates respectively, following the manufacturer's instructions. For rescue trials, cells were treated for 72 hours with apabetalone (RVX-208; Biovision) 30 ~ M. Antibodies: ABCA1 (Ab18180, Abeam. 1: 500); Caveolina-1 (Ab291O, Abcam. 1: 1000); a-tubulin (T9026, Sigma 1: 1000). DLD1 cells correspond to an adherent epithelial cell line derived from human colorectal cancer (ATCC® CCL-22pM). Stable cell lines capable of overexpressing ASCA 1 have been obtained by transducing genetically modified lentiviruses.
    Example 1: ApoA 1 protein offers protection against cellular malignancy
    In relation to the first aspect of this invention, the generation of stable DLD1 cell lines for overexpression of ApoA1 (Figure 1A) allowed the inventors to observe that, although there are no significant differences in proliferative capacity (Figure 18), those cells With elevated levels of ApoA1, the invasive capacity is diminished (Figure 1C). On the other hand, when analyzing the levels of ApoA1 expression in two cohorts of colorectal cancer patients of the University Hospital of La Paz, the inventors observed how those patients in stage U (CRC-TNM Stage 11 or Sil) express, in the tissue tumor, levels of ApoA1 that are significantly higher than the corresponding healthy tissue. However, those patients in more advanced and therefore more invasive stages have a significant decrease in ApoA1 messenger RNA levels (Figure 2A). An plasma ELlSA analysis derived from patients from the same hospital who had been diagnosed with a primary colon tumor and from patients presenting with metastases was also performed (Figure 28). Therefore, the results described suggest that ApoA 1 protein offers protection against cellular malignancy.
    Example 2: ApoA 1 protein inhibits proliferation migration and invasion of DLD1 cells
    In order to corroborate the previous results in vitro, the inventors carried out cell viability tests that allowed them to demonstrate how the administration of ApoA1 in a concentration of 40IJg / ml in a first dose of 72h and in a concentration of 20IJg / ml during the acquisition of results it results in a decrease 27 10

    Significant cell proliferation (Figure 3A). Following the same scheme as soon as
    upon treatment, the inventors carried out migration and invasion tests. In this case, those cells treated with ApoA 1 showed diminished their ability to migrate to the serum-rich region or invade the extracellular matrix (Figure 38).
    Example 3: RVX-20B inhibits proliferation of DLD1 cells
    The same trials were repeated replacing ApoA 1 with an RVX treatment.
    208. In this case, DLD1 cells were pre-treated during the 72h prior to the start of the experiment with an RVX-208 concentration of 30IJm and were subjected to an RVX-208 treatment of 15IJM during the development of the experiments. First, as shown in Figure 4A, such treatment caused a significant decrease in proliferative capacity. On the other hand, the treated cells present alterations in the levels of gene expression that are used as markers of the characteristic epitheliomenechymal transition of tumor cells; that is: increase in E-cadherin expression levels and decrease in vimentin levels (Figure 48).
    Example 4: ApDA 1 protein inhibits proliferation migration and invasion of DLD1 cells
    that express ABCA 1
    When compared to the control situation, DLD1_A8CA1 cells that express elevated levels of the A8CA1 transporter (Figure 5A) showed greater proliferative capacity (Figure 58), as well as a significant increase in migration and invasion capabilities (Figure 5C). When treated with 40IJg / ml of apolipoprotein A1, DLD1_ABCA1 cells reduced their proliferative capacity to levels comparable to the control situation (Figure 6A). In addition, the increase in migratory and invasive capacities was also controlled thanks to the treatment (Figure 68).
    Example 5: RVX-20B inhibits the proliferation of DLD1 cells expressing ABCA1
    The inventors were able to confirm these results thanks to the treatment with RVX-208 of DLD1 cells expressing A8CA1 under the conditions already detailed in Example 3. In this case, DLD1 cells that overexpress A8eA1 were pre-treated during the 72h prior to Start of the experiment with an RVX-208 concentration of 30IJm and underwent a treatment with RVX-208 of 15IJM during the development of the experiments. First, as shown in Figure 7A, such treatment caused a

    significant decrease in proliferative capacity. In addition, the increase in migratory and invasive capacities was also controlled thanks to the treatment (Figure 7B). On the other hand, the treated cells show alterations in the levels of gene expression that are used as markers of the epithelial-mesenchymal transition characteristic of tumor cells; that is: increase in Ecadherina expression levels and decrease in vimentin levels (Figure 7e, graph on the left). In this experiment, the levels of Caveolin-1, a protein whose elevated levels have been associated with increases in migration and cell invasion, were also analyzed. Through a Western blot test, the inventors were able to verify that the treatment
    10 with RVX-208 is able to decrease the levels of said protein (Figure 7C, right panels).
    Example 6: RVX-208 acts through increasing levels of ApoA 1
    15 In order to confirm that the effects of RVX-208 on the recovery of the cellular phenotype were due to increased expression in ApoA-1, the inventors inhibited the expression of said protein by transfection of a small complementary RNA, ShApoA1 (Figure 8). 24 hours after the Iransfection with the vector for expression of ShApoA 1, the inventors began treatment with 30IJM of RVX-208. Feasibility and
    Cell proliferation in OL01 cells (O_NoORF) and in 0101 cell lines expressing high levels of ApoA1 (0_ApoA1) were analyzed with the xCELUgence system. In both cases, those cells treated with RVX-208 and in which the expression of ApoA1 (Scr + Rvx-208) had not been inhibited, proliferation rates were significantly lower. In addition, protein interference with shApoA 1 causes
    25 a significant increase in the proliferation levels of control cells. Both results demonstrate that the antiproliferative effects of RVX-208 are due to an increase in the expression levels of Apolipoprotein A 1.
     11-13-2017
    权利要求:
    Claims (10)
    [1]
    1. Use of an apolipoprotein A activator for the preparation of a medicament for the treatment or prevention of cancer, wherein the cancer is a colorectal cancer, and where the apolipoprotein A activator has the following structure:
    0 ",
    °
    [2]
    2. Use according to claim 1 wherein the cancer is a primary tumor. 10
    [3]
    3. Use according to claim 1 wherein the cancer is a metastasis.
    [4]
    Four. Use according to any one of claims 1 to 3 where the cancer is characterized by being in a patient who has a high level of
    The expression of the ASeA1 gene with respect to a reference value, for presenting a high level of a metabolite resulting from the activity of ASCA 1 with respect to a reference value and / or for presenting a high level of a parameter associated with the activity of ASCA 1.
    Use according to claim 4 wherein the level of expression of ASCA1 in the primary tumor is at least twice the reference value.
    [6]
    6. Use according to claim 4 wherein the metabolite resulting from the activity of ASCA 1 is cholesterol.
    [7]
    7. Use according to claim 4 the parameter associated with the activity of ASCA 1 is the half-life of caveolin-1.


    [8]
    8. Use according to anyonefromclaims 4to7 inwhere the value of
    reference is the level of expression of ABeA 1 in a non-tumor tissue, the level
    of the metabolite resulting from ABeA1 activity in a healthy subject that does not
    suffers from cancer or the half-life value of caveolina-1 in a subject that does not
    5 suffer from cancer
    [9]
    9. MethodforselecthetreatmentfromapatientwithCancerthat
    comprises determining in a biofluid of said patients the levels of ApoA 1,
    where reduced levels of ApoA 1 with respect to a reference value
    10 indicate that the therapy of choice is a therapy based on an activator of the
    apolipoprotein A, where the cancer is a colorectal cancer, and where the
    Apolipoprotein A activator has the following structure:
    NH
     0 "" ° 
    [10]
    10. Method according to claim 9 wherein the cancer is a primary tumor.
    [11]
    eleven. Method according to claim 9 wherein the cancer is a metastasis.
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    同族专利:
    公开号 | 公开日
    US20200378993A1|2020-12-03|
    ES2681124B1|2019-06-19|
    WO2018162984A1|2018-09-13|
    EP3594683A1|2020-01-15|
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    ES201730304A|ES2681124B1|2017-03-08|2017-03-08|MEDICAL USES OF APOLIPOPROTEIN A AND ACTIVATORS OF THE SAME|ES201730304A| ES2681124B1|2017-03-08|2017-03-08|MEDICAL USES OF APOLIPOPROTEIN A AND ACTIVATORS OF THE SAME|
    US16/491,948| US20200378993A1|2017-03-08|2018-03-08|Medical uses of apolipoprotein a and activators thereof|
    PCT/IB2018/000317| WO2018162984A1|2017-03-08|2018-03-08|Medical uses of apolipoprotein a and activators thereof|
    EP18719637.3A| EP3594683A1|2017-03-08|2018-03-08|Medical uses of apolipoprotein a and activators thereof|
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